[CANCER RESEARCH 49. 4879-4886. September I. I989|
Role of Ligand in Antibody-directed Endocytosis of Liposomes by Human
T-Leukemia Cells1
Katherine K. Matthay,2 Anna M. Abai, Suzanne Cobb,1 Keelung Hong, Demetrios Papahadjopoulos, and
Robert M. Straubinger4
Department of Pediatrics ¡K.K. M., S. C.], Cancer Research Institute [K. K. M., A. M. A., K. H., D. P.], and Department of Pharmacology [R. M. S., D. P.],
University of California, San Francisco, California 94143
ABSTRACT
The rate of uptake and intracellular processing of ligand-directed drug
carriers may depend heavily on the endocytic pathway of the target
antigen. We examined the role of the target antigen and type of antibodyliposome linkage in determining endocytosis of liposomes by three human
I -cell leukemias, Jurkat, CEM, and Molt-4. Liposome-cell binding and
internalization over time were studied using two independent assays for
intracellular delivery of liposome contents: a new fluorescence assay
using a pH-sensitive fluorescent dye; and a growth inhibition assay for
delivery of cytotoxic drug, methotrexate-7-aspartate. Liposomes targeted
against the transferrin receptor showed greater surface binding, internal
ization, and growth inhibition than liposomes targeted against the T-cell
surface antigens, CD2, CD3, or CDS. Furthermore, liposomes made by
conjugating the targeting antibody directly to the liposome surface were
more efficiently internalized and retained than were liposomes linked to
antibody-coated cells via Protein A. Selection of the type of antibodyliposome conjugate as well as the appropriate surface receptor to facilitate
endocytosis is essential in antibody-directed drug treatment of cancer.
INTRODUCTION
The particular cell surface antigen which provides the target
for antibody-directed cytotoxic therapy is of prime importance
in determining the subsequent fate of the immunoconjugate
after it binds to the cell. It is likely that the rate of antigen
internalization and recycling is a vital factor in the uptake of
the cytotoxic agent and its delivery to the compartment in
which it exerts its toxic effect.
Recent research suggests that uptake of liposomes by cells
depends on endocytosis via coated pits, followed by intracellular
processing, similar to that of a number of macromolecules
(1, 2). We and others have utilized this fact to design therapy
of low nonspecific toxicity by encapsulating a drug or toxin
which is relatively impermeant on its own and therefore de
pendent on endocytosis of the liposome for uptake (3-11). We
have shown specific enhancement of growth-inhibitory effect of
one such agent, methotrexate-7-aspartate,
when encapsulated
in antibody-targeted liposomes, both with direct antibody-liposome conjugates (3, 4), and with the indirect combination of
antibody-coated tumor cells with Staphylococcus aureus Protein
A liposomes (5). Liposomes conjugated to Protein A proved to
be 100-fold more effective than unencapsulated drug for growth
inhibition of a murine T-cell leukemia, AKR/J SL2, when cells
were coated with specific antibody, and 10-fold more effective
Received 9/15/88; revised 2/15/89. 5/9/89; accepted 5/16/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by USPHS Grants CA39448 (K. K. M.) and CA35340 (D. P.)
awardec by the National Cancer Institute, Department of Health and Human
Services.
2To 'vhorn requests for reprints should be addressed: Department of Pediatrics.
Box 0106. 653 M, University of California, School of Medicine, San Fran
cisco, C \ 94143.
3 Recipient of Pediatrie Clinical Research Center Summer Fellowship, USPHS
Grant RR01271.
4 Present address: Department of Pharmaceutics. 539 Cooke Hall. State Uni
versity c f New York at Buffalo. Amhersl. NY 14260.
than the liposomes using a nonspecific antibody (5). However,
when this system was extended to human leukemia lines (12),
the Protein A liposomes in combination with anti-T-cell anti
bodies proved to be relatively ineffective. The Protein A lipo
somes were also ineffective with antiidiotype antibodies against
a B-cell lymphoma, exhibiting specific cell association but no
internalization. It was only when an anti-Dr antibody was used
with the B-cell lymphoma that the Protein A liposomes had
a growth-inhibitory effect greater than that with nonspecific
antibody.
Because of the variability of liposome efficacy among the
human cell lines, we have further explored the importance of
both the type of liposome conjugate (Protein A versus direct
antibody-liposome) and the role of the target antigen in pro
moting endocytosis of liposomes. The transferrin receptor was
selected as a possible mediator of liposome uptake because of
its well-defined fate in the endocytic pathway. Transferrin that
is bound to its receptor is internalized via a coated-pit pathway
(13, 14). Dissociation of iron from transferrin occurs in the
acidic endosomal compartment (15-17), and apotransferrin and
its receptor are recycled to the cell surface, where the ligand is
released. Recycling thus allows the receptor to undergo further
cycles of endocytosis. The continuous endocytosis and recycling
of the transferrin receptor (18, 19) may make it an efficient
means of endocytosis of liposomes bearing transferrin or anti
bodies to the transferrin receptor. Preliminary studies suggest
the utility of these receptors in targeted therapy. There are
reports of conjugation of transferrin itself to liposomes, result
ing in successful uptake by cells (20, 21 ), and other reports of
transferrin or antitransferrin receptor antibody conjugated to
toxins enhancing uptake by tumor cells (22, 23). In the studies
reported here, we have compared the uptake of liposomes
mediated by antibodies against the transferrin receptor to the
uptake via T-cell-specific surface antigens.
Two independent assays of endocytosis were used in these
studies: a recently developed fluorescence assay using pyranine
or HPTS,5 a pH-sensitive fluorescent dye (24, 25), as a marker
of entry into the endosomal compartment (26, 27); and the
other a growth inhibition assay for efficacy of methotrexate-7aspartate, a drug which is relatively impermeant on its own,
and therefore dependent on liposome uptake to enter the cyto
plasm (3).
We have also studied the effect of varying the type of anti
body-liposome conjugate and type of cell receptor target on
liposome endocytosis using these two assays. Protein A-liposomes with antibody-coated cells were compared to direct an
tibody-liposome conjugates.
MATERIALS AND METHODS
Phosphatidylcholine from egg yolk was obtained from Avanti (Bir
mingham, AL) and used without further purification. Cholesterol was
'The abbreviations used are: HPTS. 8-hydroxypyrene-1.3,6-pyrenetrisulfonatc; REV, reverse-phase evaporation vesicles; SPA. Staphylococcus aureus Pro
tein A; Ab. monoclonal antibody; FITC. fluorescein isothiocyanate: IC50, 50%
inhibitory concentration.
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LIPOSOME ENDOCYTOSIS
IN T-ACUTE LYMPHOCYTIC
recrystallized 4 times from cold methanol. The 4-(p-maleimidophenyl)butyrylphosphatidylethanolamine
was synthesized and purified
as described (28). All lipids were stored at —¿
70°Cin chloroform solution
under argon in sealed ampuls until use. Methotrexate-7-aspartate was
synthesized and kindly provided by Dr. J. R. Piper, Southern Research
Institute, Birmingham, AL (29). Pyranine (HPTS) was purchased from
Molecular Probes (Eugene, OR) and S. aureus Protein A from Sigma
(St. Louis, MO).
Monoclonal Antibodies. Monoclonal antibodies were prepared and
purified as previously described (3). Hydridoma SS.1 (anti-sheep erythrocyte, irrelevant control) was supplied by Dr. M. Cohn of the Salk
Institute, San Diego, CA. The remainder of the antibodies were gener
ously supplied already purified. Leu 1 (anti-CD5) (30) was provided by
Dr. T. Reichert, Becton Dickinson, Mountain View, ÇA;B3/25 (antitransferrin receptor) (31, 32) by Dr. I. Trowbridge of the Salk Institute,
San Diego, CA; and OKT3 (anti-CD3) (33), OKT9 (antitransferrin
receptor) (34), and OKT11 (anti-CD2) (35) by Dr. G. Goldstein, Ortho
Pharmaceutical, Kari tan, NJ; and 14.2a (anti-GD2, irrelevant control)
by Dr. R. Seeger, UCLA, Los Angeles, CA. All antibodies are of the
IgG2 subclass and reactive with Protein A, except the antitransferrin
receptor antibodies, which are IgGl.
Cell Lines. The human T cell leukemia lines used for this investiga
tion were Jurkat, CEM (36), and Molt-4 (37), kindly provided by Dr.
M. Cowan at the University of California, San Francisco, and were
grown in RPMI medium supplemented with 2 HIM glutamine, 10%
fetal bovine serum, and antibiotics in the presence of 3% CO2.
Liposome Preparation. A mixture of phosphatidylcholine, choles
terol, and 4-(p-maleimidophenyl)butyrylphosphatidylethanolamine
(10:5:1 molar ratio) was suspended in a solution of 50 mM methotrexate-7-aspartate or 50 mivi HPTS in isotonic buffer containing
2-(jV-morpholino)ethanesulfonic
acid and 2-(7V-morpholino)propanesulfonic acid, pH 6.7. The liposomes were then prepared by re
verse-phase evaporation as previously described (3). The liposomes
were extruded (38) 5 times through two O.OS-^m-pore polycarbonate
membranes under sterile conditions using a high-pressure extruder
(Lipex Biomembranes, Inc., Vancouver, Canada). The REV were then
conjugated for 18 h at 25°Cwith thiolated SPA (39) or Ab (40). The
conjugated REV were removed from unbound protein by flotation on
a metrizamide gradient (3) with modifications previously described (5).
Liposomes were then passed over a Sephadex G50 (Pharmacia) column
into isotonic 2-(Ar-morpholino)ethanesulfonic
acid/2-(7V-morpholino)propanesulfonic acid/NaCl, pH 7.5, to remove unencapsulated drug
and metrizamide, concentrated to 5 mM lipid in an Amicon YM-10
membrane. After sterilization by passage through a 0.2-^m filter, lipo
somes were analyzed for lipid, drug, and protein content (3). Liposome
size was estimated by triplicate measurements of laser light scattering
on a Coulter Model N4 submicron particle analyzer (41). Liposome
characteristics were as follows: mean drug:lipid, 0.037 ±0.007 mol/
mol; protein:lipid, 29.6 ±16.1 g/mol for Ab-REV and 35.1 ±14.3 g/
mol for SPA-REV. Before conjugation to antibody, the liposome di
ameter was 0.099 ±.016 ¿im.After conjugation to antibody, the mean
diameter increased to 0.178 ±0.078 j/m.
Liposome-Cell Association Assay. The pH-dependent fluorescent dye,
HPTS, was encapsulated in the liposomes to monitor their progress
from the neutral extracellular compartment to the low pH of the
endosomal or lysosomal compartments.
Leukemia cells were suspended in growth medium at 0°Cwith the
relevant antibody for 30 min for assays using SPA-REV, then washed
free of excess antibody. Subsequently, liposomes containing HPTS (20
nmol of lipid) were incubated with 2 x IO6cells in a volume of 0.2 ml
for 45 min at 0°C.Unbound liposomes were removed by centrifugation
of cells through a dextran gradient, and the cells were then incubated
for an additional designated period at 37°Cin growth medium. At the
appropriate time points, cells were washed with chilled Dulbecco's
phosphate-buffered saline to remove any released HPTS or liposomes,
then examined by fluorescent microscopy or fluorimetry. In an alternate
experimental design to simulate conditions of the growth inhibition
studies, 1 x 10s cells were plated per well in 24-well plates. Liposomes
containing HPTS were added at two concentrations in quadruplicate
(2.5 and 25 nmol/well). After 72 h, when the cell concentration had
LEUKEMIA
reached 106/ml, the unbound liposomes were removed by centrifugation
through dextran, and the cell association fluorescence assay was done
as above.
Fluorescent Microscopy and Fluorimetry. Fluorescence microscopy
was used for visual evaluation of cell association and endocytosis. The
pH-independent fluorescence of HPTS can be viewed by exciting with
wide band violet (350 to 410 nm) provided by the Hoechst filter cube,
reflected through long wave pass dichroic mirror (455 nm) and long
wave pass barrier filter (445 nm). The second filter cube, FITC, selects
narrow blue band (450 to 490 nm) for excitation with long pass dichroic
mirror (510 nm) and long pass barrier filter (515 nm), such that only
the fluorescence of HPTS at neutral or basic pH can be seen. The
appearance of bright punctate fluorescence seen with the Hoechst cube
but not with the FITC cube reflects HPTS in the low pH compartment
(Ref. 27; Footnote 5). Photographs of identical fields were taken with
phase contrast, Hoechst, and FITC at each time point.
Kinetic measurements of total fluorescence emission at 510 nm with
excitation at the desired wavelengths were made on a SPEX Fluorolog
2 equipped with a photon counting device. Sample temperature was
controlled with a circulating water bath at 20°C.Leukemia cells (2 x
10") were suspended in phosphate-buffered saline (2 ml) and gently
mixed with a magnetic stirbar in the cuvet. The samples were excited
at 403 nm (HPTS at low pH), 413 (total HPTS, invariant with pH),
and 450 (HPTS at neutral or basic pH), and the emission spectra were
recorded. Because endosomal and lysosomal compartments were acidic,
the measured 450/413 ratio will lie between the ratio at pH 7.4 (2.0)
and at pH 6 (0.2). Therefore, the fraction of HPTS-containing lipo
somes within the endosomal (low pH) compartment can be calculated
from the formula
^measured
~
Fraction at low pH
where R is the ratio of fluorescence emission at 510 nm after excitation
at 450 nm compared to excitation at 413 nm. For each liposome
preparation, a 1 nmol aliquot was used as a fluorescence standard to
allow calculation of the amount of cell-associated liposomes. In addi
tion, cell autofluorescence was subtracted. The value for /?I„WPH
was
obtained by incubation of cells with free 1 mM HPTS for 24 h, washing,
and then measuring the 450/413 emission ratio. The value for /îpH7.4
was obtained from a similar procedure, but measuring the 450/413
emission ratio after cell lysis with 0.1 % Triton X-100, so that all HPTS
would be at neutral pH. RpH1Awas consistently 2.0 for all cell lines
tested, corresponding to pH 7.4; AiowpHvaried with each line as follows:
Jurkat, 0.6 to 0.7; CEM, 0.3 to 0.5; Molt-4, 0.5. Using the known pH
dependence of HPTS emission ratios, this corresponds to an average
pH of the endosomal compartment for Jurkat of 6.7; CEM, 6.4; Molt4, 6.5. Details of the HPTS fluorimetry and fluorescent microscopy
techniques, along with results validating this assay to document endo
cytosis in living cells, are reported by Straubinger et al.''
Growth Inhibition. Leukemia cells were plated at 10" per well in 24well Costar plates, 1 ml/well. When SPA-liposomes were tested, the
cells were pretreated with the relevant antibody at 0°Cfor 30 min. They
were treated in triplicate with the desired concentration of drug or
liposomes and then counted with a Model Fn Coulter Counter after
72-h incubation. The following calculation was made.
The IC.«was determined from a plot of the percentage of growth
versus log,o drug concentration.
% of growth =
final cell no. —¿
initial cells
control cell no. —¿
initial cells x 100
RESULTS
Effect of Ligand on Endocytosis of Liposomes: Fluorescent
Microscopy. Three human T-cell leukemia lines, CEM, Molt4, and Jurkat, were studied with either soluble antibody in
6 R. M. Straubinger, D. Papahadjopoulos, and K. Hong. Kinetic analysis of
endocytosis and intracellular fate of liposomes using pyranine(8-hydroxy-1,3,6pyrenetrisulfonate) as a probe, submitted for publication.
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LIPOSOME ENDOCYTOSIS
IN T-ACUTE LYMPHOCYT1C LEUKEMIA
combination with Protein A-bearing liposomes, or with the
direct antibody-liposome conjugate, using anti-T-surface anti
gen or antitransferrin receptor antibodies for targeting. Photo
micrographs of identical fields were taken under phase-contrast
illumination and then under fluorescent illumination with the
350- to 410-nm and 450- to 490-nm filter in order to evaluate
endocytosis of the HPTS-containing liposomes at Time 0, 0.5,
1,2, A-,and 24 h. The appearance of bright punctate fluorescence
seen with the 350- to 410-nm filter but not with the 450- to
490-ri m filter reflects HPTS in the low pH compartment (27,
42). Since the three different leukemia cell lines gave parallel
results, detailed results of microscopy are shown only for the
Jurkat cell line (Figs. 1 and 2).
A narked difference was apparent in the total cell-associated
fluorescence when the indirect targeting system of specific antiT-cell antibodies plus liposomes bearing Protein A was com
pared to the direct conjugates of the same antibodies to the
liposome surface (Fig. 1). The SPA-REV resulted in much
Anti-CD2
lower total cell-associated fluorescence alter 1 to 4 h of incu
bation, although initial binding in the cold was equivalent to
the direct conjugates. Anti-CD5 (Leul) in combination with
the SPA-REV showed a pattern consistent with capping and
shedding of the fluorescent liposomes. At the end of an incu
bation period at 0°C,the liposomes were distributed uniformly
over the cell surface. After 0.5 h of incubation at 37°C,surface
fluorescence had clustered at one pole of the cell; by 4 h,
considerable loss of fluorescence occurred (Fig. 1); and by 24
h, no cell-associated fluorescence was detectable. By contrast,
the direct anti-CD5-liposome conjugate resulted in a higher
degree of retention of cellular fluorescence. Substantial endo
cytosis of liposomes bearing the anti-CD5 occurred, evidenced
by the pattern of HPTS fluorescence (Fig. 1). The leukemia
cells with anti-CD2 (OKT11) and anti-CD3 (OKT3) also gave
lower fluorescence with SPA-liposomes than with antibodyliposomes after a 4-h incubation (Fig. 1). Although endocytosis
occurred with anti-CD2 and anti-CD3 antibodies with both
Anti-CD3
Anti-CD5
Phase
SPA-REV
Hoechst
(350-410
nm)
Phase
Ab-REV
Hoechst
(350-410
nm)
Fig. 1. Cell association and endocytosis of antibody-targeted liposomes with Jurkat T-cell leukemia. Cells (2 x 10") were incubated at O'C with the relevant
antibody when appropriate and then with HPTS-containing liposomes (20 nmol) conjugated to antibody or Protein A at O'C for 45 min. Unbound liposomes were
washed off, and the cells were incubated for an additional 4-h period at 37'C. HPTS fluorescence was recorded with the Hoechst filter cube (total HPTS. 350 to 410
nm). Slice total fluorescence was often too faint to photograph with the FITC cube (450 to 490), particularly with the SPA-liposomes. these photos are omitted.
4881
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LIPOSOME ENDOCYTOSIS
IN T-ACUTE LYMPHOCYTIC
2 h
LEUKEMIA
24 h
Phase
Fig. 2. Cell association and endocytosis of
antitransferrin
receptor-targeted
liposomes
with Jurkat T-cell leukemia. Cells were incu
bated with HPTS-containing liposomes and
photographed at the indicated times as in Fig.
1. HPTS fluorescence was recorded with the
Hoechst filter cube (total HPTS, 350 to 410
nm) and the FITC filter cube (HPTS at neutral
pH, 450 to 490 nm). Internalizaron is shown
by a punctate pattern evident at 350 to 410 nm
but diminished at 450 to 490 nm.
Hoechst
(350-410 nm)
FITC
(450-490 nm)
direct and indirect liposomes, as shown by the appearance of
bright spots seen with the 350- to 410-nm filter that disappeared
with the 450- to 490-nm filter, the degree of cell-associated
fluorescence was much less with the SPA-REV and was undetectable by 24 h (not shown). The antitransferrin receptor
antibody was not examined with SPA-REV, since it is an IgGl
and does not bind well to Protein A.
The target antigen also had a significant effect on association
and endocytosis of the Ab-REV. Only the antitransferrin recep
tor-bearing liposomes stained 100% of cells. The antitransferrin
receptor (B3/25 or OKT9) liposomes and the anti-CD2
(OKT11) liposomes resulted in very bright total fluorescence
by microscopy as well as in the greatest degree of endocytosis
with complete internalization by 24 h (Fig. 2). Both anti-CD2
and anti-CD3 liposomes showed rapid endocytosis and little
surface fluorescence, with a punctate pattern evident by 0.5 h
and prominent by 4 h (Fig. 1). Overall uptake was less with
anti-CD3 than with the antitransferrin receptor-conjugated li
posomes. The anti-CD5-REV showed some capping and slower
endocytosis as judged by the later development of punctate
fluorescence brighter with the 350- to 410-nm filter, with lower
overall cell-associated fluorescence than anti-CD2 or antitrans
ferrin receptor-liposomes.
The control (irrelevant antibody;
SS. l ) showed no cell-associated fluorescence with any leukemia
line, as expected.
Anti-CD2 and anti-CD3 did not mediate binding of lipo
somes to CEM cells, nor did anti-CD3 with Molt-4, since these
cell lines exhibit little reactivity with these antibodies when
tested by indirect immunofluorescence by ourselves and others
(40).
Cell-Liposome Binding and Intracellular Delivery by Fluori
metrie Measurements. The total cell-associated liposomes and
the proportion of those taken into a low pH compartment
following endocytosis were evaluated by measurement of fluo
rescence emission at 510 nm with excitation wavelengths of
413 and 450 nm. The change in 450/413 reflects the change in
mean pH to which the liposomes are exposed. HPTS fluores
cence with excitation at 450 nm is pH dependent, while fluo
rescence with excitation at 413 nm is not. The relative endo
cytosis of the liposomes over time can be estimated by the
decrease in the 450/413 ratio. This ratio will decrease as
liposomes containing HPTS leave the neutral extracellular
compartment and enter the low pH compartment of the endocytic vesicle. Fig. 3/4 shows the more rapid and complete
endocytosis of liposomes targeted against the transferrin recep-
TIME
(H)
Fig. 3. Change in 450/413 ratio of HPTS-containing antibody-targeted lipo
somes by human T-leukemia Jurkat cells. Progressive uptake of liposomes into
the low pH compartment of the cells is shown by the decrease in the 450/413
excitation ratio of liposome-encapsulated HPTS measured on the fluorimeter.
HPTS-containing liposomes (20 nmol) conjugated to antibody (Ab-REV) or
Protein A (SPA-REV) were incubated with 2x10" leukemia cells (pretreated
with relevant antibody where appropriate) at O'C for 45 min. Unbound liposomes
were washed off, and the cells were incubated at 37°Cfor periods of 0.5, 2, and 4
h before measuring fluorescence emission at 510 nm with excitation at 413 and
450 nm. A, Jurkat cells with anti-CDS (Leu 1) + SPA-REV (O), anti-CD-5-REV
(•),antitransferrin receptor-REV, B3/25-REV(x). and OKT9-REV (+). B, Jur
kat cells with anti-CD3(OKT3)-REV (A), anti-CD3 + SPA-REV (A), antiCD2(OKTI 1)-REV (•),and anti-CD2 + SPA-REV (D).
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LIPOSOME ENDOCYTOSIS
IN T-ACUTE LYMPHOCVHC
tor compared to anti-CD5 with either SPA-REV or Ab-REV.
The anti-CD5 direct and indirect liposomes have a similar
average pH after 2 h. The superiority of the direct anti-CD5
conjugate in total amount of liposomes internalized as assessed
by low pH fluorescence (Fig. 4) and by more effective growth
inhibition (Table 1) therefore must be due to more efficient
binding and retention of the liposomes at the cell surface. The
reason for the difference in liposome retention may be the rapid
capping and shedding observed to occur by microscopy with
the indirect Protein A conjugate (Fig. 1). Fig. 3D shows the
signif cant degree of internalization of the anti-CD2 anti-CD3REV with the Jurkat cells. Again, little difference is seen in
average pH of the direct and indirect liposomes, emphasizing
the need to look at total fluorescence as well as relative acidi
fication. Fig. 4 and Table 1 show that a greater amount of
directly conjugated anti-CD2 and CD3 liposomes is internal
ized than the Protein A conjugate, similar to the case of the
anti-CD5. The kinetic 450/413 assays of internalization of
antitrunsferrin receptor and anti-CD5 liposomes for the CEM
and Molt-4 cells were comparable to those with the Jurkat, so
the results are not shown here, although the 450/413 ratios
were used to calculate results for Fig. 4.
While Fig. 3 depicts the change in the 450/413 ratio over
time, :he measurement of both total cell-associated fluorescence
and lew pH fluorescence is necessary for any useful correlation
with total drug delivery to the cells. The total cell-associated
liposomes (as assayed by fluorescence at excitation of 413 nm)
SSI
OKTll
OKT3
Leu I
83/25
OKT9
CEM
SS.l
OKTll
CEM
Fig. 4. Total cell association and intcrnalization of fluorescent liposomes by
human T-ccll leukemia after a 4-h incubation. Leukemia cells were incubated at
0°Cfor45 min with liposomes containing HPTS (20 nmol/2 x 10*cells; 100 /IM
lipid). washed, and then incubated at 37°Can additional 4 h. After removal of
milioni! I liposomes, the cell-associated liposomes and liposomes at low pH were
measured by fluorimetry. Results are expressed as 11111.>l
of REV (lipid). as
measured by fluorescent contents, per 2x10* leukemia cells. The .v-axis shows
the targ:t antibody used, where SS.l is an irrelevant control. OKTII is anti-CD2.
OKT3 ii anli-CD3, Leu 1 is anti-CD5. and B3/25 and OKT9 are antitransferrin
receptor. •¿.
liposomes at low pH (internalized): 0, total cell-associated liposomes.
LEUKEMIA
Table 1 Growth inhibition of human T-acute lymphocytic leukemia with
antibody-directed liposomes containing methotrexate-y-asportate
Liposome
conjugate"SPA-REVAb-REVFree
(,,M)CEM1.15
0.89'0.55
±
±0.901.70
±0.410.56
±0.312.08
1.033.23
±
±0.921.30
±0.102.1
±0.202.
5
±0.402.20
10
0.201.10
±
0.460.17
±
±0.050.08
0.010.03
±
±0.010.02
±0.010.02
0.790.10
+
±0.050.02
0.010.01
±
±0.401
0.400.94
.00 ±
0.300.05
±
0.030.03
±
0.010.03
±
0.010.02
±
0.010.10
±
0.010.05
±
±0.010.15
0.010.12
±
0.010.23
±
B3/25Jurkat2.38
drugAntibody*IrrelevantAnti-CD2Anti-CD3Anti-CD5IrrelevantAnti-CD2Anti-CD3Anti-CD5An
±0.091C«,
±0.05Molt-42.30±0.02
" SPA-REV are Protein A conjugated to vesicles made by reverse-phase evap
oration; Ab-REV are vesicles conjugated directly to monoclonal antibody.
* Irrelevant antibody is SS. 1, anti-sheep erythrocytc; anti-CD2 is OKTI l;antiCD3 is OKT3; anti-CD5 is Leu 1; anti-TFR are the antitransferrin receptor
antibodies OKT9 or B3/25 as indicated.
' Mean ±SD of 3 to 7 triplicate experiments.
compared to the liposomes at low pH for the various target
antibodies are shown at the end of a 4-h incubation at 37°C
(Fig. 4). These fluorimetry results parallel the observations by
microscopy using the same experimental format. The total cellassociated fluorescence and the fluorescence from the acidified
liposomes were both greater with Ab-REV than with SPAREV. The order of efficacy of the respective antibodies for
internalization by Jurkat cells at 4 h is anti-CD2 > anti
transferrin receptor > anti-CD5 > anti-CD3. Although the total
cell-associated liposomes were approximately equal for the antiCD5 and the antitransferrin receptor conjugates, the latter
mediated enhanced liposome endocytosis. It is also important
to note that the two liposome preparations bearing different
antitransferrin receptor monoclonals, B3/25 and OKT9, were
not equivalent. B3/25 liposomes were consistently more effec
tive in promoting endocytosis, suggesting that minor differences
between antibodies against the same target may also affect
behavior of the immunoliposome conjugates. Antitransferrin
receptor conjugates were both bound and internalized to a
higher degree than the anti-CD5 liposomes with the CEM and
Molt-4 cells.
Liposome association and uptake were also compared after
a prolonged exposure (Fig. 5), under conditions simulating
those used in the growth inhibition assays described below. The
targeted liposomes were incubated continuously with replicat
ing leukemia cells at two liposome concentrations, 2.5 fiM and
25 UM lipid. The lower concentration corresponds to the lipid
concentration at the IC5lifor growth inhibition, while the higher
concentration corresponds to that used in the 4-h incubation
(Fig. 4). Total cell association and internalization were greatest
for the antitransferrin receptor-targeted liposomes for all cell
lines at both liposome concentrations. For the Jurkat cells, the
antitransferrin receptor (B3/25) and anti-CD3 (OKT3) show
complete internalization of cell-associated liposomes by 72 h,
unlike the anti-CD5 (Leu 1) and anti-CD2 (OKTI 1), with which
only 45 to 76% are in the low pH compartment. The proportion
of internalized liposomes is higher for the B3/25 antitransferrin
receptor liposomes than for the OKT9-liposomes, perhaps due
to the smaller diameter of the B3/25-REV (98 nm compared
to 250 nm) or to some difference in the antibody affinity.
Increasing the liposome concentration 10-fold (25 ¿JM),
so that
it was comparable to that in the 4-h cell association experi-
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LIPOSOME ENDOCYTOSIS
IN T-ACUTE LYMPHOCYTIC
three cell lines than an irrelevant antibody (SS.l). Both antiCD2 and anti-CD3 antibody had greater growth-inhibitory
potency as direct liposome conjugates than with the indirect
Protein A liposomes with the Jurkat and Molt-4 cells. The
CEM lines expressed too little of these antigens for a meaning
ful comparison.
A dual antibody conjugate was also tested, using drug-con
taining liposomes conjugated to both anti-CD5 (Leu 1) and
antitransferrin receptor (B3/25), the two antibodies providing
the most potent Ab-REV as well as a combination of high
internalization (antitransferrin receptor) and T-cell specificity
(anti-CD5). The growth-inhibitory efficacy was not significantly
different from that of liposomes conjugated to either antibody
alone (Table 1).
Jurkat
SSI
OKTll
CEM
OKT3
Leu I
B3/25
OKT9
SS 1 OKT11 OKT3
Leu 1 B3/25
LEUKEMIA
OKT9
CEM
Z
er
DISCUSSION
Molt-4
OKT3
Lau 1 B3'25
OKT9
U fit
Fig. 5. Total cell association and internalization of fluorescent liposomes by
human T-cell leukemia after a 72-h incubation. Leukemia cells were incubated
continuously for 72 h at 37"C with liposomes containing HPTS using two lipid
concentrations, 2.5 /IM and 25 JIM.After removal of unbound liposomes, the cellassociated liposomes and liposomes at low pH were measured by fluorimetry.
Results are expressed as nmol of REV (lipid). assayed by fluorescence, per 2 x
10" leukemia cells. The .v-axis shows the target antibody used, where SS.l and
14.2a are irrelevant controls, OKTII is anti-CD2. OKT3 is anli-CD3. Leu 1 is
anti-CD5, and B3/25 and OKT9 are antitransferrin receptor. •¿
liposomes at low
pH (internalized); O, total cell-associated liposomes.
meni s. did not change the relative association and internaliza
tion, although it increased the absolute amount of liposomes
bound and at low pH by 3- to 10-fold (note the 5-fold increase
in scale of REV concentration in 25 ^M experiments).
Growth-inhibitory Effect of Liposomes Containing Cytotoxic
Drug. Intracellular delivery of the liposome-dependent drug,
methotrexate-7-aspartate,
was used as an independent assay to
compare the effect of ligand on endocytosis. The growth-inhib
itory efficacy of methotrexate-7-aspartate
encapsulated in li
posomes targeted for the human T-cell leukemias (Table 1)
paralleled the fluorescent uptake experiments, showing that the
antitransferrin receptor was the most potent targeting antibody
for liposome uptake. The liposomes conjugated directly to the
antitransferrin receptor antibody were 4- to 15-fold more effec
tive than free drug and 37- to 162-fold more effective than
those conjugated to a nonspecific antibody for all three cell
lines. The antitransferrin receptor antibody (B3/25) was con
sistently more effective as a direct antibody-liposome conjugate
than the anti-CD5 (Leu 1).
The relative potency of the various antibody-targeted lipo
somes with the Jurkat cells was the same as that in the pro
longed 72-h incubation fluorimetry assay, with antitransferrin
receptor > anti-CD5 and anti-CD3 > anti-CD2 (Table 1). The
direct conjugates proved more effective than the SPA-liposomes
for intracellular delivery (Table 1), as previously shown using
the fluorescent probe. The anti-CD5 (Leu 1) with the drugcontaining SPA-REV was no more effective against any of the
Two independent markers for endocytosis of liposomes were
used in these studies: HPTS, a pH-sensitive fluorescent dye
(25-27); and methotrexate-7-aspartate,
a growth-inhibitory
drug dependent on liposome uptake for maximal potency (3).
HPTS has high water solubility, low leakage, a pK<, in the
physiological 6-8 range, minimum interference from biological
fluids due to maximum emission at 510 nm, and well-separated
excitation spectra at neutral and low pH. Excitation at 413 nm
results in a pH-independent emission that allows total fluores
cence to be quantitated (26). Studies of encapsulated HPTS in
CV-1 cells using microscopy and fluorimetry document persist
ence of HPTS in acidic vesicles, labeling of intracellular organelles with an acidic lumen, and reversibility of fluorescence
emission with NH4C1 or monensin, which collapse cellular H+
gradients.5 This dye was encapsulated in the liposomes to
monitor their shift from the neutral extracellular compartment
to the low pH of the endosóme and lysosome. An aqueous
phase rather than lipid phase marker was used in order to
correspond to the aqueous phase cytotoxic drug.
The second assay depends on cytoplasmic delivery of the
liposome-dependent drug, methotrexate-7-aspartate.
Methotrexate-7-aspartate is a pteridine antifolate which is indistin
guishable from methotrexate in its ability to inhibit dihydrofolate reducÃ-ase, but 200-fold less toxic for L1210 cells than
methotrexate because its influx Km is at least 100 times greater
(29). However, it is equipotent to methotrexate if delivered
intracytoplasmically, thus providing an indicator of liposome
uptake.
The results demonstrate the importance of both the type of
liposome-antibody linkage (direct conjugation to antibody or
indirect association via Protein A conjugation to liposomes)
and of the particular target receptor in determining liposome
fate. The Protein A-bearing liposomes attached to cells pretreated with specific antibody generally showed much lower
total endocytosis than the equivalent direct antibody-liposome
conjugates by both HPTS internalization and growth inhibition
assay. This result is in contrast to the findings with the murine
T-leukemia, AKR/J SL2, where SPA-liposomes were 10-fold
more effectively taken up than the direct anti-Thy 1.1 (19E12)liposome conjugate (5). The disparity of these findings may be
due to either differences between human and murine cells or to
some inherent difference in the Thy 1.1 from the CD2, CD3,
and CD5 antigens. Previous studies using a human B-cell
lymphoma line also showed relatively low efficacy of the indi
rect Protein A conjugates, although even here uptake varied
according to antibody specificity, with greater internalization
of an anti-Dr conjugate than an antiidiotype (12). The fact that
4884
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LIPOSOME ENDOCYTOSIS
IN T-ACUTE LVMPHOCYTIC
the differences in the current study between the direct and
indirect liposomes were more marked for the growth inhibition
assays than for the fluorescence assays may be due to the
differences in incubation conditions of the two measurements.
In ortler to obtain accurate fluorimetry measurements, greater
concentrations of liposomes (10-fold greater than in growth
inhibition) were used at shorter incubation times than in the
growth inhibition treatments (4 versus 72 h). Over longer time
periods, cell-associated fluorescence of SPA-REV completely
disappears, as was shown by microscopy. Thus, the contents of
SPA-KEY are gradually lost from the cell, while the Ab-REV
are retained. This may be related to early leakage of contents
from :he SPA-REV or to a difference in intracellular process
ing.
The particular choice of target antigens resulted in dramatic
differences not only in amount of binding, an expected effect
of antigen density, but also in the cell surface distribution of
fluorescence and extent of endocytosis. The intensity and dis
tribution of cellular fluorescence seen illustrate the requirement
for significant binding, uptake, and retention of the liposomes.
Thus, while anti-CD3 facilitates comparatively rapid uptake
and complete internalization of cell-associated liposomes (Fig.
35), a finding recently corroborated by Gray et al. (42), the
amount of binding, endocytosis, and retention is lower than
with anti-CD5 or antitransferrin receptor and insufficient for
adequ ate drug delivery. The antitransferrin receptor-liposomes
show lhe highest degree of cell association and endocytosis, the
greatest retention of fluorescence both at 24 and 72 h, and the
most potent growth inhibition. Linear correlation coefficients
for th; absolute amount of liposomes internalized by fluori
metrie assay and the IC50 for growth inhibition were 0.97 and
0.86, respectively, for SPA-REV and Ab-REV with Jurkat cells
(derived from Fig. 2 and Table 1, excluding anti-CD2). It is
interesting that the dual conjugate, the liposome preparation
conjugated to both anti-CD5 and antitransferrin receptor, was
no mere effective than the antitransferrin receptor liposomes
alone. This finding may be due to a limited capacity of the cells
for endocytosis of the liposomes, which cannot be enhanced by
binding more liposomes. It may also be due to inability to
increase the number of liposomes bound beyond those already
attached to the transferrin receptor. Finally, if the transferrin
receptor and the CDS antigen are internalized by similar path
ways, synergy will not occur. In the latter case, increasing the
potency of the encapsulated drug would be required to enhance
efficacy.
The only target antigen that shows poor correlation of en
docytosis by the 4-h fluorescence assay and growth inhibition
assay is the CD2 (OKT11). The anti-CD2-REV show the great
est early uptake by fluorescence, but only intermediate efficacy
for growth inhibition. It has been shown that endosomes formed
by lymphoblasts during receptor-mediated endocytosis of var
ious lißandsmay be either acidified or not, depending on the
type o Fthe ligand (43-45). It is possible that the anti-CD2REV do not induce a high enough degree of acidification of the
pinocytic or endocytic vesicle, so that the drug is not as effec
tively delivered to the cytoplasm. The other possibility is that
over time, the contents of the anti-CD2-RE V may be lost from
the cytoplasm, resulting in less growth inhibition. This is sup
ported by the results of the 72-h fluorimetry experiment (Fig.
3), which shows proportionally lower fluorescence from the
cells incubated with anti-CD2-REV compared to the anti-CD5or anti transferrin receptor-REV.
The results with the various antibody-liposome conjugates
are coripatible with the known characteristics of the respective
LEUKEMIA
antigens. The complete internalization of liposomes bound to
the CD3 antigen is consistent with the known capacity of this
receptor to stimulate modulation of the T3-T-cell receptor
complex (46). Similarly, the CDS antigen has also been shown
to modulate when bound to antibody (47-49). This may be a
disadvantage for antibody therapy alone (48), but this modula
tion may enhance endocytosis of immunoconjugates, such as
immunotoxins (50) or liposomes, as in the current study. It is
possible that the rapid cross-linking and capping of the CD5antibody complex caused the shedding of liposomes observed
with the indirect SPA-REV, while the capacity for multiligand
binding of the direct Ab-REV facilitated internalization of the
liposomes with modulation of the receptor, accounting for their
excellent uptake. Our results differ from those of Youle and
coworkers (51), who found that anti-CD3-diphtheria toxin con
jugates were more effective than anti-CD5 immunotoxins with
Jurkat cells. In contrast, we showed using the same cell line
that the anti-CD5-REV were more effective than the anti-CD3REV. This discrepancy may be due to differences between
immunotoxins and immunoliposomes which may alter uptake,
such as the multivalency of the liposomes or the larger size.
The best liposome uptake was observed using the transferrin
receptor, known to internalize via a coated pit pathway (13, 14)
and undergo continuous cycles of endocytosis (18, 19).
These results suggest that use of a cell target known to
undergo spontaneous endocytosis and recycling, like the trans
ferrin receptor or other growth factor receptors, may markedly
enhance the amount of intracytoplasmic delivery of targeted
drug carriers. A similar variability in endocytic capacity of
lymphocytes for antibody-targeted liposomes has previously
been reported in a murine system using anti-H-2 Kk- and antiH-2 I-Ek antibody-targeted liposomes (52). The combination of
antibody to a target antigen which is highly endocytic with a
more cell-specific antibody may improve the efficacy of such
targeted liposome therapy, particularly for cells like T-lymphocytes, which have been shown to have a low capacity for
pinocytosis (53). Such cells have proven more resistant to
antibody-targeted liposome uptake in the past (4, 5, 12). Use
of a sensitive fluorescence assay like the one with HPTS will
provide new information about intracellular processing of tar
geted liposomes and allow comparison of the fate of the aqueous
contents of liposomes with that of antibody alone.
ACKNOWLEDGMENTS
We thank Dr. I. Trowbridge of the Salk Institute, Dr. T. Reichert of
Becton Dickinson, and Dr. G. Goldstein of Ortho Pharmaceutical for
their generous donation of the monoclonal antibodies and Dr. D.
Daleke for help in analysis of the fluorimetry assays.
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4886
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Role of Ligand in Antibody-directed Endocytosis of Liposomes
by Human T-Leukemia Cells
Katherine K. Matthay, Anna M. Abai, Suzanne Cobb, et al.
Cancer Res 1989;49:4879-4886.
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