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Experimental Therapeutics |
Departments of Pharmacology and Therapeutics [V. G., H. U. S.] and Oncology and the Cancer Center [H. U. S.], McGill University, Montréal, Quebec, H3G 1Y6 Canada
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ABSTRACT |
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INTRODUCTION |
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Paclitaxel has clinical efficacy, despite several problems associated with poor solubility and high toxicity. Clinical trials conducted in the late 1980s and early 1990s showed remarkable efficacy against advanced solid tumors such as ovarian and breast cancer (3) . More recent clinical studies showed its strong activity against a panel of other tumors (4) . Most of the side effects of taxanes occur at rapidly growing tissue such as bone marrow, hematopoietic, and gut epithelia. Because microtubule function is key for neuronal survival, neurotoxicity is also a problem for taxanes (5) .
The problem of selectivity can be addressed by using mAbs3 that target "tumor markers," which are proteins generally overexpressed on the surface of tumor cells. In passive immunotherapy, mAbs can act either as pharmacological agents (6) , as adjuvants, or as cytotoxic agents upon fixation of complement (7) and as carriers for large toxins (8) or cytokines (9) . However, mAbs are generally poor pharmaceuticals (10) and are poor cytotoxic agents.
Hence, we and others have proposed that chemical coupling of mAbs and chemotherapeutics would allow the delivery of a cytotoxic agent to the tumor cell with reduced side effects. For example, doxorubicin has been coupled to antibodies and peptide carriers (11) . However, doxorubicin could still be active in the protein-doxorubicin conjugate, and it would be more desirable to obtain a conjugate that remains inactive until after binding to the target occurs.
To bypass some of the problems above, we aimed to fulfill the following five objectives: (a) to chemically conjugate mAbs to the relatively small chemotherapeutic taxanes; (b) to afford conjugates that are highly soluble in physiological buffers; (c) chemical coupling should not affect mAb targeting function but should result in inactivation of taxane activity; (d) after binding to the target receptor mAbs should induce capping and internalization and therefore deliver the conjugate into the tumor cell; and (e) the chemical coupling has to allow the release of the taxane in its active form after the antibody-cytotoxic drug conjugate is internalized. This is achieved by coupling the taxane via a low pH-sensitive bond that is cleaved after conjugate internalization and exposure to lysosomal vesicles.
Two target cell surface markers were selected, which correspond to the receptors for nerve growth factor, the p140 TrkA tyrosine kinase high-affinity receptor (12) and the p75 low-affinity receptor (13) . TrkA and p75 receptors are expressed on normal cells such as neurons; but at low density, however, they are overexpressed in many cancer cell types. These receptors are useful markers for neuroblastoma, small cell lung carcinoma, B-cell lymphoma, and melanoma (14) . mAbs have been developed against those receptors, i.e., anti-p75 mAb MC192 (15) and anti-TrkA mAb 5C3 (16) .
We report the synthesis of conjugates paclitaxel-MC192 as an agent to target and kill cells expressing p75 receptors. We also report the synthesis of paclitaxel-rabbit-antimouse antibody as an all-purpose secondary reagent that allows selective tumor targeting with the use of any mouse primary antibody. The paclitaxel-coupled antibodies retain high affinity and specificity after conjugation, and the conjugates delivered the cytotoxic agent in its active form. Paclitaxel-antibody conjugates had in vitro cytotoxic activity better than free paclitaxel or free paclitaxel plus free mAb and also showed high selectivity and specificity toward cells expressing the targeted receptors. In vivo studies showed that paclitaxel-MC192 conjugate had a good antitumor activity, whereas free drugs had no effect at equivalent concentrations.
These studies will result in an increase or an improvement of the armamentarium and selectivity of cytotoxic agents. Combinations of other chemotherapeutic agents and other ligands using this approach will generate a severalfold increase in the number of antitumor agents.
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MATERIALS AND METHODS |
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. The solvent was then removed in vacuo, and the residue was
dissolved in CHCl3 and washed with
double-distilled H2O. Purification was achieved
by high-performance liquid chromatography on a semipreparative column
(Phenomenex); the mobile phase consisted of acetonitrile:water gradient
from 35:65 to 75:25 over 50 min.
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. The solution
was then dialyzed for 2 h against water and overnight against PBS.
The conjugate can internalize into cells by receptor-mediated and
capping mechanisms (18)
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Quantification of Conjugated Paclitaxel.
A known mass of paclitaxel-antibody conjugate was incubated for 48 h at room temperature in 0.1 M acetate buffer (pH 4) to
hydrolyze ester bonds. Paclitaxel was then extracted with chloroform
and evaporated to dryness. Quantification of this purified paclitaxel
was done by analytical high-performance liquid chromatography
(Phenomenex) on a mobile phase of acetonitrile:water from 35:65 to
75:25 over 40 min. Known concentrations of paclitaxel were used as
standard control. Generally, the measured molar ratio of
protein:coupled paclitaxel was 1:1, meaning that 1 molecule of
paclitaxel coupled to 1 molecule of antibody. Thus, theoretically, 1
mole of paclitaxel is delivered per mole of internalized antibody.
Cell Lines.
The B104 cells are a rat neuroblastoma line that expresses p75
receptors (p75+). The 4-3.6 cells are B104 cells
stably transfected with human TrkA cDNA (p75+,
TrkA+; Ref. 19
). NIH 3T3 are mouse
fibroblasts that do not express either p75 or TrkA. All cells were
cultured in RPMI 1640 supplemented with 5% fetal bovine serum,
L-glutamine, HEPES buffer, and antibiotics.
Antibodies.
mAb MC192 is a mouse IgG1 antirat p75 mAb (15)
, and mAb
5C3 is a mouse IgG1 antihuman TrkA mAb (16)
. MC192 and mAb
5C3 were purified and characterized as described (20)
and
were used in culture at 1–5 nM, which are near-saturating
concentrations for cell surface receptors. The "all purpose"
secondary rabbit-antimouse IgG (Sigma) was used in culture at a final
concentration of 30 nM.
Binding Profiles of the Conjugated Antibodies.
FACScan assays were used to measure the receptor binding properties of
the conjugated antibodies. For testing p75 receptor binding by
paclitaxel-MC192, B104 cells in binding buffer [BB: HBSS, 0.1% BSAe
(BSA), 0.1% NaN3] were incubated with the
indicated concentration of either paclitaxel-MC192 (test), intact MC192
(positive control), or mouse IgG (negative control) as primary
antibodies, followed by immunostaining with fluoresceinated goat
antimouse IgG as described (16)
. For testing binding of
the paclitaxel-rabbit-antimouse conjugate, 4-3.6 cells were incubated
as above with saturating mAb 5C3, followed by the
paclitaxel-rabbit-antimouse conjugate (test) or rabbit-antimouse
antibody (positive control). Cells were then immunostained with
fluoresceinated-goat-antirabbit IgG. All data (5000 cells/point) were
acquired on a FACScan and analyzed using the LYSIS II program. Data are
reported as mean channel fluorescence of bell-shaped histograms.
Kinetics of Paclitaxel Cytotoxicity: Single Bolus
versus Constant Exposure.
It is likely that a single bolus of paclitaxel-antibody conjugate would
be delivered because affected cells would not synthesize additional
target receptor. Therefore, we tested whether a single bolus of
paclitaxel would be an effective cytotoxic agent. Cells were exposed to
the indicated concentration of paclitaxel for 30 min at 4°C. Then,
cells were plated in a 96-well plate (Falcon); this group represents
treatment with paclitaxel present in a constant manner. The remaining
cells were washed free of excess paclitaxel prior to plating; this
group represents a single exposure to paclitaxel. The survival profile
of the cells was measured using the tetrazolium salt reagent MTT
(Sigma) 48, 72, and 96 h after plating as described
(20)
. Absorbance readings of MTT were done in an EIA Plate
Reader model 2550 (Bio-Rad) at 595 nm.
In Vitro Cytotoxicity of the Paclitaxel-Antibody
Conjugates.
For testing the paclitaxel-MC192 conjugate, cells in 96-well plates
(2500–5000 cells/well) were exposed to either MC192, paclitaxel-MC192
conjugates, or controls. Competition of paclitaxel-MC192 cytotoxicity
was done by adding a 4-fold molar excess of MC192 antibody. The
survival profile of the cells was measured with the MTT assay 72 h
after plating.
For testing the "all purpose" rabbit-antimouse reagent, cells were first exposed to primary mouse mAb 5C3, to mAb MC192, or controls (e.g., mouse IgG). Then, paclitaxel-rabbit-antimouse conjugate (test) or rabbit-antimouse antibody (negative control) were added to the cultures. The survival profile of the cells was measured with the MTT assay 48 h after plating.
Mechanism of Action of Paclitaxel-Antibody Conjugates.
B104 cells were plated, 25,000 cells/well in a 48-well plate (Falcon).
Free paclitaxel (80 nM) or paclitaxel-MC192 (40
nM paclitaxel-equivalent) was added to the well, and the
cells were incubated for 24 h. Cells were then treated with 0.01%
Triton X-100, 0.1% sodium citrate, and 1 µg DNase-free RNase
for 1 h at 0°C. Nuclei were collected after centrifugation. The
DNA was labeled with 75 µl of propidium iodide (1 mg/ml stock) in 400
µl of FACS buffer. All data (3000 cells/point) were acquired as
described above. Paclitaxel release from the conjugate occurs by
hydrolysis of the ester bond in the lysosomal compartment
(18)
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In Vivo Tumor Studies.
Nude mice (7 weeks of age, females) were used to test the effect of
paclitaxel or conjugates in tumor progression. Single-cell suspensions
of B104 cells (105/mouse) were injected s.c. in
the left flank near the rib cage. Tumor growth was monitored daily.
After 4 days, the tumor volume in all animals was 
2
mm3
. Mice were then randomized, and treatments
were initiated in four groups (n = 5 in each
group). Mice in group 1 received saline; mice in group 2 received free
paclitaxel (130 ng); mice in group 3 received free paclitaxel (130
ng) + free MC192 (10 µg); and mice in group 4 received
paclitaxel-MC192 conjugate (65 ng of paclitaxel-equivalent and 10 µg
of MC192-equivalent). All treatments were done by a total of five
injections every 2 days (for a total of 10 days). All injections were
done i.p. on the right side to prevent direct contact of the agents to
the tumor growing s.c. and to assure that systemic circulation of the
drugs was achieved. Measurements of tumor volume were taken using
calipers every day after treatment for a total of 25 days. The timeline
was: day -14, injection of tumors s.c.; days -10, -8, -6, -4, and
-2, injections of drugs or controls; and days 0–25, daily measurement
of tumor growth.
Statistical Analysis.
Statistical significance of differences in tumor growth among the
different treatment groups was determined by the Student t
test using SYSTAT 7.0 software. P < 0.05 was
considered significant.
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RESULTS |
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. These assays were done at 4°C to allow internalization of drug
comparable with that afforded by antibody-mediated delivery of
paclitaxel.
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Binding and Cytotoxicity of Paclitaxel-conjugated Antibodies.
To assess whether paclitaxel conjugation to antibodies affected
antibody binding, this property was tested in FACScan assays (Table 1)
. Conjugated paclitaxel-rabbit-antimouse lost only 20% of the
binding activity compared with unconjugated rabbit-antimouse antibody.
The binding activity of conjugated paclitaxel-MC192 was intact,
compared with unconjugated MC192. These results indicate that the
method used to conjugate paclitaxel to antibody in a 1:1 ratio does not
affect significantly the binding properties of the antibodies.
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. The paclitaxel-rabbit-antimouse conjugate was active against cells
only when a specific mouse primary antibody was present: mAb 5C3 that
binds 4-3.6 cells (Fig. 3A)
and mAb MC192 that binds B104
cells (Fig. 3B)
. Cytotoxicity was better and more selective
than equimolar doses of free paclitaxel. In control assays, conjugate
paclitaxel-rabbit-antimouse in the presence of a nonspecific primary
was not cytotoxic, and the specific primary mAbs in the presence of
unconjugated rabbit-antimouse were not cytotoxic. Similar analysis
using paclitaxel-MC192 conjugates also showed better activity and
selectivity than free paclitaxel at equimolar concentrations (Fig. 4)
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Selectivity and Specificity of Paclitaxel-MC192 Conjugates.
The selectivity of paclitaxel-MC192 was evaluated using cells that do
not express p75 (Fig. 5A)
. The results show that the conjugate was inactive, whereas
free paclitaxel exhibited dose-dependent cytotoxicity. These results
suggest that the activity of paclitaxel-MC192 conjugates is selective
toward cells expressing p75 receptors. The specificity of
paclitaxel-MC192 conjugate was investigated by ligand competition (Fig. 5B)
. At 10 nM paclitaxel-MC192
conjugate (10 nM paclitaxel-equivalent), there is
efficient killing of B104 cells. Concomitant addition of 40
nM MC192 blocks cytotoxicity by competing for the
p75 receptor target. In contrast, addition of 40
nM nonspecific mouse IgG does not affect the
activity of paclitaxel-MC192 conjugates. Cold competition of
paclitaxel-MC192 indicates that death is mediated specifically via p75
receptors. Furthermore, free paclitaxel had the same cytotoxicity
whether or not 40 nM of mouse IgG or 40
nM of MC192 antibody were added to the cultures.
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Cytotoxic Mechanism of Paclitaxel-MC192 Conjugates.
To assess whether the mechanism of action of paclitaxel-MC192
conjugates is the same as free paclitaxel, cell cycle analysis was done
in FACScan assays (Fig. 6)
. The data show that paclitaxel-MC192 conjugates arrest cells in the
G2-M phase of the cell cycle, which is consistent
with the mechanism of action of free paclitaxel. A
G2-M arrest leads to apoptosis in these cells.
MC192-treated cells cycle similar to untreated control, indicating no
effect by the antibody.
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. The results show that the conjugate was effective in reducing tumor
growth compared with the control (HBSS; t test,
P < 0.05; Fig. 7C
), whereas
paclitaxel alone or in combination with MC192 were not able to do so
(Fig. 7, A and B)
. Moreover, the conjugate
prolonged the survival of the mice on average by 30% compared with
free paclitaxel (data not shown).
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DISCUSSION |
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Kinetics of Paclitaxel Cytotoxicity.
Because the cytotoxicity of paclitaxel-antibody conjugates was expected
to be similar to that seen after short-term or single exposure to free
paclitaxel, we first assessed whether a single dose of paclitaxel could
be efficient at killing the cells. We demonstrated that paclitaxel
cytotoxicity is the same after 48 h, whether a constant or a
single dose is given in vitro. This finding underlines the
clinical experience of paclitaxel delivery, which is often delivered as
a single bolus every few weeks (4)
, unlike many
other chemotherapeutics that are most effective when delivered at low
doses over prolonged periods.
Criteria for Conjugation.
We set out to fulfill several criteria that would improve the
therapeutic index of paclitaxel: (a) to chemically conjugate
mAbs to taxanes; (b) to afford conjugates that are highly
soluble in physiological buffers; (c) chemical coupling
should not affect mAb targeting function, but should result in a
prodrug; and (d) after binding to the target receptor mAbs
should induce internalization and release the drug. All of these
criteria were fulfilled.
Paclitaxel was linked via its most reactive hydroxyl group (C2'
position) to antibodies as carriers. This esterification leads to
paclitaxel inactivation because this position is crucial for tubulin
binding (21)
; thus, the conjugate is a prodrug. The
binding activity of the antibodies were essentially preserved after
coupling; only 20% loss of binding was observed for the "all
purpose reagent" and no loss for MC192. After hydrolysis of the
conjugate, active paclitaxel is released in sufficient amounts to kill
cells. As expected, the conjugates arrest the cells in prophase, such
as paclitaxel does.
Improved Efficacy.
The cytotoxic activity of the conjugates was better than that of free
paclitaxel. This may be attributable to better transportation,
penetration, and accumulation of the drug inside the cells. Moreover,
conjugate cytotoxicity related well with the density of target
receptors on the cell lines. Although it has been suggested that in
certain systems ligand-bound p75 receptors may activate ceramide
pathways and proapoptotic signals (22)
, the carrier itself
had no direct pharmacological role. Hence, cytotoxicity is only
attributable to paclitaxel. It would be very interesting to use
carriers that are cytotoxic such as anti-HER-2 mAbs (23)
;
they may be better agents because of a synergistic or additive effect.
Improved Selectivity.
No binding of the conjugates was observed in cells that do not express
the target receptors. We selected the paclitaxel-MC192 conjugate for
in vivo experiments because it was more suitable than the
"all purpose" paclitaxel-rabbit-antimouse conjugate. The in
vivo experiments confirmed our in vitro findings. The
paclitaxel-MC192 conjugate had a significant antitumor activity against
cells expressing p75 receptor in the experimental model used, and we
observed a delay in tumor growth compared with other groups.
The efficacy of the conjugate compared with free drug was much
more evident in vivo than in vitro, probably
because in vivo the conjugate was concentrated at the tumor
site. The effective concentration of conjugate tested in
vivo was 3.5 nM, whereas free paclitaxel
was not effective at this dose. Because the effective concentration of
taxanes in humans is in the millimolar range, the therapeutic index of
the conjugate is improved severalfold. Futhermore, we
demonstrated appropriate in vivo systemic distribution and
pharmacokinetics for the conjugate.
It was not possible to assess the toxicity of the conjugate compared with that of free paclitaxel because of limitations in supply. Nevertheless, we did not observe any obvious toxicity in the treated animals, and we speculate that the paclitaxel-MC192 conjugate would be much less toxic than free paclitaxel because it spares non-target-expressing cells.
All of the reasons mentioned above make the paclitaxel-MC192 conjugate a potential candidate for the treatment of p75-expressing tumors. The conjugates are not only better cytotoxic agents than free paclitaxel, but they are also highly water-soluble, which is a great advantage considering the severe hypersensitivity reactions experienced by paclitaxel-treated patients. The properties of the conjugates may make them interesting therapeutic agents to add to the chemotherapeutic armamentarium. Indeed, the Food and Drug Administration has recently approved an antibody-directed cytotoxic antibiotic for the treatment of acute myeloid leukemia.
Paclitaxel is frequently given in combination with antibodies but not physically bound to them; here, we report the first synthesis of paclitaxel-antibody conjugates. A general method is proposed to selectively target cancer cells by concentrating cytotoxic drugs at the tumor site and inside the tumors. Furthermore, it would be interesting to couple cytotoxic drugs to small peptidic (10) or nonpeptidic ligands (24) of tumor markers to overcome obstacles (such as proteolysis, immunogenicity, and poor penetration of solid tumors) inherent to antibodies and proteins (14) when used as therapeutics.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the
Medical Research Council of Canada (to H. U. S.). 
2 To whom requests for reprints should be
addressed, at McGill University, Pharmacology and Therapeutics, 3655
Drummond St. #1320, Montréal, Quebec, H3G 1Y6 Canada.
3 The abbreviations used are: mAb, monoclonal
antibody; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; FACS, fluorescence-activated cell sorter.
Received 5/26/00. Accepted 10/30/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(hTNF) chemically conjugated to monoclonal antibody. J. Drug Target, 5: 109-120, 1998.[Medline]
B by nerve growth factor through the neurotrophin receptor p75. Science (Washington DC), 272: 542-545, 1996.[Abstract]
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,
constant presence of paclitaxel) or after washing off the
unincorporated paclitaxel (
, single 30-min exposure to paclitaxel).
Absorbance (A) of MTT assays were quantitated and
standardized to untreated cells (100%), using the formula
Atest/Auntreated
cells x 100 (n = 4). Data are representative of three experiments; bars,
SE. Similar kinetics of survival were obtained with 4-3.6 cells (data
not shown).
