Reversal of Vinca Alkaloid Resistance by Anti-P-Glycoprotein MonoclonalAntibody HYB-241 in a Human Tumor XenograftLana S. Rittmann-Grauer,' Milicent A. Yong, Vikki Sanders, and Daniel G. Mackensen
Hybritech Incorporated, San Diego, California 92121
ABSTRACT
A panel of monoclonal antibodies (MAbs) to P-glycoprotein wasdeveloped by immunization of mice with multidrug-resistant human neu-roepithelioma and neuroblastoma cells. All the anti-P-glycoprotein MAbsreacted with the extracellular portion of P-glycoprotein. The MAbs wereexamined for their ability to enhance accumulation of actinomycin D,vincristine, vinblastine, and doxorubicin in the human mdrl transfectantcell line, BRO/pFRmdrl.6. HYB-241, an Igd anti-P-glycoprotein MAli.was the most effective modulator, increasing actinomycin D levels in thetransfectant line by 6-fold, vincristine by 2-fold, and vinblastine levels by3-fold. None of the MAbs were capable of modifying the accumulationof doxorubicin. HYB-241 lowered the 50% inhibitory concentrationvalues of actinomycin D by 11-fold, vincristine by 6-fold, and vinblastineby 2-fold. No effect on the 50% inhibitory concentration values ofdoxorubicin or gramicidin were seen. '"In-labeled HYB-241 localized in
human tumor xenografts of BRO/pFRmdrl.6 in nude mice (25% injecteddose/g at 120 h). Mice with established drug-resistant xenografts weretreated with antibody 24 h prior to the injection of \ inca alkaloid atconcentrations known to be non-growth inhibitory. The addition of HYB-241 at 25 mg/kg per injection prior to drug resulted in a significantinhibition of growth of this drug-resistant tumor.
INTRODUCTION
P-glycoprotein is a transmembrane glycoprotein responsiblefor the ATP-dependent efflux of a broad spectrum of structurally and functionally distinct drugs from multidrug-resistantcells. A variety of small molecules capable of modulating P-glycoprotein have been described in the literature. These includecalcium channel blockers (l), calmodulin inhibitors (2), antiar-rythmics (3, 4), antimalarials (5), and other lysoosmotropicagents (6), steroids (7), antiestrogens (8), and cyclic peptideantibiotics (9). All of these compounds are effective modulatorsin vitro. They lower the ICso2 values of a variety of drugs
included in the MDR family, and they increase intracellulardrug concentrations in resistant cells. The mechanism responsible for this reversal of resistance is believed to be competitionbetween the modulator and drug for binding to the ATP-dependent efflux pump, P-glycoprotein (10, 11). The clinicalutility of any modulator, however, depends not only on itsability to reverse drug resistance at low concentrations but alsoon whether it has a low toxicity in vivo. The cardiac toxicityseen during the clinical evaluation of verapamil as a chemosen-sitizing agent pointed out the need for less toxic modulators(12, 13). Verapamil has been shown to be cytotoxic to cellsindependent of its ability to increase cancer drug accumulationin cells (14). Recent reports (15, 16) have indicated additionalsites of action, since verapamil can modulate resistance in cells
Received 8/27/91; accepted 1/24/92.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' To whom requests for reprints should be addressed, at Hybritech. Inc., P. O.
Box 269006. San Diego, CA 92126.2The abbreviations used are: 1C»,50% inhibitory concentration; ELISA,
enzyme-linked immunosorbent antibody technique: MAb, monoclonal antibody:MDR, multiple drug resistance; PBS, phosphate-buffered saline (10 mM sodiumphosphate, ISO mM sodium chloride, pH 7.5).
lacking P-glycoprotein. Two other antiaaryhthmic drugs, quin-idine and amiodarone, have also entered clinical trials as che-mosensitizing agents (17). Both drugs have produced a numberof adverse clinical side effects (18).
Our approach has been to identify MAbs which interactspecifically with P-glycoprotein, in an attempt to produce non-toxic P-glycoprotein modulators. The long serum half-life andextended residence time of MAbs on tumors may offer anadvantage over small-molecule modulators. In recent clinicaltrials (13, 19) continuous infusion of verapamil was required inorder to maintain optimal serum concentrations, since verapamil has a high systemic clearance and a half-life of 3-7 h (20).
In this paper we describe the production and characterizationof a panel of MAbs which recognize P-glycoprotein. The MAbswere evaluated for their ability to modulate P-glycoproteinactivity in vitro. HYB-241, the most potent modulator of P-glycoprotein in v/'fro, was studied further in human tumor
xenografts in nude mice.
MATERIALS AND METHODS
Cells. The multidrug-resistant human neuroepithelioma and neuroblastoma cell lines MC-IXC/VCR and SH-SY5Y/VCR (21), as well asthe drug-sensitive lines from which they were derived, MC-IXC andSH-SY5Y, were gifts from Dr. June Biedler (Sloan Kettering CancerCenter, New York). The multidrug-resistant human leukemia cell lineCEM/VLBioo (22) and its parent drug-sensitive line CCRF-CEM weregifts from Dr. William Beck (St. Jude Children's Hospital, Memphis,
TN). The human melanoma cell line BRO and the mdr I transfectantBRO/pFRmdrl.6 clone 1.1 cell line (23) were supplied by Piet Borst(The Netherlands Cancer Institute). The BRO/pFRmdrl.6 cell line wasobtained through transfection of a human mdr 1 complementary DNAinto the BRO cell under the control of a cytomegalovirus promoter andhepatitis B virus polyadenylation signals. All human lines were maintained in RPMI-I640 (Gibco, Grand Island, NY) supplemented with 2mM i ulula niim- and 10% fetal calf serum (growth media) in a humidified atmosphere of 5% COz/95% air at 37°C.The mouse myeloma
cell line P3-X63-Ag8.653 (24), a non-Ig-producing myeloma, was maintained in autoclavable minimal essential medium (Irvine Scientific,Irvine, CA) with 8% horse serum and 2% fetal calf serum. The hybrid-omas were maintained in the same media supplemented with 0.1 mMhypoxanthine, 0.4 M aminopterin, and 0.16 mM thymidine.
Drugs and Radioisotopes. Vincristine sulfate and vinblastine sulfatewere obtained from Eli Lilly (Indianapolis, In.). Actinomycin D, doxorubicin, etoposide, and gramicidin were purchased from Sigma (St.Louis, MO). All radiolabeled drugs were purchased from Amersham(Arlington Heights, IL). "'InClj was purchased from Mallinckrodt (St.
Louis, MO).Antibody Production. Six-week-old female BALB/c mice from
Charles River Biotechnology Services (Wilmington, MA) were immunized with 5 x IO6 MC-IXC/VCR cells i.p. which had been scraped
from the surface of tissue culture flasks. Three weeks later mice receiveda second i.p. injection of 5 x IO6MC-IXC/VCR cells. Four days priorto fusion, the mice received a final i.v. boost of 5 x IO6cells. This sameschedule was followed for mice immunized with SH-SY5Y/VCR cells.Splenocytes from the immunized mice were fused with the nonsecretingmouse myeloma cell line P3-X63-Ag8.653 by treatment with polyethylene glycol according to the method of Gerhard et al. (25) as modifiedfrom the method of Kohler and Milstein (26).
REVERSAL OF VINCA ALKALOID RESISTANCE BY MONOCLONAL ANTIBODY
Antibody Screening. The initial screening of hybridoma cultures wasdone by indirect ELISA using a horseradish peroxidase conjugate ofgoat anti-mouse immunoglobulin (Cappel, Málveme,PA). Tumor cellmonolayers of the drug-sensitive and drug-resistant cells were preparedby plating 10,000 cells/well in 96-well microtiter plates (Becton Dickinson, Lincoln Park, NJ) and incubating the plates at 37°Cin a COj
incubator overnight. The following day cells were fixed with 0.01%glutaraldehyde for 45 min at room temperature. After the fixative wasremoved and the plates were washed three times with PBS, the plateswere blocked with 10% bovine serum albumin for at least 45 min.Hybridoma supernatants (50 ^1) were added and allowed to incubatefor l h at 37°C.Plates were washed with PBS and incubated with 50
n\ of peroxidase-conjugated goat anti-mouse immunoglobulin diluted
1:1000 in PBS with 10% horse serum. Following five washes with PBS,positive clones were identified by the addition of 100 >i\of a solutioncontaining 1 mg/ml 0-phenylenediamine, 0.1% hydrogen peroxide, 50mM citrate, and 100 rriMsodium phosphate buffer, pH 5.0. The reactionwas quenched by the addition of 50 p\ 4 N sull'urie acid, and the plates
were read at 490 nm. donates which produced a 5-fold or greaterELISA signal on at least one drug-resistant line in comparison to itsdrug-sensitive parental line were expanded. Hybridoma cells were injected into BALB/c mice for ascites production (27). Antibodies werepurified from the ascites fluid by protein A chromatography (28).
Isotype. The subclasses of each of the MAbs were determined in anELISA as described above, where subclass-specific secondary' antibodies
were utilized (Fisher, Springfield, NJ).Immunofluorescence. Approximately 1 x IO6 cells were incubated
with 100 n\ of MAb at 10 Mg/ml in microtiter plates for l h at roomtemperature. Cells were washed three times with PBS and resuspendedin 75 n\ of fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin (Boehringer Mannheim, Indianapolis, IN) diluted 1:75 ingrowth media and incubated for 1 h. The washed cells were resuspendedin 1.0 ml PBS, and the cell suspension was analyzed at a rate of 300-600 events/s on a fluorescent activated cell sorter 400 flow cytometer(Becton Dickinson, Mountain View, CA).
Antigenic Modulation. MAbs were added to exponentially growingCEM/VLBioo cells to a final concentration of 100 Mg/ml. The cells plusMAb were maintained in tissue culture flasks at 37°Cin a CO2 incu
bator. At 1,2, 4, 6, and 24 h following the addition of MAb to thecells, an aliquot of cells (1 x IO6cells) was removed and stained witheither HYB-241 or the anti-human T-cell MAb T101 (29) as describedabove ("Immunofluorescence"). Loss of antigen from the cell surface
was monitored by flow cytometry.Drug Accumulation. BRO/pFRmdrl.6 cells in log phase growth were
harvested from tissue culture flasks using mild trypsinization, counted,and checked for viability with trypan blue. The cell concentration wasadjusted to 3-5 x 10* cells/ml of growth media, and the cells wereplated out at 1 ml/well into sterile 12-well plates (Costar, Cambridge,MA). Following attachment of the cells to the wells the growth mediumwas removed and replaced with 1.0 ml of fresh growth media with orwithout 100 Mg/ml of antibody and incubated for 30 min at 37°C.
Radiolabeled drugs were added to wells in triplicate at the followingconcentrations: [3H]actinomycin D (3 Ci/mmol) at 0.3 ^Ci/ini (1 M);[3H]vincristine sulfate (2 Ci/mmol) at 0.5 ^Ci/ini (I M); [3H]vinblastinesulfate (l 1.7 Ci/mmol) at 0.5 ^Ci/ini (0.44 M); or ['"CJdoxorubicin (55
mCi/mmol) at 0.25 ^Ci/ini (4.7 M). After incubation for varyingamounts of time at 37°Cin the dark, cells were washed with cold PBS
and released from the wells by mild trypsinization. Cells were transferred to scintillation vials, and 5 ml of CytoScint (ICN, Irvine, CA)were added to each vial. Radioactivity was measured by liquid scintillation spectrometry.
Growth Inhibition Assay. Between 3 x 10' and 5 x IO3exponentiallygrowing BRO/pFRmdrl.6 cells in 200 n\ were plated per well of a 96-well microtiter plate. One day later the growth medium was removedand replaced with growth media with or without 100 Mg/ml of antibodyfor 30 min at 37°Cfollowed by the addition of drug. Twofold serial
dilutions of each drug were made in a single microtiter plate in triplicate. Following a 3-day incubation at 37"C at 5% CO2, cells were fed
by the addition of 100 ßlof growth media. One day later (5 days afterplating) the cell medium was removed and replaced with 100 M!of 0.5%
Biodistribution in Mice with Human Tumor Xenografts. HI«)pFRmdr 1.6 cells (5 x 106cells/animal) were injected s.c. into the flanks
of 6-8-week-old female nude mice (obtained from HarÃanSpragueDawley, Indianapolis, IN). At day 21, animals with 0.2-0.3-g tumorswere selected for experiments. Diethylenetriaminepentaacetic acid-conjugated antibodies (1 Mg)containing 10 /<<'i '"In were injected into the
tail vein (0.1 ml in PBS), and groups were separated (5 animals/group)for sacrifice at various time points. The procedures for sacrificing,specimen collection, counting, and data reduction have been describedpreviously (30).
Growth Inhibition of MDRI Transfectant Tumors in Nude Mice.BRO/pFRmdrl.6 cells (5 x 10* cells/animal) were implanted by s.c.inoculation into the flanks of 6-8-week-old female nude mice. Whentumors reached 0.1-0.2 g, treatment was begun. The control groupreceived i.v. injections of sterile PBS (500 M!)once each week for 3-4weeks. The experimental group received i.v. injections of HYB-241 (25mg/kg in 500 M'PBS) followed 24 h later by either vinblastine (5 mg/kg) or vincristine (2 mg/kg). The treatment with antibody followed 24h later by drug was repeated once each week for the duration of theexperiment (3-4 weeks). A group which received antibody only (25 mg/kg i.v.) as well as a group receiving drug only every 7 days were includedin each experiment. Tumor size as well as weight of the animals weremeasured 2-3 times a week.
RESULTS AND DISCUSSION
A wide variety of small-molecule modulators of P-glycopro-tein have been described in the literature (1-9 ). These reagentsare highly effective in vitro in reducing the IC50 values of avariety of drugs in resistant cells as well as increasing theintracellular concentrations of chemotherapeutic drugs. Limited success has been achieved in vivo in reversing drug resistance due to the intrinsic toxicity of many of these small-molecule modulators. Although the results were encouragingin the Arizona trial where verapamil was added to a multidrugregimen in P-glycoprotein-positive multiple myeloma and produced responses in 2 of 5 patients (13) and in a later trial inlymphoma (19) which produced responses in 13 of 18 patients,optimal concentrations of verapamil required for the reversalof resistance in vitro could not be administered because of itspotent effects on the cardiovascular system. MAbs may represent more specific and less toxic modulators of P-glycoprotein.A variety of MAbs to P-glycoprotein have been described in theliterature. These include C219 (31), JSB-1 (32), MRK16, andMRK17 (33). Only MRK16 and MRK17 MAbs bind to cellsurface epitopes of P-glycoprotein and therefore have potentialtherapeutic value. MRK16 has been shown to modulate P-glycoprotein activity and reverse resistance to vincristine anddaunorubicin in vitro (34). We have produced a panel of MAbsto P-glycoprotein and studied their ability to modulate theactivity of this ATP-dependent efflux pump in vitro and in vivo.
Mice were immunized with either the human multidrug-resistant neuroblastoma cell line MC-IXC/VCR, which is 6000-
REVERSAL OF VINCA ALKALOID RESISTANCE BY MONOCLONAL ANTIBODY
fold resistant to vincristine, or the human multidrug-resistantneuroepithelioma line SH-SY5Y/VCR, which is 2500-fold resistant to vincristine. Two fusions produced 1139 hybridomas.donates were assayed by ELISA against drug-resistant andparental drug-sensitive cells. Ten clones consistently demonstrated greater reactivity with at least one drug-resistant cellline in comparison to the drug-sensitive control cells. Table 1shows the binding ratios of each of these MAbs on two sets ofdrug-resistant and parental drug-sensitive cell lines, MC-IXC/VCR and MC-IXC and SH-SY5Y/VCR and SH-SY5Y . EightMAbs reacted with the mdrl transfectant and not the parentalline BRO, which indicates that these antibodies are directedagainst the mdrl gene product P-glycoprotein. MAbs HYB065and HYB195 are not directed against P-glycoprotein, since theyfailed to bind to the transfected cell line. These MAbs mayrecognize novel drug-resistant related molecules. All of the anti-P-glycoprotein MAbs are IgGi, with the exception of HYB-162, which is an IgG2b, and HYB-243, which is an IgM.
The ELISA data generated on the glutaraldehyde-fixed cellssuggested that the panel of MAbs may be recognizing a cellsurface component of P-glycoprotein. Fig. 1 shows the typicalshift in mean intensity of fluorescence produced by three of theanti-P-glycoprotein MAbs, HYB-241, HYB-612, and HYB-237, when the MAbs were reacted first with the drug-sensitiveline CCRF-CEM and then with the drug-resistant line CEM/VLBioo and then examined by flow cytometry. All of the MAbsbound to intact CEM/VLBjoo cells, suggesting that they allrecognize the extracellular loop portions of P-glycoprotein. Nobinding to CCRF-CEM cells was seen in this assay.
Previously published experiments (35) indicated that bothH YB-241 and H YB-612 were capable of increasing intracellularconcentrations of actinomycin D and vincristine in MC-IXC/VCR cells. We were interested in determining whether HYB-241, HYB-612, or any of the other anti-P-glycoprotein MAbswould have similar effects on a cell line with lower levels ofresistance, such as the BRO/pFRmdrl.6 cell line, and whetherthe MAbs would modulate the activity of other drugs in theMDR family. The accumulation of actinomycin D over time inthe presence of varying amounts of HYB-241 is shown in Fig.2 . In the presence of 1.25 Mg/ml actinomycin D, BRO/pFRmdrl.6 cells reached steady-state levels of drug by 2 h andremained at this level at 6 h. The addition of 0.1 ng/m\ HYB-
241 had no effect on drug accumulation, but the addition of 1Mg/ml or more of HYB-241 significantly increased levels ofdrug which accumulated in the cells. The greatest increase in
H YB-24\cHYB-612rHYB-034HYB-057HYB-237HYB-374HYB-162HYB-243HYB-195CHYB-065IgG,IgG,IgG,IgG,IgG,IgG,IgG2bIgMIgMIgM4017277977415015512+19+13+11+6
+11+3+8+51
" Antibody binding ratios, resistant:sensitive.'Antibody reacts with the human MDRI transfectant cell line, BROlMDRI
and not the parental nontransfected cell line by ELISA.' Indicates the immunogen which produced this monoclonal antibody was the
cell line SH-SY5Y/VCR. All others were produced with MC-XIC/VCR asimmunogen.
300
200
100
0300
200
100
0300
HYB-241
4N' v 313
HYB-612
' I
I
339
200
100-
HYB-237
IBS
"10° 10' 101 10s
MEAN INTENSITY OF FLUORESCENCE (MIF)
Fig. I. Flow cytometric analysis of anti-P-glycoprotein MAbs on viable drug-resistant cells. MAbs bound to the drug-sensitive line CCRF-CEM ( ) andthe drug-resistant line CEM/VLB100 ( ). Numbers above each peak, meanintensity of fluorescence.
actinomycin D levels (7-fold) was achieved at 50 ng/m\ MAbat 6 h .
Fig. 3 illustrates the effect of each of the MAbs as well as 20M verapamil on the accumulation of vincristine, vinblastine,and actinomycin D in BRO/pFRmdrl.6 cells. HYB-241 at 50Mg/ml was the most effective MAb at increasing intracellularlevels of actinomycin D (7-fold), vinblastine (3-fold), and vincristine (2-fold). Levels of actinomycin D and vinblastine whichaccumulated in the transfectant cells in the presence of HYB-241 were comparable to levels of drug which accumulated inthe drug-sensitive BRO cell line (data not shown). In contrast,levels of vincristine which were obtained in the transfectant linein the presence of HYB-241 were 2-fold less than the levels ofvincristine which accumulated in the drug-sensitive BRO line.HYB-241 was as effective as 20 M verapamil in increasing theaccumulation of vinblastine in the transfectant, while verapamilwas more effective in increasing levels of actinomycin D andvincristine in these cells. The other anti-P-glycoprotein MAbsmodulated the accumulation of actinomycin D and the Vincaalkaloids to lesser degrees. None of the MAbs affected theaccumulation of doxorubicin (data not shown). The similarityin drug modulation seen with the MAb panel was not surprising,since competition studies suggested that all the MAbs boundto the same or overlapping epitopes (data not shown). Thedifferences in degree of modulation are probably related todifferences in MAb affinities. The two drug-resistance-relatedMAbs HYB-065 and HYB-195, which failed to bind to theMDRI transfectant, had no effect on accumulation of any ofthe drugs examined.
One mechanism which could account for the ability of a MAbto modulate drug accumulation in drug-resistant cells would bethrough antibody-mediated internalization of antigen (36).
REVERSAL OF VINCA ALKALOID RESISTANCE BY MONOCLONAL ANTIBODY
Fig. 2. Effect of HYB-241 concentration on the accumulation of |3H]actino-mycin D in BRO/pFRmdrl.6 cells. Cells (5 x 10') were incubated with [3H]
actinomycin D at 0.3 nC\/m\ (l ¿iiu)for 1. 2, 3, and 6 h in the presence of MAbHYB-241 at 100 ng/ml (•),50 Mg/ml (O), 10 ng/ml (A), 1 ng/m\ (•),0.1(D), or PBS (A).
GDJ352
HYB065
HYB195
HYB194
HYB243
HYB037
HYB034
HYB162
HYB374
HYB237
HYB057
HYB612
HYB241
VERAPAMIL
Vincristine
VinblastineActinomycin-D
10
FOLD INCREASE
Fig. 3. Effect of anti-P-glycoprotein MAbs on the accumulation of actinomycinD, vincristine. and vinblastine in BRO/pFRmdrl.6 cells. Cells (5 x 10') werepreincubated with MAb at 50 >ig/ml for 30 min followed by the addition of |'H]actinomycin D at 0.3 //Ci/ml, |JHlvincristine at 0.5 nCi/ml. or [3H]vinblastine at
0.5 fiCi/ml. Cells were washed and counted for radioactivity after 4 h. The effectof verapamil (20 UM) on BRO/pFRmdrl.6 cells and the effect of each of thedrugs on BRO cells were studied. Values represent the means of threedeterminations.
Down-regulation of P-glycoprotein on the cell surface couldlead to the increased accumulation of drugs, especially thosewith lower affinities for P-glycoprotein-binding sites. In orderto test this hypothesis, CEM/VLB,oo cells were incubated withsaturating amounts of HYB-241, and at various time intervalsthe cells were assayed for the presence of P-glycoprotein ontheir cell surface. Over a 24-h time period no loss of P-glycoprotein was observed from the surface of the cells (Fig. 4). TheMAb T101 (29), known to immunologically modulate its antigen, CDS, was included as a positive control. This result suggests that antigenic modulation is not responsible for the reversal in resistance seen with HYB-241.
Based on the drug accumulation results which indicated thatHYB-241 may be the most potent MAb modulator of our panel,
we extended our studies of this MAb to examine its ability tolower ICso values. The IC50 values for each of the drugs wasdetermined for both the drug-sensitive and drug-resistant trans-fectant lines. The transfectant line was resistant to vincristine(410-fold), vinblastine (63-fold), actinomycin D (12-fold), eto-poside (5-fold), gramicidin (24-fold), and doxorubicin (3-fold).In agreement with drug accumulation experiments, the additionof HYB-241 resulted in the greatest decrease in IC.io withactinomycin D. HYB-241 at 100 Mg/ml reduced the IC50 ofactinomycin D 11-fold from 4.0 to 0.36 n\i in mdrl-transfectedcells. The actinomycin D IC50for the mdrl-transfected cell line
120 -i
OU
100 -
80 -
60
40
HYB-241
10 20 30
TIME (h)Fig. 4. Kinetics of HYB-241-induced in vitro antigen modulation. Points, mean
loss of fluorescence at 37°Cversus time for CEM/VLB10o cells incubated withexcess HYB-241 (D) or T101 (•)MAbs and then stained by indirect immunofl-uoresence. Bars, SD.
100
90
5d
80
70
60
50
40
30
20
10
0.01 0.1 10 100
ACTINOMYCIN-D (nM)
Fig. 5. Potentiation of actinomycin D toxicity in BRO/pFRmdrl.6 cells withHYB-241. The IC50sof actinomycin D in BRO/pFRmdrl.6 and BRO cells weredetermined in the presence or absence of 100 HI: ml HYB-241. Cells werepreincubated with HYB-241 for 30 min prior to the addition of actinomycin D.O, BRO/pFRmdrl.6 cells; •¿�.BRO/pFRmdrl.6 cells preincubated with 100 ng/ml HYB-241; A. BRO cells; A. BRO cells preincubated with 100 ng/ml HYB-241. Values are the mean ±SD (ears) of three determinations.
REVERSAL OF VINCA ALKALOID RESISTANCE BY MONOCLONAL ANTIBODY
Table 2 Reversal of drug resistance in BROtMDRl cells by HYB-241 MAb VLB MAb VLB MAb VLB
DrugActinomycin
D(nM)4-HYB-241'+GDJ-352'Vincristine
(nM)+HYB-241+GDJ352Vinblastine
(nM)+HYB-241+GDJ-352Doxorubicin
(nM)+HYB-241+GDJ-352Etoposide
liAi >+HYB-241+GDJ-352Gramicidin
(u\i i+HYB-241+GDJ-352BRO
cellsICwaa0.34(0.31-0.37)'0.23(0.19-0.27)ND0.61
(0.50-0.71)0.58(0.41-0.75)ND0.49
(0.32-0.66)0.60(0.32-0.88)ND86(71-102)72
(60-85)ND0.76(0.47-1.11)
1.11(0.27-1.94)ND2.2(1.8-2.6)
3.1 (1.8-4.4)NDDMF*1.5N
D1ND0.7ND1.1ND0.7ND0.7NDBROtMDRl
cellsIC504.0(3.0-5.0)'0.36(0.13-0.60)2.8
(2.2-3.4)250(157-343)42.0(33-52)192(96-286)31
(25-36)15(11-21)19(11-26)290(262-319)269
(233-305)233(190-276)3.4
(2.0-4.9)7.6(7.0-8.5)3.9(2.2-5.6)53
(32-73)39(22-57)53
(26-79)DMF111.461.32.11.61.21.20.40.91.4120
-
18-16
•¿�14
-TUMOR
12-VOLUME10:(cm3)o1
' 8-6-4
•¿�2-0-4Fig.
7. Inhibitiin nude mice byartine.
A, 25 mg/kgonce weekly; A.5once
weekly; •¿�.Plions; bars, SE.
* ICjo values represent the drug concentration at which the growth of the cells
is 50% of that seen in the control cell wells.* DMF, dose modifying factor (IC50of drug divided by the IC50of drug in the
presence of antibody).c 95% confidence intervals.''The anti-p-glycoprotein monoclonal antibody HYB-241 was added at 100
fig/ml to the cells 30 min prior to the addition of drug.'GDJ-352, an anti-gardnarella negative control antibody, was added at 100
¿tg/mlto the cells prior to the addition of drug.
in the presence of MAb was not significantly different from theIC50 of the drug-sensitive BRO line (Fig. 5). HYB-241 at 100Mg/ml did not alter the IC50 of the drug-sensitive BRO line. Inorder to determine if the reduction in IC50 of actinomycin Dwas a specific effect of HYB-241, an IgG, anti-Gardnerellaantibody, GDJ352, was included in the same experiment. Noreduction in the IC50 of the drug was observed in the presenceof 100 Mg/ml of the irrelevant MAb (data not shown). Theability of HYB-241 to lower the IC50 values of other drugs isshown in Table 2. The IC50 values for each of the drugs wasdetermined in the presence or absence of MAb. The dose-
modifying factor was determined by dividing the IC5o of thedrug by the IC5o of drug plus MAb. The negative controlantibody GDJ352 was included in each of the studies to confirmthat the effect seen was specific for the anti-P-glycoproteinMAb. The IC50 values for vincristine and vinblastine werereduced 6- and 2-fold, respectively. HYB-241 had no effect onthe IC50 of doxorubicin or gramicidin. HYB-241 interestingly
6 8 10 12 14 t6 18 20 22
DAYS AFTER IMPLANTATION
Fig. 7. Inhibition of growth of multidrug-resistant BRO/pFRmdrl.6 tumorsin nude mice by anti-P-glycoprotein MAb HYB-241 in combination with vinblastine. A, 25 mg/kg of HYB-241 i.v. followed 24 h later by 5 mg/kg vinblastine i.v.once weekly; A, 5 mg/kg vinblastine i.v. once weekly; O, 25 mg/kg HYB-241 i.v.
PBS control i.v. once weekly. Points, means of five determina-
increased the IC5o value for etoposide by 2.2-fold. The mechanism for this increase in resistance in the presence of an anti-P-glycoprotein antibody is currently being studied.
The ability of the MAb to localize to drug-resistant BRO/pFRmdr 1.6 tumors in vivowas studied using ' ' ' In-labeled H YB-241. The radiolabeled antibody was injected into tumor-bearingnude mice, and at various time points thereafter mice weresacrificed, and the amount of radioactivity in each of the organsand in the tumor was determined. Fig. 6A shows that by 24 hafter i.v. injection of ' " In-labeled HYB-241,16% of the injected
dose per gram was bound to the tumor. The amount of MAbcontinued to accumulate at the tumor site, and at 120 h post-injection 25% of the injected dose per gram was bound to thetumor. '"In-labeled MOPC 21 (37), an irrelevant antibody of
the same isotype, bound less than 5% of the injected dose pergram at 120 h postinjection (Fig. 6B). The "'In-labeled HYB-241 did not localize to the drug-sensitive BRO tumors (datanot shown).
Based on the results of the biodistribution study which indicated that HYB-241 localized at the tumor 24 h postinjectionand remained at the tumor at high concentrations at 120 h, aprotocol was developed to study the effect of a 24-h predose ofHYB-241 followed by Vinca alkaloid administration on thegrowth of an established drug-resistant tumor. Doses of Vincaalkaloid were chosen which were ineffective at inhibiting growthof the drug-resistant BRO/pFRmdrl.6 tumor but which were
Fig. 6. Biodistribution of MAbs in nudemice bearing mdrl transfectant tumors. Die-thylenetriaminepentaacetic acid-conjugatedMAbs were labeled with "'In and injected into
the tail veins of nude mice bearing s.c. tumorsof about 0.3 g. At various time points, animalswere sacrificed (5 mice/group), and tissueswere taken for gamma well counting. After thetissues were weighed, the percentage of theinjected dose/g tissue was calculated. I. MAbHYB-241; B, MAb MOPC 21 (irrelevantMAb). The values represent the means ±SD(bars) of five determinations.
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effective in inhibiting growth of the drug-sensitive BRO tumor.Predosing animals with 500 ^g of HYB-241 (25 mg/kg) i.v. 24h prior to Vinca alkaloid weekly for 3 weeks inhibited thegrowth of the drug-resistant tumor. As shown in Fig. 7 vinblas-
tine treatment at 5 mg/kg i.v. once per week did not significantlyinhibit the growth of this tumor. The injection of 500 fig ofHYB-241 24 h prior to the same dose (5 mg/kg) of vinblastinesignificantly decreased the growth of the tumor. HYB-241 alonehad no effect on the growth of the tumors. Similar inhibitionin growth of the tumors was obtained when HYB-241 waspreadministered to mice prior to injection of weekly doses ofvincristine which alone had no effect (data not shown). Incontrast, HYB-241 was unable to potentiate the activity ofdoxorubicin in vivo.
In summary, these results indicate that HYB-241 may be aneffective chemosensitizer of vincristine, vinblastine, and acti-
nomycin D in the treatment of human tumors. Antibodies mayoffer some unique advantages in the clinic as modulators ofdrug resistance due to their long residence time at the tumorsite and long serum half-life. MAbs may also produce fewerside effects than the small-molecule modulators currently beingexamined in the clinic. Humanized MAbs substantially reducethe risk of adverse immunological response in patients. Recombinant humanized immunoglobulins are further characterizedby long in vivo half-lives in the range of 48-71 h (38, 39). Thespecificity of MAbs is another advantage over the more broadlyreactive small-molecule modulators. Potential toxicities due tothe blocking of P-glycoprotein in normal tissues by modulatorsand the accumulation of toxic compounds in normal tissuesmay not occur with antibody modulators because P-glycoprotein may not be physiologically accessible to antibody in thenormal tissues where it is expressed (40). Studies are currentlyunder way to study the effect of HYB-241 on Vinca alkaloidaccumulation in normal tissues.
The mechanism by which HYB-241 modulates the pump ispurely speculative at this time. The MAb does not modulatethe antigen immunologically, and therefore it appears that theability of the MAb to reduce IC50 values and increase drugaccumulation is not due to the loss of P-glycoprotein from thesurface of the cells. Alternatively, the binding of MAb to theextracellular portion of P-glycoprotein may induce a confor-mational change in the molecule which has an impact on theability of the molecule to transport some drugs (actinomycinD, vincristine, vinblastine) but not others (doxorubicin). Thisselectivity in the antibody's ability to modulate drug activity
may provide further insight into the structure/function relationship of P-glycoproteins.
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