antitumor effect of actinomycin d entrapped in liposomes...

(CANCER RESEARCH 43, 5328-5334, November 1983]

Antitumor Effect of Actinomycin D Entrapped in Liposomes Bearing Subunitsof Tumor-specific Monoclonal Immunoglobulin M Antibody1

Yoshiyuki Hashimoto,2 Minoru Sugawara, Takashi Masuko, and Hiroshi Hojo

Department of Hygienic Chemistry, Pharmaceutical Institute, Tohoku University, Sendai 980, Japan

ABSTRACT

Sonicated liposomes containing actinomycin D in the membranes were chemically coated with the subunits of monoclonalimmunoglobulin M (IgM) antibody against a mouse mammarytumor-associated antigen (MM antigen) and examined for theirin vitro and in vivo antitumor effects against MM46 (MM+) andMM48 (MM") tumors of C3H/He mouse origin. The antibody-

bearing, actinomycin D-containing liposomes (chemoimmunoli-posomes) were selectively bound to MM* tumor cells and

showed much more in vitro cytotoxicity against the tumor cellsthan that shown by free actinomycin D. The in vivo antitumoreffect of the chemoimmunoliposomes was tested on the mammary tumor cells (5 x 10" to 5 x 106) transplanted i.p. into

syngeneic mice. A single i.p. injection of the chemoimmunoliposomes containing 0.3, 0.5, or 1 /ug of actinomycin D into MM46tumor-bearing mice resulted in the cure of some mice and a

prolonged survival time in the rest of the mice as compared toresults in controls. In this test, free actinomycin D, anti-MM IgMantibody, and bovine serum albumin-coated liposomes contain

ing actinomycin D were marginally effective or ineffective. Toexamine a systemic antitumor effect of chemoimmunoliposomes,mice were inoculated with MM46 tumor cells and then treatedwith a single i.v. injection of liposomes 4 days later. If the micewere pretreated with an i.v. injection of unmodified multilamellarliposomes, an injection of the chemoimmunoliposomes containing 1 ng of actinomycin D resulted in a significant inhibition oftumor growth. Both free actinomycin D and bovine serum albumin-coated liposomes containing actinomycin D were ineffective

against the s.c. tumor. These results indicate that an antitumordrug entrapped in the membranes of small sonicated liposomesbearing antitumor monoclonal antibodies can be delivered toantigenic tumor cells and exert more efficient antitumor activitythan does the free drug.

INTRODUCTION

The selectivity of drug action on tumor cells is one of thefundamental virtues of cancer chemotherapy. The recent development of monoclonal antibodies against a variety of animal andhuman tumors facilitates the utilization of antibodies as specificcarriers to deliver drugs to antigenic tumor cells. In this light,either antitumor drugs or toxins are modified with antitumorantibodies, and the antitumor effect of the conjugates has beentested in animal models. Another approach to alleviate the sideeffects and to enhance the activity of antitumor drugs is the use

of liposomes as drug carriers. A variety of antitumor drugs, whenencapsulated in liposomes (4-6, 11, 14, 15, 17, 20-22, 24, 28,

30) or introduced into liposome membranes (7, 9, 10, 23), aremore intensely incorporated into tumor cells and can inhibit thegrowth of tumor cells both in vitro and in vivo. In these aspects,antibody-coated liposomes can be provided as the antigen-

specific and efficient carriers to deliver the antitumor drugs.Antibody modification of liposomes can be carried out by the

direct adsorption of antibodies to liposomes (29) or by thechemical coupling of antibodies with liposomes (2, 3, 16, 27). Inorder to couple monoclonal antibodies to liposome surfaces, weutilized SH3-bearing subunits of IgM antibody, which were cou

pled to liposomes containing SH-reacting maleimide groups (8).

The liposomes prepared by this method showed a specific andefficient targeting to the antigenic cells (8). As an antitumor drugto be introduced into antibody-bearing liposomes, we selected

actinomycin D, which is known to be a potent cytotoxic, lipophilicantitumor drug. This selection was based in part on the findingsof Papahadjopoulos ef al. (20), showing that actinomycin Dencapsulated in liposomes can exert stronger in vitro antitumoractivity than does the free actinomycin D and that it is alsoeffective in actinomycin D-resistant tumor cells. In this paper, we

report that actinomycin D introduced into the membranes ofliposomes bearing subunits of tumor-specific, monoclonal IgM

antibody showed a strong cytotoxicity against antigenic tumorcells both in vitro and in vivo.

MATERIALS AND METHODS

Chemicals

Cholesterol, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidyl-

ethanolamine, and actinomycin D were purchased from Sigma ChemicalCo., St. Louis, Mo. All the lipids used were checked for purity by thin-

layer chromatography using chloroform:methanol:water (70:30:5, v/v)as solvent and were found to contain only a trace amount of contaminants. The lipids dissolved in chloroform were divided into small portionsand stored until use under nitrogen in sealed ampuls at -20Â°. [3H]-Actinomycin D (20 mCi/mmol) and [3H]thymidine (40 Ci/mmol) were

obtained from The Radiochemical Centre, Amersham, Buckinghamshire,England, and cholesteryl [1-14C]oleate (50 mCi/mmol) was from New

England Nuclear, Boston, Mass.

Mice and Tumor Cells

Male C3H/He mice were obtained from Shizuoka Agricultural Cooperative for Experimental Animals, Hamamatsu, Japan, and were used at

' Supported in part by a Grant-in-Aid for Cancer Research from the Ministry of

Education. Science and Culture and a grant from the Ministry of Health and Welfare,Japan.

2To whom requests for reprints should be addressed, at the Department of

Hygienic Chemistry, Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980. Japan.

Received February 23, 1983: accepted July 14, 1983.

3The abbreviations used are: SH, sulfhydryl group: IgM, immunoglobulin M;

IgMs, monomeric subunit of IgM; chemoimmunoliposome, antibody-coated liposome containing actinomycin D; BSA, bovine serum albumin; chemo-BSA-liposome,bovine serum albumin-coated liposome containing actinomycin D; MBPE, N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine; PBS, phosphate-bufferedsaline (137 mM NaCI:2.7 mw KCI:1.5 mw KH2POÂ«:8.1mw Na2PO4, pH 7.2); chem-oliposome, liposome containing actinomycin D; immunoliposome, antibody-coatedliposome; MLL, antibody- and drug-free multlilamellar liposome.

5328 CANCER RESEARCH VOL. 43

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Antibody-coated Liposomes as Drug Carrier

6 to 7 weeks of age. In each experiment, mice of similar age and weight(within 3 g difference) were selected. They were housed in plastic cagesand maintained in an air-conditioned room.

MM46 and MM48 tumor cells originating from spontaneous mammarytumors of C3H/He mice were donated by Dr. T. Tachibana, Institute forTuberculosis, Leprosy and Cancer, Tohoku University, Sendai, Japan.The tumors were maintained by serial i.p. transfers in syngeneic mice.The tumor cells were harvested from the ascites, washed with medium,and used for experiments.

Antibody and the Subunit

A cell clone, 2-11 -G, which secretes monoclonal IgM antibody, has

been established by Seto ef al. (25) from the somatic cell hybrids betweenNS-1 mouse myeloma cells and spleen cells of a C3H/He x BALB/c F-,mouse that was hyperimmune to MM46 tumor cells. 2-11 -G IgM antibodyrecognizes a tumor-associated antigen (MM antigen) that distributes on

the cell surface of several lines of C3H/He mouse mammary tumors (25).We confirmed that the MM antigen recognized by 2-11-G IgM antibody

was present in MM46 cells but absent in both MM48 cells and normalspleen cells of C3H/He mice as determined by complement-dependent

cytotoxicity assay and an indirect membrane immunofluorescence test(8). Detailed procedures for preparation of the subunits of IgM antibodies(IgMs) were described previously (8). Briefly, 2-11-G hybridoma cellswere cultured in Dulbecco's modified Eagle's medium containing 10%

fetal calf serum. IgM antibody in the pooled culture supernatants wasprecipitated by adding ammonium sulfate and then purified on a Sepha-cryl S-300 column. IgMs was prepared from the purified IgM antibodyby a reduction with cysteine (19) and purified on a Sephacryl S-300

column (8).

Preparation of Protein-coated, Actinomycin D-containing Liposomes

Chemoimmunoliposomes and chemo-BSA-liposomes were preparedas follows. MBPE was synthesized from m-maleimidobenzoyl-W-hydrox-

ysuccinimide ester and dipalmitoylphosphatidylethanolamine as described previously (8). Actinomycin D was added to chloroform solutioncontaining cholesterol (17.5 Â»imol),dipalmitoylphosphatidylcholine (25Â¿jmol),and MBPE (2.5 Â¿Â¿mol).Following the addition of 5 ml of 123 (TIMNaCMO mw NaH2PO4, pH 6.8, a dry lipid film prepared from the mixturewas vortexed for 10 min and then sonicated in the cup horn of a BransonModel W-185 sonifier at Power Setting 7 for 60 min. During sonication,

a N2 atmosphere was maintained over the liposome suspension, whichwas kept on crushed ice to keep the suspension temperature under 25Â°.

Aggregated and large liposomes were removed by centrifugation at10,000 x g in a swing rotor for 30 min, and the upper 4 ml of the total5-ml liposome suspension were carefully pipeted. The recovery of the

liposomes was 65 to 70% in terms of the lipid amount. This liposomesuspension (4 ml) was then treated with BSA (2 mg in 2 ml PBS) or 2-11-G IgMs (2 mg protein in 2 ml PBS) for 60 min at 37Â°and then with

cysteine (1.25 mg/ml) for 30 min in order to block the maleimide groupsremaining on the liposome surfaces. The liposome suspension was thenpurified by means of dextran density gradient centrifugation as describedpreviously (8). Briefly, a density gradient solution was made from theliposome suspension (6 ml) and an equal volume of 20% dextran (Pharmacia Dextran 70) in PBS. After centrifugation at 150,000 x g for 16 to20 hr, the gradient solution was divided into 10 fractions (each 1.2-ml

fraction), and the second fraction from the top, which contained themajority of the liposomes, was used for our experiments.

Chemoliposomes and immunoliposomes were prepared as above butwithout antibody treatment and without added actinomycin D, respectively. The amount of protein in the purified liposome suspension wasassayed as described previously (8).

Radiolabeled liposomes were prepared by adding cholesteryl [1-14C]-

oleate (3 ÃŸC\)to the lipid mixture. In order to examine the recovery ofactinomycin D in liposomes, [3H]actinomycin D was added to the lipid

mixture together with unlabeled actinomycin D.

Liposomes Used for Blocking of Phagocytes

MLL were prepared according to the method of Souhami ef al. (26).Briefly, dipalmitoylphosphatidylcholine (35 ^mol), cholesterol (10 Mmol),and dicetyl phosphate (5 ^mol) were dissolved in chlorofornrmethanol(9:1) and evaporated to dryness in a vacuum at 25Â°. PBS (6 ml) was

added to the lipid film and vortexed for 10 min. To obtain homogeneousmultilamellar liposomes, the suspension was sonicated for 30 sec. Theliposome suspension was used within 60 min after preparation.

Targeting of Liposomes

Aliquots of 14C-labeled immunoliposomes, Chemoimmunoliposomes,

or chemo-BSA-liposomes were added in triplicate to tumor cell suspension containing 5 x 10' cells in 1 ml of Roswell Park Memorial Institute

Medium 1640 plus 10% fetal calf serum. After incubation at 37Â°for 60

min, the tumor cells were sedimented by centrifugation at 300 x g for10 min and then washed 3 times with PBS by centrifugation to removeunbound liposomes. The cell pellet was digested with Soluene (Packard),and the radioactivity was measured by a standard scintillation techniqueusing a toluene scintillation cocktail.

Cytotoxicity Assay

Aliquots of a liposome suspension or actinomycin D solution wereadded to a 1-ml tumor cell suspension containing 5x10" cells in Roswell

Park Memorial Institute Medium 1640 plus 10% fetal calf serum. Afterincubation for 60 min at 37Â°, the tumor cells were washed and then

resuspended in 1 ml of culture medium. Aliquots (0.2 ml) of the tumorcell suspension were inoculated in triplicate into wells of a Falcon No.3040 microtest plate. After incubation for 12 hr at 37Â°,the tumor cellswere pulsed with [3H]thymidine for an additional 6 hr and harvested with

a multiple cell harvester, and the radioactivity of the cells was measured.

Mouse Experiments

Injection i.p. of a Drug into Mice Bearing Peritoneal Tumor. C3H/He mice which received an i.p. injection of MM46 or MM48 tumor cellswere divided randomly into groups. One day after tumor cell inoculation,tumor-bearing mice were treated with an i.p. injection of an aliquot (0.2

ml) of a liposome preparation suspended in PBS (250 n\/m\), PBS solutionof 2-11-G IgM antibody, PBS solution of actinomycin D, or PBS. The

presence of tumor cells in the ascites was periodically examined bymeans of a smear test, in which the cells were stained with Giemsastain. The experiments were terminated 60 or 90 days after tumortransplantation. The statistically significant differences between survivaldays of experimental and control mice were assayed by the Cox-Mantel

test.Injection i.v. of a Drug into Mice Bearing s.c. Tumor. C3M/He mice

which received a s.c. injection of 106 MM46 tumor cells at the flank were

divided randomly into groups. Four days after tumor inoculation, micewere treated by an i.v. injection of an aliquot (0.2 ml) of a liposomepreparation suspended in PBS (250 /Â¿I/ml),PBS solution of actinomycinD, or PBS. One hr before the drug injection, several groups of mice weretreated by an i.v. injection of a PBS suspension (0.2 ml) of MLL. Tumorsizes were periodically measured over the skin with the use of calipers.Tumors were isolated from mice 12 days after drug treatment andweighed. The statistical differences between weights of tumors in experimental and control mice were assayed by the Student f test.

Assay of Phagocytic Activity in Mice

Phagocytic activity was examined by measuring the intravesicularclearance rate of colloidal carbon according to the method of Biozzi efal. (1). Aliquots (0.2 ml) of a suspension of carbon (Pelican ink; GÃ¼ntherWagner, Hanover, W. Germany) in 0.1% gelatin:0.9% NaCI solution wereinjected into tail veins of mice. The blood samples were taken up fromthe retroorbital venous plexus at 2,12, 22, and 32 min after the injection.

NOVEMBER 1983 5329



y. Hashimoto et al.

The blood (20 ^0 was treated with 0.1 M Na2CO3(2 ml), and theabsorbance at 660 nm was determined spectrophotometrically. Theregression lines were made on a semilogarithmicgraph, and the phago-cytic index, K, was calculated from the equation

K =log C, - log Ci

T2-T,

where C, and C2are the carbon concentrations at times 7",and T2(min),

respectively.

RESULTS

Contents of Phospholipids, Protein, and Actinomycin D inPurified Liposome Preparations

Protein-coated, actinomycin D-containing liposomes were pre

pared from MBPE (2.5 /Â¿mol),dipalmitoylphosphatidylcholine (25Â¿(mol),cholesterol (17.5 ^mol), actinomycin D, and 2 mg of 2-11 -G IgMs or BSA and were purified by a density gradient centrifu-

gation (1.2 ml, final volume). The liposome suspension contained48.9% of the added lipid and 40.5% of the added IgMs proteinor 36.0% of BSA (11.2 /jmol of phospholipid, 0.68 mg of IgMsprotein, or 0.60 mg of BSA per ml, respectively) (average of 5experiments). When chemoimmunoliposomes were prepared byadding 50,100, and 200 ^g of actinomycin D to the lipid mixture,the recoveries of actinomycin D in the purified liposome suspensions (each with 1.2 ml in the total suspension volume) were18.8, 15.0, and 14.7%, respectively (average of 3 experiments);contents of actinomycin D in the liposomes were thus 6.3,10.0,and 19.6 ng/m\, respectively. When the same amounts of actinomycin D were used for the preparation of liposomes, theamount of actinomycin D introduced into the membranes ofchemo-BSA-liposomes or chemoliposomes was equivalent to

the amount of actinomycin D introduced into the chemoimmunoliposomes (differences of less than 10%).

In Vitro Targeting of Liposomes to MM46 and MM48 TumorCells

Liposome preparations labeled with cholesteryl [14C]oleate

were incubated for 60 min with target cells, and the amounts ofcell-bound liposomes were assayed from the radioactivity. The

results are illustrated in Chart 1. Irrespective of the presence orabsence of actinomycin D in the liposome membrane, the im-

munoliposomes could selectively bind to antigenic MM46 tumorcells but not to nonantigenic MM48 tumor cells, although bindingcapacities of the liposomes to MM46 tumor cells were lessenedby introducing actinomycin D into the membranes. Neither BSA-

liposomes nor liposomes treated with cysteine alone bound toeither MM46 or MM48 tumor cells, irrespective of the presenceor absence of actinomycin D in their membranes.

In Vitro Cytotoxicity of Chemoimmunoliposomes

MM46 or MM48 tumor cells were incubated with the chemoimmunoliposomes (19.6 Â¿jgactinomycin D per 11.2 ^mol phospholipid per ml), actinomycin D alone, or a mixture of the immunoli-

posomes and actinomycin D for 60 min. After a washing, thetumor cells were cultured for 12 hr and then pulsed with [3H]-

thymidine. The chemoimmunoliposomes displayed cytotoxicityagainst MM46 tumor cells which was much stronger than thatdisplayed by free actinomycin D (Chart 2a). Cytotoxicity of acti-

20

15

* 10

5 -

6.3 12.5 25 50

Liposome suspension added (pi)

Chart 1. Binding of liposomes to MM46 and MM48 tumor cells in vitro. Aliquotsof 14C-labeled liposomes (11.2 /Â¿molphospholipid per ml) were added to tumor cellsuspension (5x10* cells in 1 ml) and incubated for 60 min at 37Â°. Unbound

liposomes were removed by centrifugation and washed with PBS, and the radioactivities were determined. Each point represents the average data of triplicatesamples. O, MM46 tumor cells with immunoliposomes; â€¢,MM46 with chemoimmunoliposomes containing 0.3 pg of actinomycin D per 50 Â¡A;A, MM46 withchemoimmunoliposomes containing 1 pg of actinomycin D per 50 pi; A, MM46 withBSA-liposomes; G, MM48 with immunoliposomes.

120

100

80

60

40

20

- b

0.12 0.25 0.5 1.0 0.125 0.25 0.5 1.0

Concentration of actinomycin D Ãpg/ml)

Chart 2. In vitro cytotoxicity of chemoimmunoliposomes. MM46 or MM48 tumorcells were incubated with liposomes or free actinomycin D or the mixture for 60min. After a washing, the tumor cells were further incubated for 12 hr and thenpulsed with [3H]thymidine. â€¢,chemoimmunoliposomes containing 1 pg of actino

mycin D per 0.56 pmol of phospholipid per 50 pi; A, free actinomycin D; â€¢actinomycin D plus immunoliposomes (0.56 pmol of phospholipid per 50 pi), a,MM46 tumor cells; b. MM48 tumor cells.

nomycin D against MM46 tumor cells was abolished by thesimultaneous addition of the immunoliposomes (Chart 2a), probably due to the masking of target cell surfaces with the cell-bound liposomes. The chemoimmunoliposomes were marginallycytotoxic against nonantigenic MM48 tumor cells as were freeactinomycin D and a mixture of the immunoliposomes and actinomycin D alone (Chart 2b).

In Vivo Antitumor Effect of Antibody-coated, Actinomycin D-containing Liposomes

Treatment i.p. against Mice Bearing Peritoneal Tumor.Throughout the following liposome treatment, 50 /Â¿Iof a purifiedliposome preparation (11.2 /Â¿molof phospholipid per ml) weresuspended in 0.2 ml of PBS and injected into a mouse.




C3H/He mice were inoculated i.p. with MM46 tumor cells (5 x104 to 5 x 106 cells/mouse). One day after tumor cell inoculation,the mice were treated by an i.p. injection of chemoimmunolipo-somes or chemo-BSA-liposomes both containing 1 ^g of acti-nomycin D. Tumor-bearing control mice were treated with PBS.Chart 3 illustrates the survival times (days) of the mice. When 5x 10", 5 x 10s, and 5 x 106 tumor cells were inoculated, mean

survival times of the control mice were 22.0 Â±2.3 (S.D.), 20.2 Â±1.3, and 18.8 Â±1.8 days, respectively; eventually, all mice dieddue to tumor growth. Treatment with the chemo-BSA-liposomesprolonged the survival of the tumor-bearing mice for a short time

as compared to survival in control mice; however, all miceeventually died due to tumor in this group also. By contrast,treatment of mice with the chemoimmunoliposomes did confer asignificant therapeutic effect against MM46 tumor-bearing miceand resulted in tumor-free mice, as judged by the absence oftumor cells in the ascites and absence of tumor nodules in theautopsied specimens. Even in the experiment in which the micewere inoculated with as much as 5 x 106 tumor cells, a single

5

4

3

2

l

5

4

I 3

o l

5

4

3

2

P<0.01 (P<0.01)

MM46

5 x 10

P<0.01 (P<0.01)

- 5 x 1(T P<0.01

5 x IO6 L

ii10

20P<0.01

(P<0.01)P<0.05

Â»â€”-ii

1 1 1130

40 50 60

Days after tumor inoculationCharts. Antitumor effect of chemoimmunoliposomes and chemo-BSA-lipo

somes on MM46 tumor cells transplanted i.p. into syngeneic mice. C3H/He micewere inoculated with the indicated numbers of MM46 tumor cell. After 1 day. themice were treated with a single i.p. injection of chemoimmunoliposomes(1 ^9 ofactinomycin D per 50 >il liposome per 0.2 ml of PBS per mouse), chemo-BSA-liposomes (1 ng of actinomycin D per 50 M!of liposome per 0.2 ml of PBS permouse) or 0.2 ml of PBS. Significant difference (Cox-Mantel test) to PBS controland to chemo-BSA-liposome-treatedmice (in parentheses) in the survival days areshown. , PBS; , chemo-BSA-liposome; , chemoimmunoliposome.


injection of the chemoimmunoliposomes led to 3 tumor-free miceof 5 mice tested, and the rest of the mice survived longer thandid both the control mice (p < 0.01 by the Cox-Mantel test) andthe mice treated with the chemo-BSA-liposomes (p < 0.01 ).

The potent and selective antitumor activity of chemoimmunoliposomes was further demonstrated by the following experiments. We examined the antitumor effects of liposome preparations containing different amounts of actinomycin D, 2-11-G IgMantibody, free actinomycin D, and chemo-BSA-liposomes onMM46 and MM48 tumor-bearing mice. Materials were injectedinto mice which had been given an i.p. injection of 5 x 104 tumor

cells, and the survival times of the mice were examined. Theresults are summarized in Table 1. This experiment substantiatedthe previous results that chemoimmunoliposomes displayed astrong therapeutic effect against antigenic MM46 tumor cells.Three, 4, and all mice of 6 mice tested became tumor free by aninjection of chemoimmunoliposomes containing 0.3, 0.5, and 1tig of actinomycin D, respectively. Mean survival time of the deadmice in these groups was prolonged more than 10 days ascompared to that of PBS control mice. In contrast, chemo-BSA-liposomes exerted only a small anti-MM46 tumor effect. The

mean survival time of mice treated with the liposomes containing1 Mg of actinomycin D was prolonged for about 6 days, but allmice eventually died. Neither 2-11-G IgM antibodies (60 to 240

/ng protein) nor free actinomycin D (1.25 to 5.0 nÃ§)were effectivein inducing MM46 tumor-free mice, and all mice died due to

tumor, although the mean survival time of the mice was prolonged for a short duration at high doses (5 /*g of actinomycin Dper mouse corresponding to a half-dose of the 50% lethal dose

in C3H/He mice).The chemoimmunoliposomes conferred no therapeutic effect

upon nonantigenic MM48 tumor-bearing mice.

Treatment i.v. against s.c. Tumor. Mice received a s.c.injection of 106 MM46 tumor cells. After 4 days, when tumors

grew to a palpable size (about 5 mm in diameter measured overthe skin), the mice were treated by an i.v. injection of a liposomepreparation, 2-11-G IgM, or actinomycin D. As has been reported

(12, 13, 18, 26), liposomes injected i.v. can be trapped byphagocytes and accumulated in host tissues. Therefore, in several experimental mice, the phagocytes were blocked by an i.v.injection of MLL (6.7 ^mol of phospholipid per 0.2 ml per mouse).This treatment resulted in a significant inhibition of the phagocytes as indicated from the carbon clearance test (Table 2).

One hr after pretreatment with MLL, the mice were treatedwith an i.v. injection of chemoimmunoliposomes or chemo-BSA-

liposomes, both containing 1 Â¿igof actinomycin D. Tumor sizesin these and other group mice were periodically measured, andthe mice were sacrificed 12 days after drug treatment. Tumornodules were isolated from the mice and weighed. Chart 4 andTable 3 show the growth curves of tumors and tumor weight,respectively.

A single i.v. injection of the chemoimmunoliposomes led to aslight inhibition of the tumor growth as compared to that in PBScontrols, but the difference was statistically insignificant. Whenpretreated with MLL, however, tumor size (diameters) in micetreated with the chemoimmunoliposomes tended to decrease for4 days after the treatment and then started to increase. Tumorweights in these mice, measured at 12 days after the drugtreatment, were significantly lower than those in either PBScontrol (p < 0.05) or mice treated with MLL plus chemo-BSA-

NOVEMBER 1983 5331



y. Hashimoto et al.

Table 1Effect of i.p. injection of 2-11-G IgM, actinomycin D, and liposome preparations on i.p. transplanted MM46

and MM48 tumorsTumor cells (1 x IO4) were injected i.p. into C3H/He mice (22 to 25 g). After 24 hr, the mice were treated

with an i.p. injection of a material (liposome preparations, 50 Â¿il)dissolved or suspended in 0.2 ml of PBS. Theexperiments were terminated 90 days after tumor injection.

MM46tumorMaterial

injectedPBS2-11-GlgM2-11-GlgM2-11-GlgMPBSActinomycin

DActinomycinDActinomycinDPBSActinomycin

DImmunoliposome6ChemoliposomeChemo-BSA-liposomeChemo-BSA-liposomeChemoimmunoliposomeChemoimmunoliposomeChemoimmunoliposomeDose(jjg)601202401.252.55.01.25(1.0)c(0.3)(1.0)(0.3)(0.5)(1.0)No.

of tumor-treemice/no.of

micetested0/30/30/30/30/60/60/60/60/60/60/30/30/30/33/6"4/86/6Extrasurvivaldays

ofdeadmice21.

0Â±2.0a22.3

Â±3.12t.3Â±1.227.0

Â±3.021

.8 Â±1.222.7Â±2.023.3+1.923.3+1.521.7

Â±1.421.9Â±2.121.3Â±0.733.0

Â±3.723.7Â±1.727.3

Â±2.732.7Â±2.733,36MM48

tumorNo.

of tumor-freemice/no.of

micetested0/60/30/30/30/30/30/3Extrasurvivaldays

ofdeadmice15.3

+0.714.7+0.515.7Â±1.715.7

Â±1.715.3

+0.616.7Â±3.116.3

Â±2.5a Mean Â±S.D.0 All liposome preparations contained 0.56 nmol phospholipid per 50 Â«Iof preparation per injection. Chemo-

BSA-liposome and immunochemoliposomes contained 30 and 34 /Â¿gof protein per 50 >Â¿of preparation perinjection, respectively.

c Numbers in parentheses, dose of actinomycin D in the injected liposomes." Biopsy of the ascites at Day 60 and autopsy at Day 90 revealed that all surviving mice shown in this table

were free from tumor.

10

Â£. 8

4 Iâ€”Drug Injection

. I . . I I

4 6 8 10 12 14 16

Days after tumor inoculation

Chart 4. Effect of a single i.v. injection of liposome preparations and othermatenals on the growth of s.c. inoculated MM46 tumors. Values represent thegrowth of tumors in Table 3 (Experiment 2). The mice were treated with a material4 days after tumor cell injection. Tumor size is expressed as a mean of the longestdiameter plus the crossing diameter; each point represents the mathematical meanof tumors from 5 or 6 mice. Standard deviations of tumor sizes are omitted. Detailsof the experiment are given in Table 3. O, PBS control; â€¢.1 ^g of free actinomycinD per mouse; A, chemo-BSA-liposomes containing 1 ^g of actinomycin D; A,chemo-BSA-liposomes containing 1 ^g of actinomycin D after MLL pretreatment;D, chemoimmunoliposomes containing 1 ^g of actinomycin D; â€¢chemoimmuno-liposomes containing 1 (Â¿gof actinomycin D after MLL pretreatment. The growthcurve of tumors treated with 3 <jg of actinomycin D and 100 ^g of 2-11-G IgM

Table 2

Inhibition of carbon clearance by pretreatment with MLL

One hr after the i.v. injection of MLL (6.7 ^mol phospholipid per 0.2 ml PBS permouse), mice were given i.v. injections of colloidal carbon solution.

Pretreatment No. of mice Phagocytic index (K)Â°(x 102)

PBSMLL

4.01 Â±0.40*2.29Â±0.51C

" See "Materials and Methods."" Mean Â±S.D.c p < 0.001 compared to PBS control (by Student's f test).

liposome (p < 0.001). In contrast, the chemo-BSA-liposomes didnot show an antitumor effect; but in the absence of MLL pretreatment, they led to the enhanced growth of tumor. An i.v.injection of either 2-11 -G IgM (100 Â¿Â¿gprotein) or free actinomycin

D (1 or 3 /ug) was ineffective against the s.c. tumors. Injection ofliposome preparation produced no side effects in mice as far asbody weight gain, movement, and appearance of the mice isconcerned.

DISCUSSION

Several antitumor drugs encapsulated in liposomes show anin vivo antitumor effect stronger than that shown by the freedrugs, probably owing to the longer retention of the drugs intumor site (10,11) and to efficient transportation of the drug intotumor cells through a fusion or membrane interaction betweenliposomes and tumor cells (10, 15). The antitumor effect of the

overlapped with that of tumors in PBS control mice and that of tumors in 1actinomycin D-treated mice, respectively.

g of




Table 3Effect of an i.v. injection of chemoimmuno- and chemo-BSA-liposomes on s.c.

MM46 tumorMM46 tumor cells (106 cells/mouse) were injected s.c. into C3H/He mice (20.5

to 23.5 g). Four days after tumor transplantation, the mice were treated by an i.v.injection of a liposome preparation (50 n\ in 0.2 ml PBS) with or without pretreatmentof i.v. injection of MLL (6.7 /imol phospholipid per 0.2 ml PBS per mouse). Tumorswere weighed 12 days after the drug treatment.

MaterialinjectedExperiment

1PBSChemo-BSA-liposomedChemoimmunoliposomeChemo-BSA-liposomeChemoimmunoliposomeExperiment

2PBS2-11

-G IgM (100^g)ActinomycinD (1Â¿ig)ActinomycinD (3^g)Chemo-BSA-liposomeChemoimmunoliposomeChemo-BSA-liposomeChemoimmunoliposomePretreat

ment with No. ofMLLmiceâ€”

555+

4+4555555+

6+6Tumor

wt(mg)159

Â±64"218

Â±41117Â±37183Â±1976

Â±39173

Â±36211 Â±24204

Â±36175Â±4121

1 Â±33162Â±33169Â±51107

Â±43%

of inhibition"Oc-36.7e26.3'-14.6'52.1"tÃ-22.0-17.9-1.1-22.06.42.3'38.2*

Tumor wt in drug-treated mice" 100 x 100Tumor wt in PBS control

Statistical significance (by Student's ( test): f versus e, p < 0.01; h versus g,p <

0.001 ; h versus c, p < 0.05; k versus Â¡,p < 0.05; k versus i, p < 0.02. Other pairswere not significant.

0 Mean Â±S.D." All liposome preparations injected contained 0.56 fimol of phospholipid and 1

tig of actinomycin D.

drugs encapsulated in simple liposomes, however, seems to beunsatisfactory in leading to complete cure of the tumor, probablybecause of a lack of selectivity of the liposomes to tumor cells.In this context, development of drug-containing liposomes that

were effective not only to local tumors but also to tumors locateddistant from the site of liposome application has long beenawaited.

We prepared the liposomes coated with subunits of monoclonal IgM antibody directed to a mouse mammary tumor-asso

ciated antigen and proved their effective and specific binding toantigenic tumor cells in vitro (8). Our method, coupling of SH-bearing subunits of IgM antibody to liposomes containing SH-

reacting maleimide groups, facilitated the preparation of theliposomes to which the antibody coupled at the Fc portion,providing antibody-coated liposomes that can specifically bind toantigen-positive cells but not to Fc receptor-bearing cells present

in host lymphoid cells (8). This is advantageous for the in vivoapplication of the antibody-coated liposomes, since, if the Fc

portion remains intact, the liposomes can be trapped by Fcreceptor-bearing normal cells. Liposomes used for antibody coat

ing were neutrally charged, small sonicated liposomes containingcholesterol, which could be more stable in the animal than werecholesterol-free preparations (11) and could be trapped in the

host tissues to a lesser extent than were negatively chargedliposomes (26).

This work demonstrated the remarkable augmentation of bothin vitro and in vivo antitumor activities of actinomycin D byentrapping the drug in the membrane of antibody-coated lipo

somes. Only one i.p. injection of the chemoimmunoliposomescontaining 0.56 u.mo\ of phospholipid (400 /Â¿gof total lipids), 34vg of antibody protein, and 1 /ug of actinomycin D led to 10


tumor-free mice of 11 mice that had been transplanted with 5 x10" antigenic tumor (MM46) cells a day before the drug injection

(accumulated results from Chart 3 and Table 2). This dose ofactinomycin D in the chemoimmunoliposomes was sufficient toeffect cure of the mice transplanted with as much as 5 x 106

antigenic tumor cells (Chart 3). As indicated by the smear test,the majority of i.p. tumor cells were found to be killed by thechemoimmunoliposomes within a few days and that after 6 daysof treatment the tumor cells completely disappeared from theascites except in the mice dying due to tumor growth (data notshown). The augmented antitumor effect of actinomycin D in theliposomes could be brought about by the specific binding of thechemoimmunoliposomes to the antigenic tumor cells as revealedfrom the result of in vitro targeting (Chart 1), the ineffectivenessof the liposomes against nonantigenic tumor cells, and the failureof an injection of either free actinomycin D (1.25 to 5 Â¿ig),chemoliposomes, or chemo-BSA-liposomes to induce a cure in

mice (Table 2). In these experiments, we did not test the effectof a mixture of free actinomycin D and immunoliposomes, sincewe had demonstrated by an in vitro assay that separate additionof actinomycin D and immunoliposomes decreased the cytotox-

icity of free actinomycin D, probably due to the blocking ofactinomycin D action while immunoliposomes were attached tothe target cells.

Although an i.p.-i.p. system may provide a relevant model for

local tumor therapy, it is desirable to have a liposome preparationthat is effective against tumors remote from the application site.Such an effect of a liposome preparation has been thought tobe difficult to obtain, since liposomes inoculated in vivo are easilytrapped by phagocytes or organs (12,13, 18, 26) and since onlya small percentage of the liposome may migrate into tumor tissuethrough blood vessels. The present work, however, demonstrated that, if the animals had been pretreated with MLL toblock the in vivo trapping for chemoimmunoliposomes, an i.v.injection of the chemoimmunoliposomes containing actinomycinD was effective against s.c. tumors. Since neither free actinomycin D nor chemo-BSA-liposomes were effective against the

s.c. tumors, the therapeutic effect of the chemoimmunoliposomes can be attributable to their specific targeting to theantigenic tumor cells, although the selective localization of theliposomes to tumor tissues must be manifested by use of morefundamental techniques.

In the present tumor therapy model, mice were treated onlyby a single injection of a fixed amount of liposomes. A moretherapeutic effect of the chemoimmunoliposomes may be expected if we perform multiple injections of the liposomes.

Although the present work appears to be the first to show aclear in vivo antitumor effect of monoclonal antibody-bearing

liposomes containing an antitumor drug, the following basicsubjects remain to be studied: (a) fate and mechanism of actionof chemoimmunoliposomes in the tumor cells; (b) effect of non-immune IgMs-coated liposomes containing the drug; (c) dose,

timing of preinjection, and lipid composition of MLL used to blockthe entrapment of chemoimmunoliposomes; (d) toxicity of chemoimmunoliposomes (chemotherapeutic index); (e) organ or tissue (including tumor) distribution and clearance of i.v. injectedliposome preparations.

ACKNOWLEDGMENTS

We thank Dr. T. Takahashi, Aichi Cancer Center Institute, Nagoya, Japan, forthe donation of 2-11-G hybridomma cells and Dr. T. Tachibana, Institute for

NOVEMBER 1983 5333



y. Hashimoto et al.

Tuberculosis, Leprosy and Cancer, Tohoku University, Sendai, Japan, for thedonation of MM tumor cells.

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1983;43:5328-5334. Cancer Res Yoshiyuki Hashimoto, Minoru Sugawara, Takashi Masuko, et al. Immunoglobulin M AntibodyBearing Subunits of Tumor-specific Monoclonal Antitumor Effect of Actinomycin D Entrapped in Liposomes

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