Cancer Research Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 115-121, January 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stan, A. C.
Right arrow Articles by Brumeanu, T.-D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stan, A. C.
Right arrow Articles by Brumeanu, T.-D.
[Cancer Research 59, 115-121, January 1, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Antineoplastic Efficacy of Doxorubicin Enzymatically Assembled on Galactose Residues of a Monoclonal Antibody Specific for the Carcinoembryonic Antigen1

Alexandru C. Stan, Dorel L. Radu, Sofia Casares, Constantin A. Bona and Teodor-D. Brumeanu2

Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have developed a novel procedure to couple enzymatically the antineoplastic agent doxorubicin (Dox) on the galactose residues of a monoclonal antibody specific for the tumor-associated carcinoembryonic antigen. The synthesis of the immunoconjugate consists of covalent attachment of the NH2 terminus of Dox to oxidized galactose residues of desialylated monoclonal antibody, followed by concurrent stabilization of Schiff bases by mild reduction with pyridine borane. The immunoconjugate preserved both antibody specificity and drug cytotoxicity. At equimolar concentrations, the immunoconjugate was 8 times more cytotoxic against two carcinoembryonic antigen-expressing carcinoma cell lines, LoVo and SW-480, than Dox alone. The intracellular drug accumulation was 8–8.5 times higher than that obtained with free Dox, and >50% of the drug delivered by the conjugate was retained for 24 h in the tumor cells. Only 4 days after treatment with a single dose of immunoconjugate carrying 2.5 ng of Dox, LoVo and SW-480 tumor transplants on the chorioallantoic membrane of embryonated hen eggs showed reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues. In contrast, the same amount of free drug induced insignificant changes in tumor progression and tumor-induced angiogenesis. Enzymatically mediated, glycosidic coupling of antineoplastic agents to antibodies specific for tumor-associated antigens may represent a novel platform for the development of more efficient anticancer agents with reduced side effects.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dox,3 an anthracycline derived from Streptomyces peucetius var. coesius, is a powerful chemotherapeutic agent (1) . The cytotoxic effects of Dox rely on its intercalation into double-stranded nucleic acids (2) , with subsequent inhibition of DNA and RNA synthesis (3) . Among other pleiotropic effects that depend upon the cell type, it is now generally accepted that the primum movens of Dox activity is primarily exerted by its effect on stabilization of DNA-topoisomerase II complexes (4 , 5) .

We and others showed that the sine qua non condition for efficient therapy with Dox depends ultimately on the amount of intracellular drug accumulation (6) .4 Several new strategies were aimed at increasing the intratumoral concentration of Dox: (a) encapsulation of Dox in polyisohexylcyanoacrylate nanospheres (7 , 8) ; (b) combined therapy with mannose, leukotriene, and bradykinins or their analogue, RMP-7, to increase selectively the transport of the drug into solid tumors (9, 10, 11, 12, 13, 14, 15) ; and (c) intratumoral or intracavitary administration of Dox, pre- or postoperation (16, 17, 18, 19) .

Immunotargeting tumors with Dox conjugated to antibodies has been considered an attractive anticancer strategy. Chemically engineered immunoconjugates of Dox showed up to 10 times increased toxicity than Dox alone. Tumor progression was inhibited by chemically coupled Dox to mAbs specific for C-erb-2 in breast carcinomas, Thy-1 in neuroblastomas, and CEA in colon carcinomas (20, 21, 22) . To increase the number of Dox molecules per molecule of immunoglobulin, the drug was chemically coupled to aminodextran, i.e., Gentran 40, and the aminodextran-Dox intermediate was chemically attached to various antitumor antibodies (23, 24, 25, 26, 27, 28) . However, despite the large number of Dox molecules carried by such preparations, i.e., 30–40 Dox molecules per molecule of immunoglobulin, the therapeutic efficacy was below expectations. The major drawbacks were considered to be the loss of pharmacological activity of Dox due to drug oxidation during the coupling reaction, as well as a deficient catabolism of aminodextran-Dox intermediates, which, in turn, impairs delivery of active drug to the nucleus (29) .

Herein, we describe a mild method to couple Dox enzymatically to the modified carbohydrate moieties of a mAb specific for CEA. The enzymatically engineered anti-CEA-Gal-Dox conjugate preserved both antibody specificity and drug cytotoxicity, and it was 8 times more cytotoxic against CEA-expressing carcinoma cells than Dox alone. Single application of anti-CEA-Gal-Dox conjugate on LoVo and SW-480 human colon carcinoma transplants on the CAM of embryonated hen eggs reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies.
T84.66A3.1A.1F2 hybridoma cell line secreting a mouse IgG1 antihuman CEA mAb was obtained from American Type Culture Collection (Rockville, MD). The antibody was purified from cell culture supernatants by affinity chromatography using a rat antimouse {kappa} chain mAb-Sepharose column. Anti-Pgp mAb (Pgp, mouse IgG1, clone 4E3; Signet Laboratories, Dedham, MA) was used for FACS analysis. FITC-(Fab')2 goat antimouse IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit anti-Dox serum (Western Chemical Research, Fort Collins, CO) was enriched for the IgG fraction by affinity chromatography on protein A-Sepharose column. Goat antirabbit IgG (Boehringer Mannheim) was radiolabeled with 125I (Amersham) according to a standard chloramine-T method. The 6.5.2 mAb, a rat IgG1 anti-T-cell receptor clonotypic mAb (30) , was purified from the cell culture supernatants by affinity chromatography on protein A-Sepharose column and was enzymatically coupled to Dox. The 6.5.2-Gal-Dox conjugate was used as specificity control for the anti-CEA-Gal-Dox conjugate.

Cell Lines.
LoVo and SW-480 human colon carcinoma cell lines were obtained from American Type Culture Collection. LoVo is an aneuploid cell line with a modal number of 49 (31) , and SW-480 is a hypotriploid cell line with 12% polyploidy, established from a human colorectal adenocarcinoma Duke’s type B (32) . Cells were grown at 37°C and 5% CO2 in DMEM (Life Technologies, Inc., Gaithersburg, MD), supplemented with 10% FCS, 1 mM sodium pyruvate, and 0.05% penicillin-streptomycin.

Synthesis of the Conjugates.
Dox-acceptor sites accessible on anti-CEA mAb and 6.5.2 mAb were determined using GAO and toluidine-horseradish peroxidase coupled assay system, as described previously (33 , 34) . Enzymatic-mediated synthesis of the conjugates was carried out according to a modified protocol for the assembly of immunogenic peptides on modified carbohydrate moieties of immunoglobulins (34) . Briefly, 1 mg of mAb was incubated overnight at 37°C with 50 milliunits each of the neuraminidase from Arthrobacter ureafaciens and that from Clostridium perfringens (Calbiochem-Novobiochem International, Inc, La Jolla, CA) in 1 ml of 0.1 M phosphate buffer (pH 5.5) containing 5 mM CaCl2. Free NANA released by the enzymes was removed by dialysis against PBS (pH 7.4). The reaction mixture was incubated for 48 h at 37°C under sterile conditions and stirred continuously with GAO (20 units; Sigma Chemical Co., St. Louis, MO), pyridine borane (80 mM; Aldrich), and 0.1 mg of Dox (Sigma). Schiff bases formed between the aldehyde groups generated by oxidation with GAO at the sixth carbon of terminal Gal residues, and the NH2 terminus of Dox was stabilized by mild reduction with pyridine borane. The conjugate was extensively dialyzed against PBS in SPECTRA/POR bags of 100,000 molecular weight cutoff (Sigma) and concentrated by speed vacuum centrifugation to 1 mg/ml with respect to immunoglobulin content. The chemical coupling of Dox to anti-CEA mAb was carried out using EDC (Imject Immunogen EDC Conjugation Kit, Pierce, Rockford, IL). For this, 1 mg of anti-CEA mAb was incubated for 4 h at room temperature, with continuous stirring, in 1 ml of conjugation buffer containing 0.1 mg of Dox and 0.1 mg of EDC. The conjugate was extensively dialyzed against PBS in SPECTRA/POR bags of 100,000 molecular weight cutoff, and the degree of coupling was calculated as follows:

where A indicates the spectrophotometric absorbance; 0.73 and (8 x 103) are the correction factors for absorbance of immunoglobulin-Dox conjugate and Dox, respectively; and 1.4 represents the extinction coefficient of mouse or rat IgG1 at {lambda}280 nm. The absorption of Dox was measured at {lambda}495 nm.

Electrophoretic Analyses.
Isoelectric focusing of the unconjugated and desialylated anti-CEA mAb and anti-CEA-Gal-Dox conjugates was carried out on precast IEF 3-10 PhastGels using the PhastSystem apparatus (Pharmacia LKB). Gels were stained with Coomassie Blue R-250 according to the manufacturer’s instructions. The anti-CEA-Gal-Dox conjugate was also analyzed by SDS-PAGE using PhastGels 4–20% gradient polyacrylamide (Pharmacia LKB) under reducing conditions. Samples were left untreated or were treated with 0.01 units/µg PGN-ase F, and then 10 µg of the conjugate were electrophoresed for 1 h at 150 V. Gels were either stained with Coomassie Blue R-250 or electrotransferred under semidry conditions for 45 min at 450 mA onto 0.45-µm Immobilon polyvinylidene fluoride membranes (Sigma) using a Multiphor II apparatus (Pharmacia LKB). Membranes were blocked overnight at 4°C with 5% fat-free milk (Carnation, Nestlé Food Company, Glendale, CA) in PBS, washed with PBS, and incubated overnight at 4°C with 10 µg/ml affinity-purified IgG fraction from rabbit anti-Dox serum in PBS, 1% BSA, and 0.05% Tween 20. Membranes were washed with PBS-0.05% Tween 20 and bound rabbit anti-Dox IgG was detected after incubation for 2 h at room temperature with 125I-goat antirabbit IgG (2 x 105 cpm/10 x 10-cm membrane) in PBS, 1% BSA, and 0.05% Tween 20, using Kodak X-OMAT films (Sigma).

Thymidine Incorporation Assay.
LoVo or SW-480 cells (5 x 104) in 200 µl of complete medium and containing 1 µCi/25 µl tritiated thymidine were incubated for 24 h with various doses of enzymatically or chemically engineered conjugates, Dox alone, or medium alone. In a parallel set of assays, graded amounts of unconjugated anti-CEA mAb (1–100 µg/ml) were added to the tumor cell cultures, 30 min before incubation with a constant amount of immunoconjugates and tritiated thymidine. After 24 h of culture, cells were harvested on filter paper, and the radioactivity was measured in a ß-scintillation chamber (Pharmacia LKB).

Confocal Laser Scanning Microscopy.
Tumor cells (1 x 103 cells in 2 ml per well) were inoculated in the 12-well plates containing coverslips and cultured for 3–4 days with daily changes of medium until the cells were tightly adherent to the glass. Coverslips were rinsed with cold PBS-1% BSA, fixed for 10 min in methanol:acetone (1:1) at -20°C, and air-dried overnight. After rehydration in PBS-1% BSA, coverslips were incubated for 1 h at room temperature with 5 µg/ml anti-CEA or anti-Pgp mAbs, rinsed with cold PBS-1% BSA, and then incubated for 1 h at room temperature with 5 µg/ml FITC-(Fab')2 goat antimouse IgG. Coverslips were rinsed with cold PBS-1% BSA and incubated for 30 min at 37°C with 3 µg/600 µl/well RNase A in PBS-1% BSA to remove RNA. To visualize nuclei, coverslips were overstained for 30 min at room temperature with 2 µg/600 µl PI in PBS-1% BSA per well; rinsed with cold PBS-1% BSA, followed by distilled water; and then mounted with Vectashield medium (Vector Laboratories, Burlingame, CA), sealed with Permount (Fisher Scientific), and analyzed in an inverted Leica confocal laser scanning microscope equipped with a fluorescence filter set for double excitation at 488/568 nm (Leica Lasertechnik, Heidelberg, Germany). Control staining was performed using an isotype control IgG1 (Sigma) as primary antibody, and FITC-(Fab')2 goat antimouse IgG as secondary antibody.

FACS Analyses.
To analyze the expression of CEA and Pgp, LoVo and SW-480 cells (2 x 105) were incubated on ice for 30 min with anti-CEA mAb (10 µg), anti-Pgp mAb (2 µg), or the same amounts of mouse IgG1 isotype control. Cells were washed with cold PBS, incubated for 30 min on ice with FITC-(Fab')2 goat antimouse IgG, washed, and fixed with 1% paraformaldehyde in PBS, and the fluorescence intensity was measured by FACS, as described previously (35) .

To determine the extent of intracellular drug accumulation, 1 x 105 tumor cells in DMEM complete medium were exposed for 24 h to 0.2 µg of Dox or to a molar equivalent of Dox carried by the anti-CEA-Gal-Dox conjugate, washed, and fixed with 1% paraformaldehyde; and the fluorescence intensity was measured by FACS. In a parallel set of assays, 1 x 105 cells were cultured for 24 h with 0.2 µg of Dox or with a molar equivalent of Dox carried by the anti-CEA-Gal-Dox conjugate, washed, and then recultured for another 24 h in DMEM alone. Cells were washed and fixed with 1% paraformaldehyde, and the fluorescence intensity was measured. The intrinsic fluorescence of Dox accumulated or retained intracellularly was acquired on an EPICS Profile II Analyzer (Coulter Corporation, Hialeah, FL) equipped with air-cooled argon ion laser emitting {lambda}488 nm at 15 mV in standard optical configuration. The mean of fluorescence intensity for the entire population of cells was measured among 5000 cells at {lambda}488 nm excitation versus {lambda}580 nm absorbance.

Tumor Growth in CAM System and Determination of Tumor Progression.
LoVo and SW-480 cells were grown in DMEM complete medium and then detached with trypsin-EDTA. Tumor cells (2 x 106 cells/2 ml) were inoculated for 12 h onto sterile blank concentration discs (1/4 inch = 6.4 mm; Difco Laboratories, Detroit, MI) in 12-well plates (Becton Dickinson), and discs were transferred to new 12-well plates containing fresh medium and cultured for another 7 days until cells formed a confluent layer. Fertilized 10-day-old white Leghorn hen eggs (Spafas Inc., Norwich, CT) were opened at the air sac, CAMs were transplanted aseptically with discs containing tumor cell inoculum, and the shells were sealed. Thirty eggs per cell line were prepared in this way. Eggs were incubated at 37°C and 98% relative humidity, and after 3 days, the 13-day-old eggs received a single-dose treatment of free Dox or conjugates applied in 25 µl of DMEM in the center of the disc. After 4 days, eggs were fixed in 10% buffered formalin solution and embedded in paraffin according to a standard procedure. Tumor progression was determined by morphometric measurements of tumor-CAM transplants using 7-µm cross-sections that were cut along the diameter of the supporting disc, then stained with H&E and scanned at x125 magnification using a three-chip charged-coupled device color camera (DXC-960 MD; Sony Electronics, Inc.) adapted to a stereomicroscope (Carl Zeiss, Jena-G{delta}ttingen, Germany). Digitized images were measured planimetrically after previous calibration of the system with a standard µ-slide (Carl Zeiss) using the software ImagePro Plus (Media Cybernetics, Silver Spring, MD). The cross-sectional areas were traced in mm2. Statistical analysis of the measurements was carried out using the SPSS/PC+ software, version 7.0 (SPSS International BV, Gorinchem, the Netherlands). The defined variable was the cross-sectional area of CAM at the site of tumor transplantation. ANOVA (one-way ANOVA) was used to determine the difference in tumor progression between the groups. Measurements of tumor-CAM transplants were averaged for each group (5 CAMs/group) and calculated according to the t test for P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Specificity of the Enzymatically Mediated Conjugation.
Neuraminidases from A. ureafaciens and C. perfringens being able to cleave, respectively, Gal-{alpha}—(2-6)-NANA and Gal-{alpha}—(2-3)-NANA bonds (36) , yielded fully desialylated mAb preparations, making accessible the Gal residues for oxidation by GAO. Mouse IgG1 anti-CEA mAb and rat IgG1 6.5.2 mAb showed four potential Gal acceptors for Dox. The coupling ratios for anti-CEA-Gal-Dox, 6.5.2-Gal-Dox, and anti-CEA-Dox conjugates were 3.7, 3.2, and 7.8 Dox molecules per molecule of immunoglobulin, respectively.

Isoelectric focusing analysis showed a slightly higher pI for the anti-CEA-Gal-Dox conjugate (6.2 and 6.5 pI), as compared to the desialylated-unconjugated mAb (6.1 and 6.3 pI), indicating a change in the net electrical charge of immunoglobulin after coupling the drug (Fig. 1aCitation , Lanes 2 and 3). Also, the molecular mass of the heavy but not light chains of anti-CEA mAb was slightly increased (Fig. 1bCitation , Lanes 2 and 3). Western blot analysis developed with rabbit anti-Dox IgG revealed the presence of Dox on the carbohydrate moieties of anti-CEA mAb but not on the conjugate treated with PGN-ase F, indicating that Dox was specifically coupled to the N-glycan moieties of mAbs (Fig. 1cCitation , Lanes 1 and 2).



View larger version (45K):
[in this window]
[in a new window]
  		Fig. 1. Electrophoretic analyses of anti-CEA-Gal-Dox conjugate. a, the isoelectric focusing of desialylated anti-CEA mAb before enzymatically mediated, glycosidic conjugation (Lane 2) and after conjugation with Dox (Lane 3). The pI markers (broad pI kit; Pharmacia Biotech) are indicated in Lane 1. b, SDS-PAGE under reducing condition of desialylated anti-CEA mAb before enzymatically mediated, glycosidic conjugation (Lane 2) and after conjugation with Dox (Lane 3). Lane 1, molecular weight markers (low molecular weight SDS-PAGE standards; Bio-Rad). The positions of heavy and light chains of anti-CEA mAb are indicated (arrows). c, Western blot analysis of anti-CEA-Gal-Dox conjugate developed with rabbit IgG anti-Dox primary antibody and 125I-radiolabeled goat antirabbit IgG secondary antibody. Lane 1, anti-CEA-Gal-Dox conjugate reduced and treated with PGN-ase F; Lane 2, anti-CEA-Gal-Dox conjugate reduced and not treated with PGN-ase F.

 
Cytotoxicity of anti-CEA-Gal-Dox Conjugates.
Inhibition of DNA synthesis in LoVo and SW-480 cells, as determined by thymidine incorporation assay, was dose dependent for both anti-CEA-Gal-Dox and anti-CEA-Dox conjugates, as well as for free Dox. On a molar basis, at half-maximal inhibition of cell growth (IC50), Dox delivered by the enzymatically engineered conjugate was 8.3 times more cytotoxic than Dox alone (Fig. 2)Citation . Both cell lines showed 15–20% growth inhibition upon incubation with 6.5.2-Gal-Dox conjugate (specificity control). Notable, although the coupling ratio for anti-CEA-Gal-Dox conjugate was 3.7, as compared to 7.8 for anti-CEA-Dox conjugate, the cytotoxicity of the enzymatically engineered conjugate was almost 4 times higher. Up to 80% inhibition of the cytotoxic effect exerted by anti-CEA-Gal-Dox conjugate was obtained on both tumor cell lines preincubated with 100 µg/ml of unconjugated anti-CEA mAb (data not shown), indicating that antibody specificity was preserved after enzymatic coupling of Dox.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. Thymidine incorporation by colon carcinoma cells upon incubation with Dox immunoconjugates. The cytotoxic effect of the enzymatically and chemically engineered conjugates was compared to Dox alone (a, LoVo; b, SW-480). Cytotoxicity of Dox delivered by the conjugates was compared on a molar basis at half-maximal cell growth (horizontal line). {rectangle}, Nil; {blacksquare}{blacksquare} 6.5.2 mAb (10 µg/ml); , anti-CEA mAb (10 µg/ml); {blacksquare}, 6.5.2 Gal-Dox conjugate; •, Dox; {diamondsuit}, anti-CEA-Gal-Dox conjugate; {diamond}, anti-CEA-Dox conjugate. Columns and data points, means of quadruplicate wells; bars, SD (ordinate).

 
Expression of CEA and Pgp on Colon Carcinoma Cell Lines.
Using confocal laser scanning microscopy, we found that both LoVo and SW-480 cells express CEA and Pgp on the membrane (Fig. 3)Citation . The multidrug resistance associated Pgp is an ATP-driven transmembrane pump that facilitates active cellular efflux of toxic chemicals (37) and, thereby, lowers cytotoxicity of the drugs. FACS analysis showed that CEA was 3 times higher expressed on LoVo than on SW-480 cells (Fig. 4Citation , top), whereas Pgp was 3 times higher expressed on SW-480 than on LoVo cells (Fig. 4Citation , bottom).



View larger version (116K):
[in this window]
[in a new window]
  		Fig. 3. Identification of CEA and Pgp on colon carcinoma cells. The expression of CEA and Pgp on the surface of LoVo and SW-480 colon carcinoma cells was analyzed by confocal laser scanning microscopy. Top, expression of CEA; bottom, expression of Pgp. Digitized microscopic images represent 1 of 8 horizontal cell sections of 0.5-µm thickness. The CEA and Pgp expressed by the tumor cells are indicated (green), as revealed by secondary antibody coupled to FITC. The nuclei are visualized (red), as stained with PI.

 


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 4. Expression of CEA and Pgp on colon carcinoma cells. The level of expression of CEA and Pgp by LoVo (top left and bottom left) and SW-480 (top right and bottom right) colon carcinoma cells was determined by FACS analysis. CEA and Pgp expression were measured as the average of fluorescence intensity (abscissa) among 5000 cells (ordinate).

 
Intracellular Retention of Dox Accumulation.
Data illustrated in Fig. 6Citation indicate that both carcinoma cell lines accumulated 8–8.5 times more drug during 24-h exposure to equimolar concentrations of Dox delivered by the conjugate than by Dox alone (Fig. 5Citation , top). After another 24 h of resting in medium alone, cells exposed to the enzymatically engineered conjugate retained 54–58.8% from the initial amount of drug accumulated (Fig. 5Citation , bottom).



View larger version (83K):
[in this window]
[in a new window]
  		Fig. 6. Inhibition of tumor progression upon treatment with various prodrugs. The macroscopic aspect, at x125 magnification, of the tumor progression on CAMs after 4 days of culturing with or without treatment with prodrugs is illustrated. a, treatment of CAM with DMEM alone; c, free Dox (250 ng/25 µl); e, free Dox (2.5 ng/25 µl); g, anti-CEA mAb (250 ng/25 µl); i, anti-CEA-Gal-Dox conjugate (250 ng/25 µl) carrying 2.5 ng of Dox; and k, 6.5.2-Gal-Dox conjugate carrying 2.5 ng of Dox. The corresponding H&E stainings of CAM cross-sectional areas are illustrated in b, d, f, h, j, and l, respectively.

 


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 5. Analysis of intracellular drug accumulation and retention by colon carcinoma cells. Colon carcinoma cells were treated with molar equivalents of Dox as carried by the anti-CEA-Gal-Dox conjugate or by free Dox, and the average of intrinsic fluorescence on 5000 cells was determined by FACS, as described previously (35) . Top, percentage of drug accumulation upon 24 h of continuous incubation of LoVo and SW-480 cells with prodrugs; bottom, percentage of drug retention after resting the cells in medium alone for another 24 h.

 
Inhibition of Tumor Progression.
Untreated or low-dose Dox (2.5 ng)-, anti-CEA mAb (250 ng)-, or 6.5.2-Gal-Dox (250 ng)-treated LoVo-CAM transplants showed massive tumor growth with strong angiogenesis and profound invasion of CAMs (Fig. 6, a and b, e and f, g and h, and k and lCitation , respectively). After 4 days from a single-dose application of 250 ng of Dox strongly inhibited both tumor progression and tumor-induced angiogenesis, leaving only small cell islets with pyknotic nuclei (Fig. 6, c and d)Citation , whereas 2.5 ng of Dox showed no detectable effect (Fig. 6, e and fCitation , and Fig. 7Citation ). In contrast, 2.5 ng of Dox delivered by anti-CEA-Gal-Dox conjugate reduced tumor progression and tumor-induced angiogenesis of LoVo-CAM transplants (Fig. 6, i and j)Citation , as well as SW-480-CAM transplants (data not shown) by 42 and 50%, respectively (Fig. 7)Citation . At 250 ng of free Dox, tumor progression was completely inhibited, but severe fibrosis and sclerosis of the CAM parenchyma and stroma were observed. In contrast, although a slightly reduction in thickness of the CAM parenchyma was detected upon treatment with anti-CEA-Gal-Dox conjugate (Fig. 7, j versus d)Citation , inhibition of tumor progression and tumor-induced angiogenesis by the enzymatically engineered conjugate was not accompanied by visible alterations of media and intima of blood vessels or by damage of the CAM parenchyma and stroma, as analyzed microscopically.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 7. Determination of tumor progression on CAM transplants treated with various prodrugs. The cross-sectional areas (ordinate, mm2) of LoVo-CAM (top) and SW-480-CAM (bottom) transplants after treatment with various prodrugs (abscissa) were determined as described. Columns, means per group; bars, SD. The percentage of tumor progression was calculated in relation to the nontransplanted CAM treated with DMEM alone. Lower values for tumor-CAM transplants treated with free Dox as compared to nontransplanted CAM indicate sclerosis of surrounding tissue below the physiological limit. The amounts of anti-CEA-Gal-Dox and 6.5.2-Gal-Dox conjugates are indicated with respect to the protein content, i.e., 250 ng of conjugate for which coupled Dox represents 2.5 ng.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study demonstrates that enzymatically mediated, glycosidic conjugation of Dox to a mAb specific for CEA tumor-associated antigen exerts efficient and specific cytotoxicity on two human colon carcinoma cell lines expressing CEA. Both enzymatically and chemically engineered immunoconjugates of Dox showed dose-dependent cytotoxicity on CEA-expressing cells. Although the chemical conjugation using EDC introduced twice as many Dox molecules per molecule of immunoglobulin than did the enzymatically mediated conjugation, the enzymatically engineered conjugate was 4 times more cytotoxic. This suggests that the enzymatically mediated coupling of Dox to the Gal residues of immunoglobulins provides a more efficient delivery of Dox to tumor cells than the chemical coupling does. Dox enzymatically assembled on the Gal residues of anti-CEA mAb exerted 8 times higher cytotoxicity against CEA-expressing carcinoma cells than the drug administered per se. The antiproliferative capacity of the enzymatic conjugate persisted for >72 h after a single exposure of 2 x 105 LoVo cells to 1 µg/ml conjugate (data not shown). A 15–20% nonspecific cytotoxicity was observed for both enzymatically and chemically engineered conjugates. This was presumably the result of endocytosis of the conjugates by fluid-phase pinocytosis.

At equimolar concentrations, Dox delivered by the enzymatically engine ered conjugate showed a remarkable 8–8.5 times higher intracellular drug accumulation than the drug administered per se. This was presumably the result of targeting the drug to CEA-expressing tumor cells. It is noteworthy that higher level of CEA expression on LoVo than on SW-480 cells did not significantly influence the extent of intracellular accumulation of Dox. For as much as 0.2 µg/ml Dox per 2 x 105 cells, the 24-h intracellular retention upon delivery by the enzymatically engineered conjugate was >50%, whereas cells exposed to the same amount of free drug retained the entire amount of drug. However, cells exposed 24 h to >1 µg/ml free Dox per 2 x 105 cells retained only 20–25% of the drug (data not shown). This implies that at 0.2 µg/ml Dox per 2 x 105 cells, the entire amount of drug may have been tightly bound to DNA. It is also likely that cell saturation with the drug may correlate to maximal amount of drug bound to nucleus, and the unbound drug is exocytosed, especially when the Pgp multidrug resistance pump is present. Interestingly, Pgp expression on LoVo (4.8%) and SW-480 (15.8%) carcinoma cells did not make a significant difference between the two cell lines in terms of the extent to which Dox delivered by the immunoconjugate was retained intracellularly. One could consider that the pathway of cellular degradation and trafficking for Dox enzymatically assembled on the Gal residues of immunoglobulins may differ from that of Dox administered per se. Dox coupled to the sugar moieties of immunoglobulin may be incrementally released by endoglycosidases, thereby allowing a more efficient targeting to the nucleus, whereas the excess of free Dox entering the cell can be easier exocytosed by Pgp pump. This may explain why expression of Pgp on LoVo and SW-480 carcinoma cells significantly lowered the amount of drug when administered per se but did not lower the intracellular retention of Dox delivered by the immunoconjugate.

CAM experiments indicated that single-dose treatment of carcinoma transplants with anti-CEA-Gal-Dox conjugate carrying 2.5 ng of Dox reduced tumor progression and tumor-induced angiogenesis by half, whereas the same dose of free drug did not. Although the inhibition of tumor progression and tumor-induced angiogenesis was not complete at this dose, the drug delivered by the enzymatically engineered conjugate induced no detectable damage to surrounding CAM tissues. In contrast, severe sclerosis and fibrosis of media and intima of blood vessels occurred rapidly in the case of high dose of free Dox (250 ng).

Our results suggest that enzymatically mediated, glycosidic coupling of Dox to the Gal residues of antibodies specific for tumor-associated antigens may offer a more efficient anticancer therapy than chemically engineered conjugates and Dox administered per se. This is mainly because: (a) specific targeting of the drug to tumor cells that minimizes the side effects rather than being distributed randomly to surrounding tissues; (b) long-term cytotoxicity due to efficient intracellular drug accumulation and retention; and (c) bypass of Pgp multidrug resistance pump by means of different pathways of intracellular trafficking.


   ACKNOWLEDGMENTS
 
We thank Veronica Gulle and Ameera Ali from the Department of Pathology, Mount Sinai School of Medicine (New York, NY) for the excellent histologies of tumor-CAM transplants and Dr. Alexander Miller for critical review of the manuscript.


   FOOTNOTES
 
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.

1 Supported by a grant (to T-D. B.) from Alliance Pharmaceutical Corporation (San Diego, CA). A. C. S. is an Assistant Professor at the Institute of Neuropathology, Hannover Medical School (Hannover, Germany) and is the recipient of a fellowship (Grant Sta 429/2-1) from the Deutsche Forschungsgemeinschaft. Confocal laser scanning microscopy was performed at the Mount Sinai School of Medicine—Confocal Laser Scan Microscopy core facility and was supported by NIH Shared Instrumentation Grant 1 S10 RR0 9145-01 and National Science Foundation Major Research Instrumentation Grant DBI-9724504. Back

2 To whom requests for reprints should be addressed, at the Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, NY 10029-6574. Phone: (212) 241-7551; Fax: (212) 828-4151. Back

3 The abbreviations used are: Dox, doxorubicin; mAb, monoclonal antibody; CEA, carcinoembryonic antigen; Gal, galactose; CAM, chorioallantoic membrane; Pgp, P-glycoprotein; FACS, fluorescence-activated cell sorting; GAO, Gal oxidase; NANA, N-acetyl neuraminic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; PI, propidium iodide. Back

4 A. C. Stan, D. L. Radu, S. Casares, and T-D. Brumeanu, unpublished results. Back

Received 8/ 7/98. Accepted 10/30/98.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Blum R. H., Carter S. K. Adriamycin. A new anticancer drug with significant clinical activity. Ann. Intern. Med., 80: 249-259, 1974.
  2. Goldman R., Facchinetti T., Bach D., Raz A., Shinitzky M. A differential interaction of daunomycin, Adriamycin and their derivatives with human erythrocytes and phospholipid bilayers. Biochim. Biophys. Acta, 512: 254-269, 1978.[Medline]
  3. Meriwether W. D., Bachur N. R. Inhibition of DNA and RNA metabolism by daunorubicin and Adriamycin in L1210 mouse leukemia. Cancer Res., 32: 1137-1142, 1972.[Abstract/Free Full Text]
  4. Wang H., Jiang Z. G., Wong Y. W., Dalton W. S., Futscher B. W., Chan V. T. W. Decreased CP-1 (NF-Y) activity results in transcriptional down-regulation of topoisomerase II{alpha} in a doxorubicin-resistant variant of human multiple myeloma RPMI 8226. Biochem. Biophys. Res. Commun., 237: 217-224, 1997.[Medline]
  5. Tewey K. M., Rowe T. C., Yang L., Halligan B. D., Liu L. F. Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science (Washington DC), 226: 466-468, 1984.[Abstract/Free Full Text]
  6. Rahman Z. U., Frye D. K., Buzdar A. U., Smith T. L., Asmar L., Champlin R. E., Hortobagy G. N. Impact of selection process on response rate and long-term survival of potential high-dose chemotherapy candidates treated with standard-dose doxorubicin-containing chemotherapy in patients with metastatic breast cancer. J. Clin. Oncol., 15: 3171-3177, 1997.[Abstract]
  7. Hu Y. P., Jarillon S., Dubernet C., Couvreur P., Robert J. On the mechanism of action of doxorubicin encapsulation in nanospheres for the reversal of multidrug resistance. Cancer Chemother. Pharmacol., 37: 556-560, 1996.[Medline]
  8. Bennis S., Chapey C., Couvreur P., Robert J. Enhanced cytotoxicity of doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres against multidrug-resistant tumour cells in culture. Eur. J. Cancer, 30A: 89-93, 1994.
  9. Sakai N., Kondo H., Shikinami A., Hirata T., Funakoshi T., Tanabe Y., Yamada H. Postoperative treatment for malignant intracranial tumors—especially concerning intermittent intra-carotid administration of Adriamycin. No. Shinkei. Geka., 12: 237-243, 1984.[Medline]
  10. Bonstelle C. T., Kori S. H., Rekate H. Intracarotid chemotherapy of glioblastoma after induced blood-brain barrier disruption. Am. J. Neuroradiol., 4: 810-812, 1983.[Abstract]
  11. Black K. L., King W. A., Ikezaki K. Selective opening of the blood-tumor barrier by intracarotid infusion of leukotriene C4. J. Neurosurg., 72: 912-916, 1990.[Medline]
  12. Nomura T., Inamura T., Black K. L. Intracarotid infusion of bradykinin selectively increases blood-tumor permeability in 9L and C6 brain tumors. Brain Res., 659: 62-66, 1994.[Medline]
  13. Inamura T., Nomura T., Bartus R. T., Black K. L. Intracarotid infusion of RMP-7, a bradykinin analog: a method for selective drug delivery to brain tumors. J. Neurosurg., 81: 752-758, 1994.[Medline]
  14. Inamura T., Black K. L. Bradykinin selectively opens blood-tumor barrier in experimental brain tumors. J. Cereb. Blood Flow Metab., 14: 862-870, 1994.[Medline]
  15. Matsukado K., Inamura T., Nakano S., Fukui M., Bartus R. T., Black K. L. Enhanced tumor uptake of carboplatin and survival in glioma-bearing rats by intracarotid infusion of bradykinin analog, RMP-7. Neurosurgery, 39: 125-133, 1996.[Medline]
  16. Shimura T., Teramoto A., Nakazawa S., Aihara K. A clinicopathological study of malignant glioma done after local administration of chemotherapeutic agents. Clin. Neuropathol., 15: 119-124, 1996.[Medline]
  17. Nakazawa S., Itoh Y., Shimura T., Matsumoto M., Yajima K. New management of brain neoplasms. 2. Local injection of Adriamycin. No. Shinkei. Geka., 11: 821-827, 1983.
  18. Shimura T., Nakazawa S., Itoh Y., Matsumoto M. Effect of local injections of Adriamycin on survival in malignant brain tumor: histopathological evaluation. Gan. To. Kagaku. Ryoho., 10: 1179-1187, 1983.[Medline]
  19. Shimura T., Nakazawa S. Intraneoplastic local injection of Adriamycin for malignant brain tumor chemotherapy: a clinicopathological study. No. Shinkei. Geka., 8: 35-42, 1980.[Medline]
  20. Jinno H., Ueda M., Enomoto K., Ikeda T., Kyriakos P., Kitajima M. Effectiveness of an Adriamycin immunoconjugate that recognizes the C-erbB-2 product on breast cancer cell lines. Surg. Today, 26: 501-507, 1996.[Medline]
  21. Zhu J., Takahashi H., Nakazawa S. Human monoclonal antibody-drug conjugates in the experimental treatment of malignant gliomas: studies in vitro and in vivo. Neurol. Med.-Chir., 34: 279-285, 1994.
  22. Hurwitz E., Arnon R., Sahar E., Danon Y. A conjugate of Adriamycin and monoclonal antibodies to Thy-1 antigen inhibits human neuroblastoma cells in vitro. Ann. N. Y. Acad. Sci., 417: 125-136, 1983.[Abstract]
  23. Shih L. B., Goldenberg D. M., Xuan H., Lu H., Sharkey R. M., Hall T. C. Anthracycline immunoconjugates prepared by a site-specific linkage via an amino-dextran intermediate carrier. Cancer Res., 51: 4192-4198, 1991.[Abstract/Free Full Text]
  24. Ohyanagi H., Ishida H., Ishida T., Soyama N., Yamamoto M., Okumura S., Kano Y., Ueda Y., Saitoh Y. A monoclonal antibody, KM10 reactive with human gastrointestinal cancer and its application for immunotherapy. Jpn. J Cancer Res., 79: 1349-1358, 1988.[Medline]
  25. Shouval D., Adler R., Wands J. R., Hurwitz E., Isselbacher K. J., Sela M. Doxorubicin conjugates of monoclonal antibodies to hepatoma-associated antigens. Proc. Natl. Acad. Sci. USA, 85: 8276-8280, 1988.[Abstract/Free Full Text]
  26. Takahashi Y., Ueno M., Mai M., Tsukada Y. Effect of a conjugate of Adriamycin and an antibody to human {alpha}-fetoprotein (AFP) on AFP-producing hepatic tumors. Gan. No. Rinsho., 34: 847-850, 1988.[Medline]
  27. Yu D. S., Chu T. M., Yeh M. Y., Chang S. Y., Ma C. P., Han S. H. Antitumor activity of doxorubicin-monoclonal antibody conjugate on human bladder cancer. J. Urol., 140: 415-421, 1988.[Medline]
  28. Deguchi T., Chu T. M., Leong S. S., Horozsewicz J. S., Lee C. L. Potential therapeutic effect of Adriamycin-monoclonal anti-prostatic acid phosphatase antibody conjugate on human prostate tumor. J Urol., 137: 353-358, 1987.[Medline]
  29. Shih L. B., Goldenberg D. M., Xuan H., Lu H. W., Mattes M. J., Hall T. C. Internalization of an intact doxorubicin immunoconjugate. Cancer Immunol. Immunother., 38: 92-98, 1994.[Medline]
  30. Weber S., Traunecker A., Oliveri F., Gerhard W., Karjalainen K. Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor. Nature (Lond.), 356: 793-796, 1992.[Medline]
  31. Drewinko B., Romsdahl M. M., Yang L. Y., Ahearn M. J., Trujillo J. M. Establishment of a human carcinoembryonic antigen-producing colon adenocarcinoma cell line. Cancer Res., 36: 467-475, 1976.[Medline]
  32. Leibovitz A., Stinson J. C., McCombs W. B., McCoy C. E., Mazur K. C., Mabry N. D. Classification of human colorectal adenocarcinoma cell lines. Cancer Res., 36: 4562-4569, 1976.[Abstract/Free Full Text]
  33. Avigad G. Oxidation rates of some desialylated glycoproteins by galactose oxidase. Arch. Biochem. Biophys., 239: 531-537, 1985.[Medline]
  34. Brumeanu T. D., Dehazya P., Wolf I., Bona C. A. Enzymatically mediated, glycosidic conjugation of immunoglobulins with viral epitopes. J. Immunol. Methods, 183: 185-197, 1995.[Medline]
  35. Luk C. K., Tannock I. F. Flow cytometric analysis of doxorubicin accumulation in cells from human and rodent cell lines. J. Natl. Cancer Inst. (Bethesda), 81: 55-59, 1989.[Abstract/Free Full Text]
  36. Corfield A. P., Higa H., Paulson J. C., Schauer R. The specificity of viral and bacterial sialidases for {alpha}(2-3)- and {alpha}(2-6)-linked sialic acids in glycoproteins. Biochim. Biophys. Acta, 744: 121-126, 1983.[Medline]
  37. Becker K. F., Allmeier H., Hollt V. New mechanisms of hormone secretion: MDR-like gene products as extrusion pumps for hormones?. Horm. Metab. Res., 24: 210-213, 1992.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stan, A. C.
Right arrow Articles by Brumeanu, T.-D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stan, A. C.
Right arrow Articles by Brumeanu, T.-D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online