Inhibition of multidrug-resistant human tumor growth in athymic mice by anti-P-glycoprotein monoclonal antibodies

In an effort to devise an effective treatment for human drug-resistant cancers, we have developed monoclonal antibodies, MRK16 and 17, reactive to the multidrug transporter protein, P-glycoprotein. The monoclonal antibodies given intravenously effectively prevented tumor development in athymic mice inoculated subcutaneously with drug-resistant human ovarian cancer cells 2780AD. Treatment with MRK16 induced rapid regression of established subcutaneous tumors and apparent cures of some animals. Complement-dependent cytotoxicity (MRK16) and antibody-dependent cell-mediated cytolysis (MRK16 and 17) were observed with these antibodies. These monoclonal antibodies may have potential as treatment tools against multidrug resistant human tumors possessing the P-glycoprotein.     

Mouse-human chimeric antibody against the multidrug transporter P-glycoprotein

In an effort to devise an effective treatment for human drug-resistant cancers, we have generated a monoclonal antibody, MRK16, reactive to the multidrug transporter P-glycoprotein. The monoclonal antibody inhibited the growth of human drug-resistant tumor cells in a xenograft model, suggesting its potential usefulness in the immunotherapy of drug-resistant cancers. In this study, we have developed a recombinant chimeric antibody in which the antigen-recognizing variable regions of MRK16 are joined with the constant regions of human antibodies. When human effector cells were used, the chimeric antibody, MH162, was more effective in killing drug-resistant tumor cells than the all-mouse monoclonal MRK16. The chimeric antibody against the multidrug transporter P-glycoprotein will be a useful agent in immunotherapy of human drug-resistant cancers.     

Mouse-Human Chimeric Antibody MH171 against the Multidrug Transporter P-Glycoprotein

We have developed a mouse-human chimeric antibody MH171, in which the antigen-recognizing variable regions of the mouse monoclonal antibody MRK17 are joined with the constant regions of human IgG1 antibodies. The MRK17 recognizes specifically the multidrug transporter P-glycoprotein and inhibits the growth of human multidrug resistant (MDR) tumor cells in vitro and in the xenograft nude mouse model system. The established chimeric MH171 antibody forms an apparently intact IgG composed of heavy and light chains covalently assembled via disulfide bonds in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and is specific to MDR cell lines with a similar affinity to the original mouse MRK17. MH171 also displays strong antibody-dependent cell-mediated cytotoxicity to the target cells in vitro , when human mononuclear cells are used as effector cells. The chimeric antibody against P-glycoprotein, MH171, should be a useful agent in the treatment of human drug-resistant tumors.     

Mouse-human chimeric antibody MH171 against the multidrug transporter P-glycoprotein.

We have developed a mouse-human chimeric antibody MH171, in which the antigen-recognizing variable regions of the mouse monoclonal antibody MRK17 are joined with the constant regions of human IgG1 antibodies. The MRK17 recognizes specifically the multidrug transporter P-glycoprotein and inhibits the growth of human multidrug resistant (MDR) tumor cells in vitro and in the xenograft nude mouse model system. The established chimeric MH171 antibody forms an apparently intact IgG composed of heavy and light chains covalently assembled via disulfide bonds in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and is specific to MDR cell lines with a similar affinity to the original mouse MRK17. MH171 also displays strong antibody-dependent cell-mediated cytotoxicity to the target cells in vitro, when human mononuclear cells are used as effector cells. The chimeric antibody against P-glycoprotein, MH171, should be a useful agent in the treatment of human drug-resistant tumors.     

Monoclonal Anti-P-glycoprotein Antibody-dependent Killing of Multidrug-resistant Tumor Cells by Human Mononuclear Cells

Mouse monoclonal antibodies (MRK16 and MRK17) against human multidrug-resistant cancer cell lines were tested for antibody-dependent cytotoxicity mediated by human blood mononuclear cells, using a 4-h ⁵¹Cr release assay. MRK16 (IgG₂₈ isotype) was shown to be more effective than MRK17 (IgG₁ isotype). Moreover, when four pairs of drug-resistant and their parent sensitive human cancer cells were tested for antibody-dependent cell-mediated cytolysis (ADCC) using MRK16, only the drug-resistant cell lines were susceptible to ADCC reaction. When highly purified lymphocytes (>99%) and monocytes (>97%) were isolated from blood mononuclear cells by centrifugal elutriation and adherence, MRK16 promoted both lymphocyte- and monocyte-mediated tumor cell killing, whereas MRK17 induced only a lymphocyte-mediated ADCC reaction. These results suggest that MRK16 of IgG₂₈ subtype may be a useful therapeutic agent in eradication of drug-resistant cancer cells expressing P-glycoprotein through ADCC reaction.     

Monoclonal anti-P-glycoprotein antibody-dependent killing of multidrug-resistant tumor cells by human mononuclear cells

Mouse monoclonal antibodies (MRK16 and MRK17) against human multidrug-resistant cancer cell lines were tested for antibody-dependent cytotoxicity mediated by human blood mononuclear cells, using a 4-h 51Cr release assay. MRK16 (IgG2a isotype) was shown to be more effective than MRK17 (IgG1 isotype). Moreover, when four pairs of drug-resistant and their parent sensitive human cancer cells were tested for antibody-dependent cell-mediated cytolysis (ADCC) using MRK16, only the drug-resistant cell lines were susceptible to ADCC reaction. When highly purified lymphocytes (greater than 99%) and monocytes (greater than 97%) were isolated from blood mononuclear cells by centrifugal elutriation and adherence, MRK16 promoted both lymphocyte- and monocyte-mediated tumor cell killing, whereas MRK17 induced only a lymphocyte-mediated ADCC reaction. These results suggest that MRK16 of IgG2a subtype may be a useful therapeutic agent in eradication of drug-resistant cancer cells expressing P-glycoprotein through ADCC reaction.     

Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein-specific monoclonal antibodies.

We have cloned a human MDR3 complementary DNA, coding for a P-glycoprotein, into a mammalian expression vector and cotransfected it with a selectable marker into drug-sensitive human BRO melanoma cells. With low frequency we obtained stable, MDR3-expressing clones. Immunocytochemical and immunoblotting analysis of these clones using the monoclonal antibody C219 indicated that human MDR3 P-glycoprotein, like human MDR1 P-glycoprotein, was mainly localized in the plasma membrane and probably glycosylated. Although a significant fraction of the cells (5-10%) in one of the MDR3-expressing clones expressed as much P-glycoprotein as a clearly drug-resistant MDR1-transfected clone, we found no resistance against a range of drugs affected by multidrug resistance. The drugs tested included vincristine, colchicine, VP16-213, daunorubicin, doxorubicin, actinomycin D, and gramicidin D. We did not detect enhanced daunorubicin efflux either in any of the MDR3-expressing cells by fluorescence microscopy. Direct selection with vincristine, actinomycin D, gramicidin D, or daunorubicin of BRO cells transfected with expression constructs containing the regular MDR3 complementary DNA, or a complementary DNA representing a major MDR3 splice variant (C(-141)), likewise failed to yield resistant clones. Thus, although human MDR3 P-glycoprotein is highly similar to human MDR1 P-glycoprotein, we found no indications that it can transport drugs. We investigated the cross-reactivity of the monoclonal antibodies C219, C494, JSB-1, HYB-241, and MRK16, recognizing human MDR1 P-glycoprotein, with human MDR3 P-glycoprotein using immunocytochemistry and immunoblotting. Apart from monoclonal antibody C219, none of the monoclonal antibodies showed detectable cross-reactivity with human MDR3 P-glycoprotein. In our hands, monoclonal antibodies MRK16 and HYB-241 were most suitable for sensitive and specific cytochemical detection of human MDR1 P-glycoprotein.     

P-glycoprotein expression in primary breast cancer detected by immunocytochemistry with two monoclonal antibodies

We have investigated P-glycoprotein (P-gp) expression in samples of primary breast cancer from 29 patients before therapy. We employed immunohistochemical techniques using two monoclonal antibodies (C219 and MRK16) and an indirect alkaline phosphatase method. Heterogeneous expression in epithelial cells was detected with both C219 (21 of 29) and MRK16 (16 of 29). A surprising finding was P-glycoprotein expression in stromal cells with both C219 (26 of 29) and MRK16 (12 of 29). Our results suggest that significant levels of P-glycoprotein expression may be present in breast cancer before exposure to drugs associated with multidrug resistance.     

Immunohistochemical identification of P-glycoprotein in previously untreated, diffuse large cell and immunoblastic lymphomas.

Expression of P-glycoprotein has been linked to multidrug resistance in cancer cell lines and human tumors. We investigated the frequency and clinical significance of P-glycoprotein immunoreactivity in 57 previously untreated diffuse large cell and immunoblastic lymphomas. Banked frozen tissue, which had been obtained prior to chemotherapy, was tested for reactivity with 2 monoclonal antibodies (MRK16 and C219) that recognize different domains of P-glycoprotein, using an immunoperoxidase technique. Thirteen of 57 lymphomas (23%) showed strong staining of greater than 50% of neoplastic cells; 15 of 57 (26%) showed labeling of a minority (11-50%) of neoplastic lymphocytes; 14 of 57 (25%) yielded equivocal results (reactivity in less than 10% of cells); and 15 of 57 (26%) were negative for P-glycoprotein. The 2 monoclonal antibodies were comparable in reactivity. Expression of MDR-1 mRNA was determined in 6 cases with sufficient available tissue, and did not correlate well with the percentages of cells reactive for P-glycoprotein by immunohistochemistry. Thirty-nine of our 57 patients completed multiagent chemotherapy. Contrary to our expectations, we found that P-glycoprotein immunoreactivity did not decrease the likelihood of response to induction chemotherapy. Median survival also was not adversely affected.     

Further characterization of the human adrenal-derived P-glycoprotein recognized by monoclonal antibody MRK 16 reacting with only human P-glycoprotein

This paper describes further characterization of the 170-180-kDa glycoprotein (P-glycoprotein) recognized by the monoclonal antibody MRK 16 in the human adrenal. By electron microscopy, P-glycoprotein was observed in the adrenal cell membranes. However, MRK 16-defined P-glycoprotein was not found in cow, pig, horse, monkey or rabbit adrenal, indicating that MRK 16 recognizes the non-homologous part of P-glycoprotein of various species. Eleven out of 16 adrenal tumors including 4 cases of primary aldosteronism and 7 cases of Cushing syndrome were intensely stained with MRK 16, whereas pheochromocytoma, non-functioning adrenocortical adenoma with no associated increase of serum adrenal-derived hormones and myolipoma of the adrenal were not. Finally, P-glycoprotein-MRK 16-protein A-Sepharose complex derived from human adrenal possessed marked ATPase activity. Taken together, these data suggest that P-glycoprotein may play a physiological role in the human adrenal.     

Further Characterization of the Human Adrenal-derived P-GIycoprotein Recognized by Monoclonal Antibody MRK 16 Reacting with Only Human P-Glycoprotein

This paper describes further characterization of the 170–180-kDa glycoprotein (P-glycoprotein) recognized by the monoclonal antibody MRK 16 in the human adrenal. By electron microscopy, P-glycoprotein was observed in the adrenal cell membranes. However, MRK 16-defined P-glycoprotein was not found in cow, pig, horse, monkey or rabbit adrenal, indicating that MRK 16 recognizes the non-homologous part of P-glycoprotein of various species. Eleven out of 16 adrenal tumors including 4 cases of primary aldosteronism and 7 cases of Cushing syndrome were intensely stained with MRK 16, whereas pheochromocytoma, non-functioning adrenocortical adenoma with no associated increase of serum adrenal-derived hormones and myolipoma of the adrenal were not. Finally, P-glycoprotein-MRK 16-protein A-Sepharose complex derived from human adrenal possessed marked ATPase activity. Taken together, these data suggest that P-glycoprotein may play a physiological role in the human adrenal.     

Detection of multidrug resistant phenotype in leukemia and lymphoma by monoclonal antibodies

Two monoclonal antibodies, MRK16 and MRK20 that recognize P-glycoprotein and P-85 kd protein on the surface of adriamycin (ADM) resistant cells, respectively, were tested for the reactivity with 40 cultured leukemia/lymphoma cell lines. F(ab')2 form is essential to avoid false reaction through Fc gamma-R. Drug sensitivity of 19 representative cell lines were also examined in vitro. From this study, it was found that these cell lines were classified into 4 groups. Group 1 (4 cell lines) was insensitive to ADM, mitoxantron (MXT), etoposide (VP-16) and vincristine (VCR), and reactive to MRK16 and MRL20. Group II (1 cell line) was insensitive to the 4 drugs, but not reactive to both antibodies. Group III (3 cell lines) was insensitive to ADM, MXT and VP-16, but sensitive to VCR, and reactive to MRK20, but not to MRK16. Group IV (all other cell lines) was sensitive to these drugs, and not reactive to both antibodies. From these results, MRK16 detects P-glycoprotein-associated multidrug resistance (MDR), while MRK20 does P 85-kd-associated another type MDR (cross resistance to ADM, MXT and VP-16, but not to VCR). MRK20 reacted with monocytes, but MRK16 did not with any WBC type. One hundred and ninety eight clinical samples obtained from blood cancer were tested for the reactivity with MRK16. MRK16 did not react with any of 98 samples obtained before treatment, but did with 9 of 100 obtained at relapse or refractory stage after chemotherapy. The results indicate that MRK16 is useful to detect drug resistance phenotype of leukemia and lymphoma.     

Tissue distribution of P-glycoprotein encoded by a multidrug-resistant gene as revealed by a monoclonal antibody, MRK 16

A monoclonal antibody, MRK 16, specific to a human myelogenous leukemia cell line, K-562, and resistant to Adriamycin, was used to determine the localization of the antigen molecules (P-glycoprotein) recognized by the monoclonal antibody. P-glycoprotein was found to be expressed very strongly in the adrenal cortex and medulla of adults and strongly in the renal tubules of the kidney and the placenta. Interestingly, P-glycoprotein was not distributed in fetal and neonatal adrenals, and thus may be closely related to adrenal maturation. A high level of P-glycoprotein expression was also seen in one case each of untreated lung cancer (one of ten) and breast cancer (one of nine). Immunoelectron microscopically, the P-glycoprotein was distributed evenly on the membranes of K-562/ADM and 2780 cells. These results imply that the presence of the glycoprotein may be useful as a marker for in vitro studies of multidrug resistance in various malignancies and as an indicator of therapeutic efficacy of ex vivo eradication of multidrug-resistant cancer cells, although other mechanisms of drug resistance may exist, and there is a possibility that this MRK 16 monoclonal antibody may not recognize all P-glycoprotein.     

High-level expression of MRK 16 and MRK 20 murine monoclonal antibody-define proteins (170,000-180,000 P-glycoprotein and 85,000 protein) in leukaemias and malignant lymphomas

Using flow cytometry and immunocytochemistry, we investigated the reactivities of two different murine monoclonal antibodies (MAbs), MRK 16 and MRK 20, specific to adriamycin-resistant K562 cells (K562/ADM) with peripheral human mononuclear cells (MNC) (mainly blastic cells and lymphocytes) from 31 patients with leukaemia or malignant lymphoma. Reactivity with MRK 16 MAb was observed in five cases and reactivity with MRK 20 MAb in 18 cases. The cases were divided into three groups according to their reactivity patterns: group I, only the proportion of MRK 16-positive cells was increased; group II, only the proportion of MRK 20-positive cells was increased; group III, both MRK 16-and MRK 20-positive cells were increased. Some cases reflected the prior administration of adriamycin, vincristine, vinblastine and VP-16, which are known to induce P-glycoprotein expression. Expression of Mr 85,000 protein was observed more frequently than that of P-glycoprotein in leukaemia and malignant lymphoma, and this was not associated with either the total dose or period of administration of anticancer drugs. The expression of Mr 85,000 protein recognised by MRK 20 was further confirmed by Western blot analysis.     

Expression of MDR1/P glycoprotein in human sarcomas.

Conflicting reports of MDR1 gene expression in human tumours are observed according to whether studies are performed at the mRNA or P-glycoprotein level. We have investigated this expression in 22 clinically drug-resistant sarcomas at the mRNA level by Northern blot (NB), Dot blot (DB), in situ hybridisation (ISH), and at the protein level by immunohistochemistry (IHC) using three monoclonal antibodies (MoAbs): C219, JSB1, MRK16. Increased MDR1 mRNA expression was detected by NB, DB, and ISH in 1/22 sarcoma (an Ewing''s sarcoma). ISH was perfectly correlated with DB hybridisation and confirmed the expression of tumoral cells alone. Specific staining of 100% of tumoral cells was obtained with the three MoAbs in the same sarcoma. Expression in tumoral cells of 12 other sarcomas was detected with MRK16, and positive staining of stromal cells with both C219 (1/22) and MRK16 (8/22) was observed. This study confirms that MDR1 overexpression occurs in human sarcomas but is not the principal mechanism of drug-resistance. Furthermore, positivity with one antibody does not necessarily imply the presence of P glycoprotein (P-gp) and a disparity may exist between the levels of P-gp and its mRNA in the same sample. So care must be taken in interpreting results and more sensitive techniques such as the polymerase chain reaction (PCR) could prove useful.     

Decreased P-glycoprotein expression in multidrug-sensitive and -resistant human myeloma cells induced by the cytokine leukoregulin.

Modulation of the expression of P-glycoprotein, a plasma membrane protein associated with multidrug resistance, was examined in drug-sensitive and drug-resistant tumor cells treated with leukoregulin, a M(r) 50,000 cytokine from human lymphocytes that rapidly permeabilizes the plasma membrane of many tumor cells facilitating the uptake of doxorubicin and other tumor-inhibitory antibiotics. P-glycoprotein expression was measured flow cytometrically by the binding of C219 or MRK16 monoclonal antibody to multidrug-sensitive human K562 erythroleukemia and 8226/S myeloma cells, compared to multidrug-resistant 8226/DOX40 myeloma cells. Cells were treated for up to 2 h with up to 80 units of leukoregulin/ml or one of a variety of unrelated cytokines including interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, colony-stimulating factor, macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, gamma-interferon, alpha-interferon, epidermal growth factor, platelet-derived growth factor AA, platelet-derived growth factor BB, insulin-like growth factor I, insulin-like growth factor II, fibroblast growth factor, or transforming growth factor beta. Leukoregulin caused a concentration-dependent decrease in P-glycoprotein expression; however, P-glycoprotein expression was unaffected by the other cytokines (< 12% decrease in expression). Leukoregulin-induced membrane permeabilization, determined flow cytometrically by intracellular fluorescein efflux, and decreased P-glycoprotein expression occurred simultaneously within 15 min in drug-sensitive and -resistant cells. Enhanced doxorubicin uptake, measured flow cytometrically by doxorubicin influx, was also present within 15 min. Leukoregulin enhancement of doxorubicin uptake and increased membrane permeability varied directly with the decrease in P-glycoprotein expression. Leukoregulin in combination with doxorubicin enhanced the inhibition of cell proliferation in 8226/DOX40 multidrug-resistant cells over expressing P-glycoprotein. In contrast, combined treatment of HL-60/MX2 multidrug-resistant human promyelocytic leukemia cells that do not overexpress P-glycoprotein in association with their multidrug resistance resulted in no greater growth inhibition than observed with HL-60/MX2 cells treated with doxorubicin alone. This is the first demonstration that a naturally occurring macromolecule with anticancer activities can modulate the expression of P-glycoprotein concomitant with enhanced drug uptake and inhibition of cell proliferation.     

In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody.

Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6. The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration. Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately. In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay. Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment. This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation.     

Monoclonal antibody MRK16 reverses the multidrug resistance of multidrug-resistant transgenic mice.

Using multidrug-resistant (MDR)-transgenic mice, whose bone marrow cells express the human MDR1 gene at a level approximately equal to that found in many human cancers, we determined the efficacy of human-specific anti-P-glycoprotein monoclonal antibody MRK16 in overcoming multidrug resistance in an intact animal. MRK16 alone (2 mg) did not significantly affect the WBC counts of the MDR-transgenic mice, but MRK16, as well as the F(ab')2 fragments of MRK16, led to a dose-dependent circumvention of bone marrow resistance against daunomycin, doxorubicin, vincristine, vinblastine, etoposide, and taxol. This sensitizing effect could not be enhanced by combining MRK16 with low molecular weight chemosensitizing agents such as verapamil, quinine, quinidine, or cyclosporin A. We also investigated the concept of specifically targeting and killing multidrug-resistant cells by using MRK16 coupled to Pseudomonas exotoxin (PE). MRK16-PE resulted in a dose-dependent killing of bone marrow cells in MDR-transgenic mice, whereas no bone marrow toxicity was observed in normal control mice. Administration of excess MRK16 prior to injection of MRK16-PE successfully blocked the effect of MRK16-PE. MOPC-PE, a non-MDR-related control monoclonal antibody conjugate, did not target and kill multidrug-resistant bone marrow cells in MDR-transgenic mice. Thus, these immunological approaches to reversing multidrug resistance appear to be both specific and effective.     

Detection of multidrug resistance markers, P-glycoprotein and mdr1 mRNA, in human leukemia cells

We have examined the expression of P-glycoprotein in clinical leukemic cell samples by using a monoclonal antibody (MRK16) against P-glycoprotein. We found that leukemia cells isolated from 3 out of 6 patients with blast crisis of chronic myelogenous leukemia were reactive to MRK16. These 3 cell lines expressed high levels of mdr1 mRNA, which codes for P-glycoprotein. The present result indicates that the clinically refractory state of the tumor may be predicted in part by determining P-glycoprotein expression using the monoclonal antibody against P-glycoprotein, and the mdr1 probe.