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.     

Inhibition of human tumor xenograft growth in nude mice by a novel monoclonal anti-HSPG isolated from human liver

Heparan sulfate proteoglycans (HSPGs) were isolated from normal human liver and α monoclonal antibody (MAb) was raised against them. Preliminary studies showed that MAb clone 1E4-1C2 was able to react with many cell lines tested, including hematopoietic cells and solid tumors. MAb1E4-1C2 was used to study whether HSPG was involved in growth and proliferation of human liver cancer using hepatocellular carcinoma (HCC) cell line (HepG2) as a model. Inhibition by MAb1E4-1C2 of HepG2 cell proliferation was studied in vitro by MTT assay. For in vivo assay, xenograft induction in athymic mice was performed. The results showed that MAb1E4-1C2 inhibited proliferation of HepG2 cells significantly, compared to isotype and medium control. MAb1E4-1C2 also suppressed the growth of tumor, resulting in smaller tumor size and weight. The investigation also showed that MAb1E4-1C2 inhibited proliferation and restricted tumor growth through the induction of apoptosis. The results suggest that HSPG might be involved in liver cancer cell proliferation. Therefore, a specific MAb that was raised against liver HSPG might be an alternative therapeutic agent for the treatment of human liver cancer.     

Inhibition of human tumor growth by intraperitoneal immunotoxins in nude mice

Intracavitary administration of immunotoxins may play a role in the control of malignant effusions. Selection of immunotoxins for this form of therapy is based on their prior evaluation in preclinical studies. Monoclonal antibodies (mAb) 454A12 (antitransferrin receptor), and 260F9 are directed against antigens which are present on tumor cells in pleural and peritoneal effusions of patients with adenocarcinoma of the breast and ovary. In the present study, immunotoxins derived by conjugating these mAb to recombinant ricin A (rRA) were shown to be cytotoxic to human ovarian adenocarcinoma HEY cells in vitro and in vivo. In the in vitro assay 454A12-rRA and 260F9-rRA were 1000-fold and 10-fold, respectively, more cytotoxic than free rRa against HEY cells, and both immunotoxins were potentiated approximately 1000-fold by monensin. For in vivo studies HEY cells were injected i.p. into nude mice at a challenge dose (3 x 10(5) cells) which produced carcinomatosis with ascites, leading to death 30 days following injection. Administration of 454A12-rRA i.p. following the challenge dose resulted in a complete cure, whereas administration of 260 F9-rRA with monensin significantly prolonged survival. The greater cytotoxicity of 454A12-rRA than 260F9-rRA against HEY cells could be accounted for by the greater number of binding sites and higher internalization rate for 454A12-rRA and mAb 454A12 than 260F9-rRA and mAb 260F9, respectively. These results suggest a potential role for 454A12-rRA and 260F9-rRA plus monensin in the intracavitary therapy of malignant effusions associated with carcinoma of breast and ovary. In the case of 260F9-rRA, this represents the first preliminary indication of the suitability of this immunotoxin for intracavitary therapy of malignancies.     

Radioimmunoimaging of human bladder tumor xenografts in nude mice by using monoclonal antibodies

Monoclonal antibodies HBJ127 and HBJ8, raised against T24 human bladder cancer cells, predominantly react with the cells in proliferating stages and with a portion of epithelial tumor cells, respectively. To investigate the in vivo localization of these monoclonal antibodies, the antibodies were labeled with radioiodine and indium-111 (111In) and injected into nude mice transplanted with human bladder tumors. The BT-11 bladder tumor had the highest concentration of radioiodinated HBJ127 and HBJ8 monoclonal antibodies, with 11.6 and 14.3% of the injected dose per gram and with a tumor-to-blood ratio of 2.6 and 1.6, respectively, at 4 days after the administration. An irrelevant monoclonal antibody did not show any specific accumulation in the BT-11 tumor. The 111In-labeled HBJ127 antibody was also localized in the tumor with a higher tumor-to-blood ratio than the radioiodinated antibody. The xenografted BT-11 tumor was successfully visualized with the radiolabeled HBJ127 and HBJ8 antibodies by scintigraphy. These monoclonal antibodies and the human bladder tumor xenografts may provide a good model for radioimmunoimaging and possibly therapy.     

Inhibition of human T-cell tumor growth by T101-ricin A-chain in an athymic mouse model

An immunotoxin prepared with the pan-T-cell, anti-CD5, antibody T101, and purified ricin A-chain (RTA) was selectively cytotoxic in vitro, inactivating protein synthesis in the human T-cell line MOLT-4 but not in the human B-cell line 8392. Modulation studies showed that the immunoconjugate was more rapidly cleared from the cell surface than unconjugated T101. Preclinical evaluation of T101-RTA was conducted in a human T-cell, athymic mouse model (Dillman et al., Cancer Res., 45:5632-5336, 1985). Tumor-bearing mice received single i.p. injections of saline, T101, UPC-10 (irrelevant IgG2a), unconjugated RTA, an irrelevant conjugate, UPC-10-RTA, a mixture of T101 plus RTA, or T101-RTA. T101-RTA was the most effective reagent. Thirty animals given injections of 33 micrograms of T101 showed reductions in tumor growth (compared to tumor growth in animals receiving phosphate-buffered saline) but no complete regressions. No decrease in tumor growth was observed with UPC-10. Animals given 12 micrograms of free RTA exhibited reduced tumor growth but only one complete regression was observed; similar results were obtained with mice given 45 micrograms of UPC-10-RTA or a mixture of 33 micrograms of T101 plus 12 micrograms of RTA. Eleven complete regressions and 18 partial regressions were produced in the 46 animals given injections of 45 micrograms of T101-RTA and tumor growth was almost completely blocked. No toxicity was observed in any experimental arm. These results suggest that T101-RTA may be administered safely and with significant antitumor effect.     

Radiolocalization of human mammary tumors in athymic mice by a monoclonal antibody

Monoclonal antibody B6.2 reacts with a protein found on the surface of primary and metastatic human mammary tumors. B6.2 immunoglobulin G (IgG) was purified, F(ab')2 and Fab' fragments were generated by pepsin digestion, and the IgG and its fragments were radiolabeled with 125I; all were successful in localizing human mammary tumors transplanted into athymic mice, with tumor:tissue ratios increasing over a 4-day period. The 125I-labeled IgG gave tumor:spleen, tumor:liver, and tumor:kidney ratios of greater than 10:1 and tumor:brain and tumor:muscle ratios of 50:1 to 110:1. The F(ab')2 fragment gave higher tumor:tissue ratios than did the IgG, with tumor:liver and tumor:spleen ratios of 15:1 to 20:1. No localization of the labeled B6.2 monoclonal antibody or its fragments was observed in athymic mice bearing a human melanoma or with isotype-identical control immunoglobulin or its fragments in athymic mice bearing the mammary tumors. Imaging experiments confirmed the ability of radiolabeled monoclonal antibody B6.2 and its fragments to detect the presence of transplanted human mammary tumor lesions of less than 0.4 cm without the aid of background subtraction manipulations.     

Reversal of Vinca alkaloid resistance by anti-P-glycoprotein monoclonal antibody HYB-241 in a human tumor xenograft.

A panel of monoclonal antibodies (MAbs) to P-glycoprotein was developed by immunization of mice with multidrug-resistant human neuroepithelioma and neuroblastoma cells. All the anti-P-glycoprotein MAbs reacted with the extracellular portion of P-glycoprotein. The MAbs were examined for their ability to enhance accumulation of actinomycin D, vincristine, vinblastine, and doxorubicin in the human mdr1 transfectant cell line, BRO/pFRmdr1.6. HYB-241, an IgG1 anti-P-glycoprotein MAb, was the most effective modulator, increasing actinomycin D levels in the transfectant line by 6-fold, vincristine by 2-fold, and vinblastine levels by 3-fold. None of the MAbs were capable of modifying the accumulation of doxorubicin. HYB-241 lowered the 50% inhibitory concentration values of actinomycin D by 11-fold, vincristine by 6-fold, and vinblastine by 2-fold. No effect on the 50% inhibitory concentration values of doxorubicin or gramicidin were seen. 111In-labeled HYB-241 localized in human tumor xenografts of BRO/pFRmdr1.6 in nude mice (25% injected dose/g at 120 h). Mice with established drug-resistant xenografts were treated with antibody 24 h prior to the injection of Vinca alkaloid at concentrations known to be non-growth inhibitory. The addition of HYB-241 at 25 mg/kg per injection prior to drug resulted in a significant inhibition of growth of this drug-resistant tumor.     

Establishment of a human B-cell tumor in athymic mice

Human B-cell tumors have been established in athymic, BALB/c mice using the EBV-positive Burkitt lymphoma cell line Namalwa. One-hundred-one of 104 animals (97%) developed tumors 10-14 days following s.c. injection of a mixture of 20 x 10(6) Namalwa and 5 X 10(6) irradiated human fibrosarcoma (HT-1080) cells. Tumors developed at the site of injection and reached approximately 300 mm2 (product of cross-sectional diameters) after 21 days; no metastases were found. Histological analysis showed that tumors consisted solely of lymphoid cells. Immunofluorescence assays demonstrated that while 85% of the tumor cells retained reactivity with the monoclonal B-cell antibody BA-1, 96% retained reactivity with antibody BA-2 and 43% with BA-3. A similar reactivity profile was observed with cultured Namalwa cells. Tumors were passaged serially 10 times without significant change in BA-1, BA-2, or BA-3 reactivity. Indirect immunofluorescence demonstrated that antibody BA-2 reached tumor cells within 2 h following i.p. injection; antigen modulation was not observed. These results demonstrate the suitability of this B-cell model for testing the in vivo efficacy and stability of anti-B-cell immunoconjugates.     

Radiolocalization of human pancreatic tumors in athymic mice by monoclonal antibody DU-PAN 1

Monoclonal antibodies that selectively bind to pancreatic tumors may be useful in the therapy and diagnosis of pancreatic carcinoma. In this study we have examined the tumor localization of radioiodinated DU-PAN 1, a mouse monoclonal antibody that is selective for a human pancreatic cancer-associated antigen. After radiolabeling, both DU-PAN 1 intact monoclonal antibody and F(ab')2 fragments retained immunoreactivity and showed high affinity for the pancreatic tumor cell line CA13 in vitro. Paired-label biodistribution studies in nude mice bearing CA13 s.c. xenografts were performed. Mice received both 131I-labeled DU-PAN 1 immunoglobulin G2a or F(ab')2 fragment and 125I-labeled mouse myeloma immunoglobulin G2a or F(ab')2 fragment. Tumor uptake for 5-micrograms doses of DU-PAN 1 immunoglobulin ranged from 4.8 to 11.83% injected dose/g. Tumor uptake values for mice given 5-micrograms doses of DU-PAN 1 F(ab')2 ranged from 3.9 to 6.9% injected dose/g. Tumor uptakes of the respective myeloma controls were lower in all cases when compared with the DU-PAN 1 preparations. Tumor localization indices for 5-micrograms doses of DU-PAN 1 immunoglobulin were 3.0 and 24 h and 2.9 at 48 h. For 5-micrograms doses of DU-PAN 1 F(ab')2, tumor localization indices were 29.9 at 24 h and 90.0 at 48 h. In most cases, tumor:normal tissue ratios were greater than 3 at all time points, indicative of tumor selectivity for both DU-PAN 1 preparations, but the ratios were considerably higher using the DU-PAN 1 F(ab')2. The F(ab')2 fragment thus displays better tumor localization characteristics when compared with the intact immunoglobulin. Protein doses of DU-PAN 1 F(ab')2 of between 5 and 10 micrograms gave the best localization, although protein doses of up to 100 micrograms could be administered before apparent tumor saturation was seen.     

Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies

Monoclonal antibodies (MAbs) were raised against partially purified Class I P-glycoprotein from multidrug-resistant Chinese hamster ovary CH^RB30 cells. Fifteen stable monoclonal hybridoma cell lines were established, and the secreted antibodies were classified into 8 groups on the basis of banding pattern on immunoblots of P-glycoprotein digested with cyanogen bromide or partially digested with proteases. One representative of each group was tested further for several activities. Six of the 8 recognized human P-glycoprotein in the multidrug-resistant SKVLB1 cell line. None of the antibodies recognized P-glycoprotein in unfixed cells, suggesting that all recognize cytoplasmic epitopes or extracellular epitopes not accessible in native P-glycoprotein. All 8 antibodies were able to immunoprecipitate P-glycoprotein from non-denaturing detergent solution. The linear epitopes of the antibodies were mapped to 11–27 amino acids. Two of the antibodies had epitopes in the linker region, 3 in the N-terminal nucleotide binding domain, 2 in the C-terminal nucleotide binding domain and 1 in the predicted cytoplasmic loop between predicted transmembrane helices 8 and 9. These antibodies, with known epitopes, could have uses for P-glycoprotein detection, structure/function studies, purification and quantitation. © 1996 Wiley-Liss, Inc.     

Effects of plant polysaccharide paliustran on the growth of human tumor transplants in athymic mice

Plant polysaccharide palyustran inhibited the growth of human lung carcinoma P-1 transplanted to athymic mice by 60% but failed to do so in human rhabdomyosarcoma. Treatment with palyustran was followed by a 2-fold decrease in lympho- and granulocyte levels, selective inhibition of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase activity in tumor cells and--in single cases--metastatic involvement of the liver.     

Inhibition of CWR22Rnu1 tumor growth and PSA secretion in athymic nude mice by green and black teas

Cancer of the prostate gland (CaP), the most common invasive malignancy and a major cause of cancer related deaths in male population in the USA, is an ideal candidate disease for chemoprevention because it is typically detected in elderly population with a relatively slower rate of growth and progression. Many dietary phytochemicals are showing promising chemopreventive effects, at-least in pre-clinical models of CaP. Our published data in cell culture and animal studies, supported by the work from other laboratories, as well as epidemiological observations and case-control studies, suggest that polyphenols present in green tea possess CaP chemopreventive and possibly therapeutic effects. This present study was designed to compare CaP cancer chemopreventive effects of green tea polyphenols (GTP), water extract of black tea, and their major constituents epigallocatechin-3-gallate and theaflavins, respectively, in athymic nude mice implanted with androgen-sensitive human CaP CWR22Rnu1 cells. Our data demonstrated that the treatment with all the tea ingredients resulted in (i) significant inhibition in growth of implanted prostate tumors, (ii) reduction in the level of serum prostate specific antigen, (iii) induction of apoptosis accompanied with upregulation in Bax and decrease in Bcl-2 proteins, and (iv) decrease in the levels of VEGF protein. Furthermore, we also found that GTP (0.01 or 0.05% w/v; given after establishment of CWR22Rnu1 tumor) causes a significant regression of tumors suggesting therapeutic effects of GTP at human achievable concentrations.     

Inhibition of growth of human tumor xenografts in athymic mice treated with ricin toxin A chain-monoclonal antibody 791T/36 conjugates

Immunotoxin constructed by conjugating ricin A chain to monoclonal antibody 791T/36 specifically inhibits growth of human tumor xenografts which express the gp72 antigen recognized by the antibody component. Dose schedule tests showed that the major response was obtained during the first 5 days of treatment and further prolonged treatment did not improve therapy. Expression of gp72 antigen on tumor cells derived from xenografts in immunotoxin-treated mice was not markedly altered indicating that treatment did not lead to the expansion of tumor antigen deficient tumor cells. The experiments indicate that treatment for short duration with immunotoxin may be the most effective protocol.     

Inhibition of growth of human tumor cells in nude mice by a metalloproteinase inhibitor

The effects of a new metalloproteinase inhibitor, BEI6627B [L-N-(N-hydroxy-2-isobutylsuccinynamoyl)-seryl-L-valine, MW: 375.2] isolated from microbial cultures, on human tumor cell growth in nude mice were investigated. BE16627B inhibited metalloproteinases in enzyme assays, as well as gelatinolysis and collagenolysis in cell cultures. BE16627B at 100 μg/ml showed no apparent cytotoxicity to human tumor cells in culture and its LD₅₀ in mice was more than 1,000 mg/kg (i.p.). The effects of BE16627B on the in vivo growth of 1 human tumor cell lines were examined: HT1080 fibrosarcoma, which overproduces metalloproteinases, and HCT116 colon carcinoma, which barely secretes metalloproteinases. When BE16627B was administered to mice at 2 mg/mouse/day by an osmotic pump implanted s.c. for 3 weeks from 1 week after i.v. inoculation of HT1080 cells, the number and size of nodules of HT1080 cells on the lung surface were reduced to 24.3 and 46.4%, respectively, of those of controls, and the increase in lung weight due to tumor-cell growth was inhibited 85.5% without body-weight loss. Moreover, BE16627B inhibited 71.2% of the growth of HT1080 cells inoculated s.c. into mice under the same conditions, but did not significantly inhibit the s.c. growth of HCT116 human colon-carcinoma cells. Thus, BE16627B inhibited metalloproteinase-dependent human tumor-cell growth as well as lung colonization without showing cytotoxicity in nude mice.     

Stimulation by EGF of the growth of EGF receptor-hyperproducing tumor cells in athymic mice

EGF receptor-hyperproducing cells of squamous carcinoma origin were inoculated s.c. into the bilatero-abdominal regions of athymic mice and a mini-osmotic pump containing EGF was implanted on teh back. After 2 weeks the tumors formed from 5 different cell lines in the presence of EGF weighed 3 to 6 times more than those formed in the absence of EGF. The cells recovered from these tumors maintained their original characteristics such as the amplified EGF receptor gene, high numbers of EGF receptors and a sensitivity to EGF-mediated inhibition of growth in vitro. These results suggest that EGF plays an important role in promoting growth of squamous-cell carcinoma in vivo.     

Growth inhibition of human melanoma tumor xenografts in athymic nude mice by swainsonine

Swainsonine, an inhibitor of alpha-mannosidases, has been shown to block experimental metastasis of B16F10 melanoma and MDAY-D2 lymphoid tumor cells in syngeneic mice. In this report we demonstrate that swainsonine also reduces the growth rate of human melanoma cells in vitro and in vivo. Graded doses of swainsonine were administered either orally or via implanted Alzet miniosmotic pumps to athymic nude mice bearing subcutaneously implanted human MeWo melanoma cells. Swainsonine at 10 micrograms/ml in the drinking water or 0.5 mg/kg/day administered by miniosmotic pump reduced the growth rate of the MeWo tumors by approximately 50% and inhibited the expression of complex-type oligosaccharides in tumors and host intestine by only 10-20%. Swainsonine doses of 4 mg/kg/day reduced expression of complex-type oligosaccharides by 85% in vivo but afforded no additional inhibitory effect. A glycosylation mutant of MeWo called 3S5 has a defect in the synthesis of complex-type asparagine-linked oligosaccharides resulting in incomplete processing similar to that observed in swainsonine-treated MeWo tumor cells. Swainsonine did not inhibit the proliferation of 3S5 cells in vitro nor the growth of 3S5 tumors in nude mice. The results suggest that expression of highly branched complex-type oligosaccharides commonly associated with the malignant phenotype may provide the tumor cells with a growth advantage.     

Inhibition of tumor growth and progression of LNCaP prostate cancer cells in athymic mice by androgen and liver X receptor agonist

Androgen-dependent human LNCaP 104-S tumor xenografts progressed to androgen-independent relapsed tumors (104-Rrel) in athymic mice after castration. The growth of 104-Rrel tumors was suppressed by testosterone. However, 104-Rrel tumors adapted to androgen and regrew as androgen-stimulated 104-Radp tumors. Androgen receptor expression in tumors and serum prostate-specific antigen increased during progression from 104-S to 104-Rrel but decreased during transition from 104-Rrel to 104-Radp. Expression of genes related to liver X receptor (LXR) signaling changed during progression. LXRalpha, LXRbeta, ATP-binding cassette transporter A1 (ABCA1), and sterol 27-hydroxylase decreased during progression from 104-S to 104-Rrel. These coordinated changes in LXR signaling in mice during progression are consistent with our previous findings that reduction of ABCA1 gene expression stimulates proliferation of LNCaP cells. To test if attenuation of LXR signaling may enhance prostate cancer progression from an androgen-dependent state to an androgen-independent state, castrated mice carrying 104-S tumors were given the synthetic LXR agonist T0901317 by gavage. T0901317 delayed progression from 104-S to 104-Rrel tumors. Based on our in vivo model, androgen is beneficial for the treatment of androgen-independent androgen receptor-rich prostate cancer and modulation of LXR signaling may be a potentially useful therapy for prostate cancer.     

Inhibition of v-fms-induced tumor growth in nude mice by castanospermine

Castanospermine, an indolizidine alkaloid isolated from the seeds of the chestnut tree Castanospermum australe, has been shown to prevent the normal glycosylational processing of the v-fms-transforming glycoprotein. v-fms-transformed cells grown in vitro in the presence of castanospermine accumulate immature forms of the glycoprotein that do not reach the cell surface. These treated cells revert to the normal phenotype. We have now extended those studies to an in vivo tumorigenicity model in the nude mouse using v-fms-transformed rat cells (SM-FRE) and dietary administration of castanospermine. Following tumor induction with s.c. injection of 10(6) SM-FRE cells, mice whose diet had been supplemented with 2.4 mg castanospermine/g food consumed approximately 0.84 mg castanospermine/g mouse/day. Furthermore, these mice had slower growing tumors that were 2.6 times smaller than tumors in control groups at the termination of the experiment 24 days postinjection. These results indicate that castanospermine is effective in slowing the growth of v-fms-transformed cells in vivo, suggesting that this drug may offer an effective therapy against certain tumors, including some arising from activated protooncogenes that encode glycoproteins such as growth factor receptors.     

Propylthiouracil-induced hypothyroidism reduces xenograft tumor growth in athymic nude mice.

BACKGROUND: Thyroid hormones are endocrine modulators of several vital processes that are crucial to tumor growth and differentiation. Several anecdotal reports in the literature suggest that some histologic types of carcinoma may remain in a dormant state for prolonged periods of time in patients with hypothyroidism, with eventual progression of the disease once the decreased thyroid function is identified and corrected. METHODS: Oral propylthiouracil (PTU) was used to induce hypothyroidism in athymic nude mice that were subsequently inoculated with lung adenocarcinoma and prostate adenocarcinoma cells. Mice were also treated with a combination of PTU and thyroxine, which resulted in hyperthyroid levels of T(4). RESULTS: Subcutaneous lung and prostate xenografts grew significantly more slowly in hypothyroid mice treated with PTU than in euthyroid or hyperthyroid mice, regardless of treatment with PTU. Tumors grew well in groups of mice that were changed from a hypothyroid state to a euthyroid state by withdrawal of oral PTU. Administration of PTU 3 weeks after tumor inoculation also caused the tumor growth to slow significantly compared with tumors in mice that did not receive PTU. Mice that received PTU and thyroxine had tumors that grew as well as the tumors in euthyroid control animals. CONCLUSIONS: Our study indicates that human lung and prostate tumors do not grow well in hypothyroid nude mice, and that rendering these animals euthyroid has a significant impact on the growth rate of these tumors. Furthermore, in vitro and in vivo data indicated that this was not a result of an interaction of the tumor cells with PTU, but rather a result of the hypothyroid state.