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 2780^AD. 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.     

Transduction of the Macrophage Colony-stimulating Factor Gene into Human Multidrug Resistant Cancer Cells: Enhanced Therapeutic Efficacy of Monoclonal Anti-P-glycoprotein Antibody in Nude Mice

To develop a therapeutic modality for overcoming multidrug-resistant (MDR) cancer with anti-MDR1 antibody, we examined the effect of macrophage colony-stimulating factor (M-CSF) gene transfection into MDR AD10 cells on therapy of MDR cancer with anti-MDR1 antihody (MRK17) in nude mice. MDR human ovarian cancer (AD10) cells were transduced with the human M-CSF gene inserted into an expression vector to establish gene-modified cells capable of producing low (ML-AD10), intermediate (MM-AD10) and high (MH-AD10) amounts of M-CSF. Systemic administration of MRK17 resulted in significant dose-dependent inhibition of subcutaneous growth of ML-AD10 tumors. In contrast, systemic administration of recombinant M-CSF in combination with MRK17 did not augment the therapeutic efficacy of MRK17 alone, but rather promoted the growth of the parent AD10 cells. To test the efficacy of in vivo M-CSF gene therapy combined with antibody, we mixed the parent AD10 cells with MH-AD10 cells producing a large amount of M-CSF, and inoculated the mixed cells subcutaneously. Treatment with MRK17 inhibited growth of the mixed cells more than that of the parent cells alone. Thus, combined therapy with anti-MDR1 mAb and M-CSF gene modification of MDR cancer cells may provide a new immunotherapeutic modality for overcoming MDR in humans.     

Cyclosporin A Enhances Susceptibility of Multi-drug Resistant Human Cancer Cells to Anti-P-glycoprotein Antibody-dependent Cytotoxicity of Monocytes, but Not of Lymphocytes

Cyclosporin A (CsA) was previously found to bind to P-glycoprotein expressed on multidrug-resistant (MDR) cancer cells. In the present study, the effect of CsA on anti-P-glycoprotein monoclonal antibody (mAb)-dependent cell-mediated cytotoxicity (ADCC) against human MDR cells was examined. The ADCC reaction was assessed by 4-h ⁵¹Cr-release assay. Highly purified lymphocytes (> 99%) and monocytes (>99%) obtained from blood mononuclear cells (MNC) of healthy donors were used as effector cells. CsA decreased the cytotoxic activity of MNC against MDR cells, but enhanced their ADCC activity in the presence of anti-P-glycoprotein mAb MRK16. Lymphocyte-mediated ADCC and natural killer activity against MDR cells were also suppressed by addition of CsA. CsA induced a significant dose-dependent increase in monocyte-mediated ADCC activity. Interestingly, pretreatment of MDR cancer cells, but not of monocytes, with CsA significantly enhanced ADCC activity mediated by monocytes, but not by lymphocytes. A CsA analog (PSC833) and FK-506, but not verapamil also increased the sensitivity of MDR cells to ADCC by monocytes. CsA did not affect the binding of monocytes to MDR cells in the presence of MRK16 mAb. These results indicate that CsA may directly enhance the susceptibility of MDR cancer cells to the monocyte-mediated ADCC reaction.     

Influence of Systemic Chemotherapy on Anti-P-glycoprotein Antibody-dependent Cell-mediated Cytotoxicity in Patients with Small Cell Lung Cancer

Anti-P-glycoprotein antibody (MRK-16)-dependent cell-mediatedcytotoxicity (ADCC by blood mononuclear cells (MNC was examinedin patients with small cell lung cancer (SCLC) before and aftersystemic chemotherapy. The effect of in vitro treatment of MNCwith interleukin (IL)-2 and macrophage-colony-stimulating factor(M-CSF) was also examined. The ADCC reaction was assessed bya 6 h ⁵¹ Cr-release assay using a multidrug-resistant (MDR)SCLC cell line (H69/VP cells). The MRK-16 monoclonal antibodywas able to augment spontaneous cytotoxicity by MNC, even inSCLC patients. Pretreatment of MNC with IL-2 significantly augmentedtheir ADCC ability in SCLC patients, while M-CSF had no effecton ADCC activity. After the first cycle of systemic chemotherapy,the ADCC activity tended to decline, but ADCC of MNC pretreatedwith IL-2 was not affected. The results suggest that anti-P-glycoproteinantibody, in combination with a cytokine such as IL-2, may betherapeutically useful against human SCLC resistant to chemotherapeuticdrugs.     

Multidrug Resistance in Cultured Human Leukemia and Lymphoma Cell Lines Detected by a Monoclonal Antibody, MRK16

Forty cultured human leukemia and lymphoma cell lines never exposed to anticancer agents in culture, apart from doxorubicin (ADM)-resistant K562/ADM, were examined for reactivity with a monoclonal antibody, MRK16 in F(ab')₂ form [MRK16-F(ab')₂], which recognizes P-glycoprotein (P-gp). The relative resistance index to various drugs was calculated by dividing the 50% growth inhibitory concentration (IC₅₀) of the test cell line by IC₅₀ of K562, which was the negative control in the antibody experiment. MRK16-F(ab')₂ reacted with four cell lines, K562/ADM, KYO-1, HEL and CMK, which had relative resistance index values of 2 or more to vincristine (VCR), vindesine, vinblastine, ADM, daunorubicin, mitoxantrone (MIT), etoposide (VP-16) and actinomycin-D (ACT-D). The level of resistance to VCR and ADM in these cell lines decreased significantly in the presence of 10 μ M verapamil in vitro . Significant expression of mRNA of P-gp gene was also detected in K562/ADM, KYO-1 and HEL. MRK16-F(ab')₂ did not react with 36 other cell lines. Among them, three cell lines, PL-21, P31/FUJ and KOPM-28, had relative resistance index values of 2 or more to anthracyclines, MIT and VP-16, but not to vinca alkaloids or ACT-D. The level of ADM-resistance in these cell lines did not decrease significantly in the presence of 10 μ M verapamil. Five cell lines, ATL-1K, HL-60, KMOE-2, ML-1 and U266, had relative resistance index values of 2 or more to some of the drugs, but not to the others, and 19 other cell lines did not. These results indicate that the reactivity of MRK16-F(ab')₂ correlates with a relative resistance index of 2 or more to all these drugs in cultured human leukemia and lymphoma cell lines.     

A Novel Multidrug Resistance in Cultured Leukemia and Lymphoma Cells Detected by a Monoclonal Antibody to 85-kDa Protein, MRK20

A monoclonal antibody, MRK20, in F(ab')₂ form [MRK20-F(ab')₂], which reacts with 85-kDa membrane protein in a doxorubicin (ADM)-resistant subline (K562/ADM) of human myelogenous leukemia cell line, K562, was examined for reactivity with 41 cultured human leukemia and lymphoma cell lines. None of these cell lines had ever been exposed to any anticancer agent in vitro except K562/ ADM. The relative resistance index to various drugs was calculated by dividing the 50% growthinhibitory concentration (IC₅₀) of the test cell line by IC₅₀ of K562 (the negative control in the antibody experiment). MRK20-F(ab')₂ reacted with seven cell lines, KYO-1 derived from chronic myelogenous leukemia in blastic crisis (CMLbc), CMK from acute megakaryoblastic leukemia, HEL from erythroleukemia, P31/FUJ from acute monocytic leukemia, KOPM-28 from CMLbc, PL-21 from acute promyelocytic leukemia and K562/ADM, MRK20-F(ab')₂ did not react with 34 other cell lines. All seven MRK20-F(ab')₂-positive cell lines had relative resistance index values of 2 or more to anthracyclines (ADM, pyrarubicin, daunorubicin), mitoxantrone, etoposide, bleomycin, and pepleomycin. There was no distinct correlation between the reactivity to MRK20-F(ab')₂ and a higher relative resistance index than 2 to vinca alkaloids, actinomycin-D, cisplatin, 4-hydroperoxycyclophos-phamide, nimustine hydrochloride, methotrexate or cytarabine. These results indicate that MRK20-F(ab')₂ detects a novel multidrug resistance to anthracyclines, mitoxantrone, etoposide, bleomycin and pepleomycin in cultured human leukemia and lymphoma cells.     

Long-lasting Accumulation of Vinblastine in Inostamycin-treated Multidrug-resistant KB Cells

Inostamycin, a novel polyether compound, reverses multidrug resistance in KB cells. The mechanism of its action was studied by use of radioactively labeled vinblastine. Inostamycin dose-dependently increased the accumulation of [³H] vinblastine in multidrug-resistant KB-C4 cells at 0.5-2 μg/ml, while it did not enhance accumulation in the drug-sensitive KB-3-1 cells. At a concentration of 1 μ/ ml inostamycin inhibited active [³H] vinblastine efflux from KB-C4 cells, but not from KB-3-1 cells, and inhibited [³H] vinblastine binding to KB-C4 membranes with an IC_5O of 0.94 μg/ml (1.3 μ M ). Furthermore, [³H] vinblastine accumulated by treatment with 1 /μg/ml of inostamycin was resistant to efflux from KB-C4 cells, even after the removal of inostamycin.     

Anti-ganglioside GM2 Monoclonal Antibody-dependent Killing of Human Lung Cancer Cells by Lymphocytes and Monocytes

Ganglioside GM₂ (GM₂) frequently appears on the cell surface of human cancers of neuroendocrine origin. A mouse-human chimeric monoclonal antibody (mAb), KM966, against GM₂ was previously found to promote the lysis of various cancer cells by human blood mononnclear cells (MNC). In this study, we analyzed the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against small cell lung cancer (SCLC) cells and examined the enhancing effect of various cytokines on the ADCC activity. The ADCC activity was assessed by 4-h ⁵¹Cr release assay. Highly purified lymphocytes (>99%) and monocytes (>90%) were separated by centrifugal elutriation from peripheral blood MNC of the same healthy donor. KM966 induced lysis of SCLC cells mediated by both lymphocytes and monocytes to similar extents, in a dose-dependent manner. Pretreatment of lymphocytes with various cytokines [interleukin (IL)-2, IL-12 and interferon-γ] and that of monocytes with macrophage-colony-stimulating factor significantly augmented the killer activity against SCLC cells in the presence of KM966 mAb. KM966 was also effective for the lysis of non-small cell lung cancer cells in direct proportion to the GM₂ expression levels. These findings suggest that combined treatment of KM966 mAb with cytokines may be therapeutically useful for in vivo killing of lung cancer cells expressing GM₂ through the ADCC reaction.     

Expression Behavior of 85-kDa Membrane Protein in Adriamycin-resistant Tumor Cells and the Inhibition of Human Tumor Growth in Athymic Mice by MRK-20 Monoclonal Antibody against the Protein

Treatment of tumors with monoclonal antibodies against tumor antigen is one of the selective modalities for cancer therapy. We examined the therapeutic effect of MRK-20 (IgG₁), a murine monoclonal antibody against resistance-associated 85-kDa membrane protein. The 85-kDa protein is expressed on the surface of multidrug-resistant cells induced by adriamycin. This protein is also expressed in some multiple-drug-resistant cells, including atypical multidrug-resistant cells. The protein, once lost during long-term culture without adriamycin, was rapidly induced by treatment with adriamycin but not with vinblastine or etoposide, suggesting a close relationship of the protein with adriamycin resistance but not with multidrug resistance. The antibody MRK-20 suppressed the growth of subcutaneously implanted tumors expressing the 85-kDa protein. Adriamycin-resistant human ovarian tumor 2780AD and innately resistant human erythroleukemia HEL cells in athymic mice were completely cured by treatment with MRK-20 antibody when the antibody was administered i.v. 2 days after s.c. tumor implantation. On the other hand, MRK-20 did not show any effect on the growth of the 85-kDa protein-negative A2780 human ovarian tumor. These results indicate that the effect of MRK-20 is highly specific to cells expressing 85-kDa protein.     

Diversity of Cellular Molecules in Human Cells Detected by Monoclonal Antibodies Reactive with c-myc Proteins Produced in Escherichia coli

Six clones of monoclonal antibodies, MYC-1 to -6, were prepared by using two kinds of truncated c- myc proteins, p23 and p42, produced in Escherichia coli as immunogens. Analysis with enzyme-linked immunosorbent assays and immunoblotting assays with peptides produced in Escherichia coli showed that 5 clones of monoclonal antibodies, MYC-1 to -4 and -6, were reactive with c- myc protein encoded by exon 2. The remaining one clone, MYC-5, was reactive with the portion of c- myc protein encoded by exon 3. All monoclonal antibodies were also reactive with phosphorylated c- myc protein produced by insect cells infected by the baculovirus expression vector with the human c- myc gene. With immunoblotting assays using cellular lysates, MYC-1 and -3 detected bands at the levels of 58 kDa and 60 kDa, MYC-5 detected a band at 56 kDa and MYC-6 detected bands at 68 kDa and 75 kDa. All of these bands were detectable in nuclear extracts of HL-60 and Colo320, both of which have amplified c- myc genes, and also the extract of RmycYl which is the c- myc gene transfectant into 3Y1 rat cells. None of them was detectable in peripheral blood mononuclear cells and 3Y1, both of which lacked activated c- myc genes. This indicates that these nuclear proteins are either c- myc gene products or molecules closely related to the c- myc gene. The remaining two clones, MYC-2 and -4, detected a band at the level of 85 kDa in cytoplasmic extracts of all the above-mentioned cells independent of the presence of the c-myc gene. This suggests that 85 kDa protein might be irrelevant to the c- myc gene. The 56 kDa protein was detectable by MYC-5 in phytohemagglutinin-stimulated peripheral blood mononuclear cells as well as leukemic cells of some patients.     

抗乳腺癌单克隆抗体的制备及其应用

目的：制备抗人乳腺癌单克隆抗体（McAb），用于临床肿瘤的免疫病理诊断与分型研究，为药物导向诊断和治疗奠定基础。方法：以乳腺癌细胞系ZR75免疫Balb/c小鼠，取其脾细胞与小鼠骨髓瘤细胞SP2／0进行融合，经选择培养、筛选及克隆化，获得恒定分泌抗ZR76单克隆抗体的杂交瘤细胞系BM109。结果：单抗BM109的Ig亚类为IgG1，BM109抗原主要表达在靶细胞膜上，为分子量37KD的蛋白质抗原。在乳腺癌的免疫反应率为86．7％（26／30），与胃癌、食管癌、卵巢癌等6种肿瘤的免疫反应率为9．5％（4／42），而对乳腺纤维瘤、囊性增生症和周围正常乳腺组织未见阳性反应。结论：所获一株抗乳腺癌单克隆抗体，具有较好的特异性，可望进一步用于乳腺癌的诊断、治疗及其发生发展的研究。     

Correlation of Gene-specific Damage with Cisplatin between Human Adenocarcinoma Cells and Peripheral Blood Mononuclear Cells Analyzed by Polymerase Chain Reaction-stop Assay

We investigated gene-specific damage in adenocarcinoma cells, obtained from pleural effusions of 9 primary lung cancer patients, induced by incubation with cisplation for 3 h in vitro . The 2.7 kb fragment of the hypoxanthine phosphoribosyltransferase (HPRT) gene was amplified by the polymerase chain reaction (PCR) to quantify the DNA damage. A 7-fold difference in the extent of gene-specific damage among the patients was observed. Mononuclear cells (MNC) were obtained from freshly isolated blood from the same patients before they received chemotherapy. These cells were also incubated with cisplatin in vitro , and PCR amplification of the HPRT gene was carried out. A 4-fold variation of DNA damage among the patients was observed. Moreover, there was a linear correlation between the extents of the DNA damage in the tumor cells and MNCs (R²=0.676, P =0.0016). These results suggest that the PCR-stop assay could be used to detect interindividual variations in the extent of gene-specific damage in both tumor cells and MNC from the same patients induced by cisplatin treatment. In conclusion, MNC could be used to analyze cisplatin-induced gene-specific damage in cancer patients whose tumor cells are inaccessible.     

Immunohistochemistry of Human Gastrointestinal Cancer-associated Antigen Detected by Monoclonal Antibody PA 8-15

A murine monoclonal antibody (MAb), PA 8-15, was produced against a newly established human pancreatic adenocarcinoma cell line, SUIT-2. With the avidin-biotin immunoperoxidase technique, PA 8-15 MAb reacted strongly with 27 of 28 formalin-fixed paraffin-embedded pancreatic adenocarcinoma tissues. All six gall bladder carcinomas and all nine biliary tract carcinomas also showed positive reactions. In addition, PA 8-15 MAb reacted with gastric carcinomas (6/10), colorectal carcinomas (7/11), and some other primary adenocarcinomas. The distribution of PA 8-15 antigen on the same tissues of pancreatic cancer was different from those of CA 19-9 and DU-PAN-2 antigens and CEA. PA 8-15 MAb stained only the epithelium of the pancreatic duct, gall bladder and bile duct in normal adult tissues, and some normal fetal glandular epithelial cells. However, PA 8-15 MAb was not reactive with inflammatory or benign tumors of the digestive system except for the epithelium, as was seen in normal adults. Reactivity of PA 8-15 MAb with tissue specimens largely disappeared after treatment with neuraminidase, while oxidation with periodate or trypsin digestion did not alter the staining intensity, indicating that antigenic determinants may be at least partly of sialylated carbohydrate nature. These results suggest that PA 8-15 MAb detects a new oncofetal antigen in gastrointestinal cancers, especially of the pancreatico-biliary tract.     

Production and Characterization of a Human Monoclonal Antibody Recognizing a New Antigen Expressed on Some Lymphoid Cells

A cell line secreting a human monoclonal antibody was established by Epstein-Barr virus transforming B cells derived from an enlarged cervical lymph node excised from a patient bearing a carotid body tumor. The reactivity of the monoclonal antibody, designated as mNISP, was tested on various cells and cell lines. An antigen defined by the mNISP was expressed on some Burkitt's lymphoma cell lines and on a non-T non-B acute lymphoblastic leukemia cell line. Furthermore, this antigen was expressed on leukemic cells from 2 of 8 patients with chronic myelocytic leukemia, 2 of 10 patients with acute myeloblastic leukemia, one of 13 patients with acute lymphoblastic leukemia, and two patients with adult T cell leukemia, but it was not expressed on normal T, B and adherent (macrophage) cells. In addition, mNISP reacted with T cells obtained from human T-cell leukemia virus type I carriers. We found that the antigen defined by mNISP was distinct from any previously reported antigen in terms of its pattern of cellular expression and molecular weight, suggesting that mNISP recognizes a new antigen expressed on some lymphoid cells.     

Antibody-dependent Cytotoxicity Mediated by Chimeric Monoclonal Antibody Nd2 and Experimental Immunotherapy for Pancreatic Cancer

In a previous study, mouse monoclonal antibody (MoAb) Nd2 (m-Nd2, mouse IgGl) labeled with ¹³¹ I exhibited efficacy in in vivo radioimmunotherapy against pancreatic cancer. In this study we prepared mouse/human chimeric antibody Nd2 (c-Nd2, human IgG1) for clinical use and examined whether c-Nd2 induced antibody-dependent cell-mediated cytotoxicity (ADCC). Cytotoxicity to pancreatic cancer (PC) cell lines, including Nd2 antigen-positive (SW1990, RWP-1, Capan-1) and Nd2 antigen-negative (Panc-1, MiaPaca-2, Capan-2) lines, was evaluated by mixed human leukocyte and tumor cell culture (MLTC) at an effector cell to target cell (E/T) ratio of 50 with or without Nd2. Cytotoxicities to SW1990 with no antibody, m-Nd2 and c-Nd2 (1 μg/ml) were 26.7%, 38.0% and 55%, respectively; to RWP-1, 28%, 41% and 70%; to Capan-1, 26%, 30% and 52%; to Panc-1, 24%, 28% and 30%; to MiaPaca-2, 18%, 20% and 27% and to Capan-2, 29.7%, 35.0% and 40.6%. Cytotoxic capacity during MLTC with c-Nd2 was significantly higher than during MLTC with m-Nd2 or with no antibody. These findings indicated that cytotoxicity to Nd2-positive PC cells during MLTC is induced by ADCC. Intraperitoneal injection of c-Nd2 inhibited the tumor growth of SW1990 xenografted subcutaneously in nude mice and prolonged the survival of nude mice in which SW1990 tumor was transplanted orthotopically at the tail of the pancreas. These findings suggested that, because of its ability to induce ADCC, c-Nd2 may be clinically useful for the immunotherapeutic treatment of pancreatic cancer.     

Establishment of a Human Monoclonal Antibody to Hanganutziu-Deicher Antigen as a Tumor-associated Carbohydrate Antigen

We have established a human-human hybridoma producing a monoclonal antibody against a tumor-associated carbohydrate-specific antigen, Hanganutziu-Deicher (HD) antigen. Human spleen lymphocytes from a patient with esophageal varices complicated with liver cirrhosis were cultured in serum-free medium and co-stimulated with both anti-human μ-chain antibodies and supernatants of concanavalin A-stimulated human spleen cell culture (ConA sup). The activated lymphocytes were subsequently primed in vitro with particulate HD3 antigen and fused with a parent hybrid myeloma cell line, KR-12. A hybridoma, 1F43E31G7 produced anti-HD human monoclonal antibody (IgM λ). This monoclonal antibody reacted strongly with N-glycolyl-neuraminyl α2-3 lactosylceramide (HD3) and slightly with N-glycolylneuraminyl α2-3 lactoneotetraosylceramide (HD3), but did not react with N-glycolylneuraminyl α2-3 lactoneohexaosylceramide (HD7), N-acetylneuraminyl α2-3 lactosylceramide (GM3) and other derivatives of HD3 prepared by chemical modification of the sialic acid residue of HD3, which indicates that the monoclonal antibody is directed precisely toward the terminal sialic acid and whole structure of HD3.     

Synergistic Effect of Cyclosporin A and Verapamil in Overcoming Vincristine Resistance of Multidrug-resistant Cultured Human Leukemia Cells

Reversal of vincristine (VCR) resistance by cyclosporin A (CyA) or the combination of CyA and verapamil (VER) was investigated by using four P-glycoprotein (P-gp)-associated human multidrug-resistant (MDR) cell lines (K562/ADM, KYO-1, HEL and CMK). Drug sensitivity was expressed as 50% inhibitory concentration (IC₅₀). The degree of reversal of resistance was expressed as x -fold decrease by dividing the IC₅₀ value without modifier(s) by that with modifier(s). CyA overcame P-gp-associated MDR significantly in all four MDR cell lines. Reversal of VCR resistance by CyA appeared to be dose-dependent. In the case of low-grade MDR cell lines (KYO-1, HEL and CMK), CyA at the low concentration of 0.5 μg/ml was still effective. The degree of reversal of VCR resistance in this condition was greater (6.3- to 16-fold decrease) in the low-grade MDR cell lines than in a high-grade MDR cell line (K562/ADM) (2.9-fold decrease). At a high concentration (5 μg/ml) of CyA, however, it was greater (240-fold decrease) in the high-grade MDR cell lines than in the low-grade MDR cell line (20- to 100-fold decrease). This indicates that concentration of CyA required for overcoming drug resistance in MDR cells was dependent on the degree of drug resistance. CyA overcame VCR resistance more efficiently than VER. The combination of CyA and VER enhanced reversal of VCR resistance in a supra-additive or at least an additive manner and overcame VCR resistance at low concentrations of both modifiers that are clinically achievable with safety.     

Characterization of OM-B Monoclonal Antibody-defined Antigen Associated with Mucinous Type Human Ovarian Tumor

Partial characterization of the OM-B antigen associated with mucinous-type ovarian tumors was conducted. This antigen was defined by OM-B monoclonal antibody, which was raised against a mucinous-type ovarian tumor, and was present in all the mucinous-type tumors tested, but only a fraction of serous-type tumors. The OM-B crude antigen preparation fractionated from cystic fluids had a density of 1.40–1.43 g/ml, with a high neutral sugar content. Molecular mass ( M r) estimated by gel filtration was more than 2,000,000. Trypsinization of the antigen preparation under appropriate conditions resulted in two major bands and one minor band with molecular sizes of less than M r 250,000, as detected by immunoblotting. Immunoaffinity chromatography was then conducted and the amino acid composition of the purified product was determined; the high contents of serine, threonine and proline are characteristic of a mucin. Binding inhibition enzyme-linked immunosorbent assay was developed to measure OM-B antigen activity in cystic fluids and sera from patients with mucinous-type tumors. The antigen was easily detected in most cystic fluids, but not in sera, suggesting that improvement in the sensitivity of this assay is necessary before its utilization for serum diagnosis will be feasible.     

Effector Cell Analysis of Human Multidrug-resistant Cell Killing by Mouse-Human Chimeric Antibody against P-Glycoprotein

A mouse-human chimeric monoclonal antibody (mAb), MH162, against P-glycoprotein was previously found to be more effective than an all-mouse mAb (MRK16) in lysis of multidrug-resistant (MDR) tumor cells by blood mononuclear cells. The present study was performed to identify the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against MDR cells. The ADCC reaction was assessed by a 6-h ⁵¹Cr release assay. Highly purified lymphocytes (>99%), monocytes (>99%) and neutrophils (>96%) were obtained from peripheral blood of the same healthy donors. A comparison of these three effector cell populations showed no difference between MH162 and its all-murine counterpart MRK16 in MDR cell lysis by monocytes or neutrophils. But MH162 was more effective than MRK16 in lymphocyte-mediated lysis of the MDR cells. The lymphocytes responsible for this ADCC had CD16+ Fc receptors. Pretreatment of monocytes with colony-stimulating factors (IL-3, GM-CSF and M-CSF) caused significant increase in their MH162-mediated lysis of MDR cells. Another anti-P-glycoprotein chimeric mAb (MH171) was also more effective than its murine counterpart MRK17 in lymphocyte-mediated lysis of MDR cells. These findings suggest that mouse-human chimeric mAbs may be useful therapeutically for in vivo destruction of MDR cancer cells by the ADCC reaction.     

杂色曲霉素对人外周血单个核细胞TAP1及LM P2基因表达的影响

目的:探讨杂色曲霉素(ST)对体外培养的人外周血单个核细胞(HPBMc)抗原加工递呈相关基因TAP1、LMP2表达的影响.方法:采用RT-PCR和FCM方法,分析5种浓度ST处理对体外培养的HPBMc TAP1、LMP2基因在mRNA和蛋白水平表达的影响.结果:RT-PCR检测结果表明,ST处理24h后,低浓度ST组(0.125mg/L、0.25mg/L)HPBMc TAP1 mRNA相对表达量明显高于对照组,而较高浓度组(0.5mg/L、1mg/L和2mg/L)TAP1 mRNA则明显低于对照组,尤以1mg/L和2mg/L组为著.FCM定量分析表明,低浓度ST组HPBMc TAP1蛋白平均表达量略低于对照组,但无统计学意义,而较高浓度组TAP1表达则明显低于对照组(P＜0.05).在0.125mg/L到1mg/L浓度范围内,各ST处理组HPBMc LMP2蛋白表达量和mRNA的相对表达量均高于对照组.结论:ST对体外培养的HPBMc TAP1mRNA表达和蛋白表达的影响按ST剂量不同而变化,在0.125～1mg/L浓度范围内ST在mRNA及蛋白水平均可剂量依赖地提高体外培养的HPBMc LMP2的表达.