Association between dietary folate intake and clinical outcome in head and neck squamous cell carcinoma.

BACKGROUND: The association between dietary folate intake, two polymorphisms in methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TYMS), and survival in head and neck squamous cell carcinoma (HNSCC) patients is not clarified. PATIENTS AND METHODS: We conducted a retrospective cohort study of 437 HNSCC patients treated at Aichi Cancer Center. We evaluated the survival impact of pretreatment dietary folate intake, which was estimated using a food-frequency questionnaire, and two polymorphisms, MTHFR C677T and a 6-bp insertion/deletion in the 3'-untranslated region of TYMS, using multivariate proportional hazard models. RESULTS: Patients with high folate intake (≥320 µg/day; n=144) had significantly higher survival than patients with low or medium folate intake (<320 µg/day; n=278; 79.1% versus 68.2%, respectively, P=0.020). This association was consistent with multivariate analyses adjusted for established prognostic factors (hazard ratio 0.56; 95% confidence interval 0.37-0.84). MTHFR and TYMS polymorphisms did not show significant association with survival, although the TYMS 6-bp insertion allele showed potential association with a reduced risk of death. Notably, no significant interaction was observed between folate intake and the two examined polymorphisms. CONCLUSIONS: High pretreatment dietary folate intake was identified as an independent prognostic factor associated with improved clinical outcomes in HNSCC patients. Further study is warranted.

The mechanism for the activation of latent TGF-beta during co-culture of endothelial cells and smooth muscle cells: cell-type specific targeting of latent TGF-beta to smooth muscle cells.

Transforming growth factor-beta (TGF-beta) is secreted in a latent form and activated during co-culture of endothelial cells and smooth muscle cells. Plasmin located on the surface of endothelial cells is required for the activation of latent TGF-beta (LTGF-beta) during co-culture, and the targeting of LTGF-beta to the cellular surface is requisite for its activation. In the present study, the cellular targeting of LTGF-beta was examined. We detected the specific binding of 125I-large LTGF-beta 1 isolated from human platelets to smooth muscle cells but not to endothelial cells. A mAb against the latency-associated peptide (LAP) of large LTGF-beta 1 complex, which blocked the binding of 125I-large LTGF-beta 1 to smooth muscle cells, inhibited the activation of LTGF-beta during co-culture. The binding of 125I-large LTGF-beta 1 could not be competed either by mannose-6-phosphate (300 microM) or by the synthetic peptide Arg-Gly-Asp-Ser (300 micrograms/ml). These results indicate that the targeting of LTGF-beta to smooth muscle cells is required for the activation of LTGF-beta during co-culture of endothelial cells and smooth muscle cells. The targeting of LTGF-beta to smooth muscle cells is mediated by LAP, and the domain of LAP responsible for the targeting to smooth muscle cells may not be related to mannose-6-phosphate or an Arg-Gly-Asp sequence, both of which have been previously proposed as candidates for the cellular binding domains within LAP.

Distribution of lung adenocarcinoma-associated antigens in human tissues and sera defined by monoclonal antibodies KM-52 and KM-93.

Two monoclonal antibodies to human lung adenocarcinoma, KM-52 and KM-93, were generated by the novel immunizing procedure using mice rendered tolerant to the normal human lung (N. Hanai et al., Cancer Res., 46: 4438-4443, 1986). KM-93 recognized sialylated carbohydrate epitope on the antigen different from CA19-9 and DU-PAN-2, while KM-52 recognized the protein antigen. Both antigens were different from carcinoembryonic antigen, alpha-fetoprotein, and beta 2-microglobulin. Distribution of KA-52 and KA-93, the antigens recognized by KM-52 and KM-93, respectively, in various tissues and sera was investigated. In immunoperoxidase staining, KM-93 reacted strongly and frequently with tumor cells of lung adenocarcinoma and partially with those of lung squamous cell carcinoma, large cell carcinoma, and small cell carcinoma. In normal adult and fetal tissues, KA-93 was expressed on the surface of a small number of cells of the lung, pancreas, liver, kidney, and bone marrow. KM-52 reacted selectively with tumor cells of adenocarcinoma among four different histological types of lung carcinoma. In normal adult and fetal tissues, KA-52 was distributed on a small number of cells of the lung, stomach, intestine, and pancreas. Of the two monoclonal antibodies, KM-93 could be used in detecting the antigen in sera of patients with lung cancer. The KA-93 level in sera was determined by the sandwich-type enzyme-linked immunosorbent assay. Serum with a high KA-93 level was found in 34 of 70 patients with lung adenocarcinoma (48.6%), one of 67 healthy adults (1.5%), and none of 32 patients with benign diseases (0%). Combined detection by KA-93 with KA-32, a new tumor marker of lung squamous cell carcinoma (N. Hanai et al., Cancer Res., 46: 5206-5210, 1986), elevated the positive percentage in patients with lung squamous cell carcinoma (52.7%) and with lung adenocarcinoma (59.5%). These results suggested that KM-52 and KM-93 would be potential monoclonal antibodies in immunohistological diagnosis and serum diagnosis of lung adenocarcinoma, respectively.

Distribution of a squamous cell lung carcinoma-associated antigen, KA-32, in human tissues and sera defined by monoclonal antibody KM-32.

The distribution of a variant of blood group A antigen recognized by a murine monoclonal antibody, KM-32, generated against human squamous cell lung carcinoma was investigated in various tissues and sera. By immunoperoxidase staining, the antibody was found to react with a number of lung carcinoma tissues of squamous cell carcinoma, adenocarcinoma, and small cell carcinoma, and several other tumor tissues. Positive staining was also observed in a small number of cells of some normal tissues, such as bronchiolar epithelium, gastrointestinal glands, and convoluted tubules of the kidney. The antibody could also be used in detecting macromolecular antigens, designated KA-32, in sera of patients with lung cancer. The antigen level in serum was determined by an inhibition assay using purified KM-32. The higher level of inhibition was seen in sera from over half of patients with lung cancer and patients with benign diseases when compared with those in sera from healthy adults. Purification of the antigen in serum was performed by gel filtration chromatography, immunoaffinity chromatography, and polyacrylamide gradient gel electrophoresis. Purified antigen exhibited a glycoprotein nature, and its molecular weight was estimated at more than 500,000.

Generation of monoclonal antibodies against human lung squamous cell carcinoma and adenocarcinoma using mice rendered tolerant to normal human lung.

Four murine monoclonal antibodies against human lung carcinoma were generated using a novel immunization procedure. BALB/c mice were rendered neonatally tolerant to normal human lung tissues and subsequently immunized with human lung tumor tissues. The lower level of antibody-reactivity to the tolerogen was seen in the sera of mice rendered neonatally tolerant as compared with the level of reactivity in the sera of nontolerant mice. The mice which maintained a sufficiently tolerant state were selected for hybridoma production. Two monoclonal antibodies, KM-32 and KM-34, were developed from mice immunized with lung squamous cell carcinoma tissues and two other monoclonal antibodies, KM-52 and KM-93, were developed from mice immunized with lung adenocarcinoma tissues. Distribution of antigens detected by the monoclonal antibodies were investigated by enzyme-linked immunosorbent assay using membrane fractions prepared from a number of tumorous and normal tissues and various human normal and tumor cell lines. KM-32 recognized a carbohydrate antigen expressed predominantly on lung squamous cell carcinoma cells and its carbohydrate structure appeared to be associated with blood group A antigen. KM-93 recognized a sialylated carbohydrate epitope on the antigen expressed on lung adeno-carcinoma cells and a few other tumor cells. KM-34 and KM-52 detected protein or glycoprotein antigens and they showed predominant reactivity to lung squamous cell carcinoma and lung adenocarcinoma, respectively. KM-32 and KM-34 antibodies showed complement-dependent cytotoxicity against a lung tumor cell line. These results suggest that the tolerance technique is useful for efficient screening of murine monoclonal antibodies specific to human tumors. The efficacy of these four monoclonal antibodies in diagnosis and therapy of lung cancer will be the subject of a sequential study.

Association between sialyl Lewis(a) expression and tumor progression in melanoma.

Twenty-three primary and 27 metastatic melanoma lesions and 17 pigmented nevi lesions were tested utilizing the immunoperoxidase reaction with anti-sialyl Lewis(a) (sLea) and anti-sLex mAbs.sLea was expressed in 9, 25, and 5 and sLex was expressed in 6, 11, and 2 of these lesions, respectively. Expression of sLea in melanocytic tumors is associated with tumor progression. Serum levels of sLea and sLex were analyzed by a sandwich assay using mAbs in 25 melanoma patients. Only 2 patients at stage 4 showed higher levels of sLea and sLex than did normal control subjects. Moreover, sLea and sLex were expressed in 1 and 2 of 5 human melanoma cell lines, respectively, and expression of sLex and sLex was not modulated by cytokines. These findings suggest that the expression of sLea in melanocytic tumors is correlated with disease progression.

Chimeric anti-ganglioside GM2 antibody with antitumor activity.

Ganglioside GM2, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin, has been focused on as a target molecule for passive immunotherapy. GM2 is thought to be one of the T-cell-independent antigens and to elicit only IgM antibody responses in rodents and humans. We have previously established two murine anti-GM2 monoclonal antibodies with high specificity and strong binding activity, KM696 and KM697, both of which are of the IgM class. Variable heavy and light chain complementary DNAs of these two murine monoclonal antibodies were cloned and used in the construction of mouse/human IgG1 chimeric antibodies, KM966 and KM967, respectively, in this study. One of the chimeric antibodies, KM966, retained strong and specific reactivity with GM2 and showed the similarity of the binding activity with tumor cell lines to that of the original murine monoclonal antibody. Indirect immunofluorescence staining of tumor cell lines with the chimeric KM966 revealed that the antigen was expressed in substantial amounts on pulmonary tumor cells and leukemia cells as well as neuroectodermal origin tumor cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, respectively, chimeric KM966 was fully effective in killing GM2-expressing tumor cells. In addition, i.v. injection of chimeric KM966 markedly suppressed the establishment of human tumor xenografts in nude mice. Taken together, chimeric KM966 is the first antibody of the human IgG class to ganglioside GM2 and has strong antitumor activity both in vitro and in vivo. It is likely that chimeric KM966 will be a useful agent for passive immunotherapy of human cancer.

High frequency of fibroblast growth factor (FGF) 8 expression in clinical prostate cancers and breast tissues, immunohistochemically demonstrated by a newly established neutralizing monoclonal antibody against FGF 8.

Fibroblast growth factor (FGF) 8, also known as androgen-induced growth factor, was originally isolated from an androgen-dependent mouse mammary Shionogi carcinoma SC-3 cell line, in which it was shown to have androgen-regulated properties. We previously demonstrated that Fgf 8 transcripts were detected in several human prostate and breast cancer cell lines and that recombinant FGF 8 was mitogenic to an androgen-sensitive prostate cancer LNCaP cell line. In this study, to characterize the roles of FGF 8 in clinical hormone-responsive cancers, we established a monoclonal antibody against FGF 8. In Western blots, this antibody specifically interacted with a FGF 8b isoform that was identical between mouse and human but was not identical to other murine 8a and 8c isoforms. In a cell growth assay using SC-3 cells, the newly established anti-FGF 8 antibody blocked androgen- and FGF 8-stimulated growth but not basic FGF-stimulated growth. Immunohistochemical analyses by use of the established anti-FGF 8 antibody demonstrated that FGF 8 was frequently expressed in human prostate cancers, appearing in 40 of 43 cases (93%), whereas both prostatic hyperplasia specimens and normal prostate tissues included in biopsy specimens were negative for FGF 8 expression. On the other hand, FGF 8 was detected in normal ductal and lobular epithelial cells in breast tissues. FGF 8 was also frequently expressed in various breast diseases, including fibroadenomas (5 of 5 cases, 100%), intraductal papillomas (3 of 3 cases, 100%), ductal hyperplasias (3 of 6 cases, 50%), and breast cancers (8 of 12 cases, 67%). Androgen receptors were also immunohistochemically detected in FGF 8-positive prostate cancers (40 of 40 cases, 100%) and FGF 8-positive breast diseases (17 of 19 cases, 89%). These findings strongly suggest that FGF 8 is involved in hormone-related tumorigenesis of the prostate and breast.

Apoptosis induction of human lung cancer cell line in multicellular heterospheroids with humanized antiganglioside GM2 monoclonal antibody.

The chimeric antiganglioside GM2 monoclonal antibody (MAb) KM966, which showed high effector functions such as complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), potently suppressed growth and metastases of GM2-positive human cancer cells inoculated into mice. To further improve the therapeutic efficacy of the anti-GM2 MAb in humans, we constructed a humanized anti-GM2 MAb, KM8969. The humanized KM8969 was more efficient in supporting ADCC against GM2-positive human cancer cell lines than the chimeric KM966, whereas complement-dependent cytotoxicity was slightly reduced in the humanized KM8969. In addition, the humanized KM8969 was shown to exert potent ADCC mediated by both lymphocytes and monocytes. To investigate the effect of the humanized KM8969 on the biological function of GM2 in the condition physiologically mimicking formation and growth of cancer masses, the heterospheroids composed of normal human dermal fibroblasts and GM2-positive human lung cancer cells were developed. Interestingly, the humanized KM8969 gave rise to growth inhibition of heterospheroids without dependence of the effector functions. Morphological and immunocytochemical analysis suggested that the inhibitory effect was due to the apoptosis of GM2-positive cancer cells in the heterospheroids. The result indicates that GM2 captured by the antibody on the cell surface loses its physiological function that plays a critical role in maintaining the three-dimensional growth of cancer cells in contact with its own cells or other type of cells in a microenvironment. The humanized KM8969, which can destroy the cancer cells via blocking functional GM2 on the cell surface as well as the effector functions, would have extraordinary potential in human cancer therapy.

Specific localization, gamma camera imaging, and intracellular trafficking of radiolabelled chimeric anti-G(D3) ganglioside monoclonal antibody KM871 in SK-MEL-28 melanoma xenografts.

The chimeric monoclonal antibody KM871, directed against the G(D3) antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with (125)I via tyrosine residues and with (111)In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A"-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with (111)In or (125)I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G(D3)-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed. (111)In-labeled CHX-A"-DTPA-KM871 showed a maximum tumor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of (111)In-labeled CHX-A"-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G(D3) monoclonal antibody to G(D3)-expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.

Lymph node density predicts lung metastases in oral squamous cell carcinoma.

The association between lymph node density and survival free of lung metastases in oral squamous cell carcinoma (SCC), has not been investigated so far to our knowledge. Lymph node density ≧ 0.07 has been reported by a multicentre international study to be a significant predictor of shorter survival in patients with oral SCC who have invaded nodes. We investigated whether a lymph node density of ≧ 0.07 correlates with shorter overall survival, survival free of distant metastases, and survival free of lung metastases, in patients with oral SCC and invaded lymph nodes. Thirty-five patients with histologically-confirmed invaded lymph nodes werestudied. Their density was calculated as the ratio of the number of invaded lymph nodes:total number of nodes. A density of ≧ 0.07 correlated significantly with shorter overall survival (p<0.02), survival free of distant metastases (p<0.01), and survival free of lung metastases (p<0.01) on log rank testing. On testing by Cox's proportional hazards model of multivariate survival analysis with adjustment for the pathological stage (pstage IV/pstage III), and invaded surgical margins or extracapsular spread, or both, we found that lymph node density ≧ 0.07 was associated with significantly shorter survival (p<0.02). We conclude that lymph node density predicts lung metastases in patients with oral SCC.

Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues.

We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat. To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied. cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli. Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end. The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies. Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity. They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability. Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition. The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis. There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

Specific targeting, biodistribution, and lack of immunogenicity of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma: results of a phase I trial.

PURPOSE: KM871 is a chimeric monoclonal antibody against the ganglioside antigen GD3, which is highly expressed on melanoma cells. We conducted an open-label, dose escalation phase I trial of KM871 in patients with metastatic melanoma. PATIENTS AND METHODS: Seventeen patients were entered onto one of five dose levels (1, 5, 10, 20, and 40 mg/m2). Patients received three infusions of KM871 at 2-week intervals, with the first infusion of KM871 trace-labeled with indium-111 (111In) to enable assessment of biodistribution in vivo. Biopsies of metastatic melanoma sites were performed on days 7 to 10. RESULTS: Fifteen of 17 patients completed a cycle of three infusions of KM871. No dose-limiting toxicity was observed during the trial; the maximum-tolerated dose was therefore not reached. Three patients (at the 1-, 5-, and 40-mg/m2 dose levels) developed pain and/or erythema at tumor sites consistent with an inflammatory response. No normal tissue uptake of 111In-KM871 was observed, and tumor uptake of 111In-KM871 was observed in all lesions greater than 1.5 cm (tumor biopsy 111KM871 uptake results: range, 0.001% to 0.026% injected dose/g). The ratio of maximum tumor to normal tissue was 15:1. Pharmacokinetic analysis revealed a 111In-KM871 terminal half-life of 7.68 +/- 2.94 days. One patient had a clinical partial response that lasted 11 months. There was no serologic evidence of human antichimeric antibody in any patient, including one patient who received 16 infusions over a 12-month period. CONCLUSION: This study is the first to demonstrate the biodistribution and specific targeting of an anti-GD3 antibody to metastatic melanoma in patients. The long half-life and lack of immunogenicity of KM871 makes this antibody an attractive potential therapy for patients with metastatic melanoma.

Osteoclast-like cells express receptor activity modifying protein 2: application of laser capture microdissection.

Receptor activity modifying proteins (RAMPs) act as receptor modulators that determine the ligand specificity of receptors for the calcitonin (CT) family. The purpose of this study was to analyze the expression of RAMPs in osteoclast-like cells using the laser capture microdissection (LCM) technique. Mouse bone marrow and spleen cells were co-cultured on a film designed for LCM. After 10 days, 250 osteoclast-like cells were captured using the LCM system. Total RNA from these cells was used to synthesize cDNA and RT-PCR analysis was performed. Osteoclast-like cells expressed CT receptor (CTR), CT receptor-like receptor (CRLR) and RAMP2, but did not express RAMP1 or RAMP3. These results indicated (1) that a pure population of osteoclast-like cells can be prepared by LCM and gene expression of this population can be analyzed by RT-PCR and (2) that RT-PCR shows that osteoclast-like cells express RAMP2, CTR and CRLR, suggesting the potential for adrenomedullin binding to osteoclast-like cells. This is the first report that osteoclast-like cells express RAMP2.

Dissection and optimization of immune effector functions of humanized anti-ganglioside GM2 monoclonal antibody.

A mouse/human chimeric monoclonal antibody (MAb) KM966, specific for the cell-surface tumor antigen ganglioside GM2, was humanized by the complementarity determining regions (CDRs) grafting method. Not only the amino acid residues in the CDRs but also several in the framework regions (FRs) were changed from the human to the murine residues. A humanized variant, huKM796H/Lm-28, containing eight and five amino acid alterations in variable light (VL) and variable heavy (VH) FRs, respectively, showed a 9-fold reduction in complement-dependent cytotoxicity (CDC) compared to the chimeric KM966, despite tight antigen binding and potent antibody-dependent cellular cytotoxicity (ADCC). Several additional variants were subsequently constructed to improve the CDC of the antibody. One of the variants, designated KM8969, which differs by three amino acids, exhibited a CDC within 3-fold of the chimeric KM966. In addition, humanized KM8969 bound GM2 antigen 1.25-fold more tightly than the chimeric KM966 and showed 5-fold higher ADCC than the chimeric KM966. These results clearly show that the humanized KM8969, having the optimized immune effector functions and theoretically minimal immunogenicity, is an ideal candidate to test the effectiveness of anti-GM2 MAb in human cancer therapy. Taken together, the results obtained here indicate that the ADCC and CDC of an antibody can be dissected independently via engineering of the antibody variable region.

Transducin-mediated, isoform-specific interaction of recombinant rat nucleoside diphosphate kinases with bleached bovine retinal rod outer segment membranes.

The properties of the binding of recombinant rat nucleoside diphosphate (NDP) kinase isoforms alpha and beta (NDP kinase alpha and beta, respectively) to bleached bovine retinal rod outer segment (ROS) membranes were investigated. It was found that: (1) both NDP kinase isoforms interacted with ROS membranes in a pH-, cation- and GTPgammaS-dependent manner; (2) the retinal G-protein transducin was an obligatory factor for the interaction; (3) the apparent affinity of NDP kinase alpha for ROS membranes was about 100-fold higher than that of NDP kinase beta; and (4) an alpha-isoform-specific peptide, corresponding to the sequence of the N-terminal third (variable region), had the ability to displace bovine NDP kinase from ROS membranes. The results suggest the possible involvement of NDP kinases in cellular regulation via interaction with G-proteins and provide a structural basis for the possible differential roles of mammalian NDP kinase isoforms in the cell.

Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5-trisphosphate receptor: application to the studies on inflammatory cells.

Monoclonal antibodies were raised that specifically recognize the COOH-terminal sequences and the loop sequences between the fifth and the sixth transmembrane spanning regions of human inositol 1,4,5-trisphosphate receptor (IP3R) type 1, 2 and 3. Western blot analysis using Jurkat cells, mouse cerebellum, COS-7 expressing IP3R type 3 cDNA showed that those monoclonal antibodies reacted specifically with each of these three IP3R subtypes and that they do not cross-react. These antibodies could be used for the specific immunoprecipitation of IP3Rs. Using these monoclonal antibodies, the expression profiles of IP3R-subtype proteins were found to be different among inflammatory cells such as macrophages, polymorphonuclear cells, mast cells, eosinophils, splenocytes, thymocytes and megakaryocytic cells. Usually, more than one type of IP3R were expressed in a cell simultaneously. The observation of CMK cells under immunofluorescence confocal microscopy revealed that IP3R type 1 and type 2 are located at different subcellular fractions.

A new vector for the high level expression of chimeric antibodies in myeloma cells.

We previously reported the expression of a mouse/human chimeric anti-ganglioside GD3 antibody, KM871 (IgG1,kappa) in mouse myeloma SP2/0 cells under the control of the ecotropic Moloney virus long terminal repeat by the co-transfection of chimeric heavy (H) and light (L) chain vectors (Shitara et al. (1993) Cancer Immunol. Immunother.). To establish an efficient and high level expression system for the chimeric antibody, we did comparative study on vector systems and host cells. An improved expression vector, named 'a tandem vector, pChi641HLGM4' was constructed, in which both of the chimeric H and L chain gene transcription units and a dihydrofolate reductase (dhfr) gene transcription unit were inserted. When two kinds of mouse myeloma cell lines, SP2/0 and P3U1, were used as host cells, frequency of the incidence of antibody-producing transfectants was markedly increased by the use of the tandem vector compared with the use of the mixture of each chimeric H vector and L chain vector. To select out appropriate host cells, transfection frequency and antibody production level were compared among SP2/0, P3U1 and rat myeloma YB2/0 cells by transfection of the tandem vector. YB2/0 cell was shown to have the highest potential in both the transfection frequency and the antibody production. Introduction of the tandem vector into YB2/0 cells and the subsequent amplification with 50-200 nM methotrexate gave rise to several clones that stably secreted 70-100 micrograms/10(6) cells per 24 h of the chimeric antibody. This productivity of the antibody is one of the highest levels which have been achieved by other investigators using transfected myeloma cells. Using this system it took only 2-3 months to establish the transfectant clones which stably produced the chimeric antibody.

Immunoglobulin class switch of anti-ganglioside monoclonal antibody from IgM to IgG.

Ganglioside GM2, which is one of the major gangliosides expressed on cell surface of neuroectodermal-origin human tumors, has been focused on as a target molecule for passive immunotherapy. One of the problems in this area was that monoclonal antibodies (mAbs) raised against GM2 were of IgM class even if donors of B cells were varied in mouse, rat or human. We stimulated two kinds of mice hybridomas having membrane-bound anti-GM2 IgM on their surface with GM2 incorporated in synthetic liposomes in the presence of the mouse thymocytes to accelerate the class switch of immunoglobulins (Igs). After the stimulation, protein A-reactive clones were sorted out using a cell sorter. We finally isolated two class switch variants generating mouse IgG3, designated KM796 and KM750, from original hybridomas producing mouse IgM anti-GM2 mAbs, KM696 and KM697, respectively after over 20-time repetitions of the sorting. ELISA with 11 common gangliosides revealed that one of the variant, KM750, retained the same binding specificity to N-acetyl GM2 as that of the parental IgM, KM697. By ELISA using panel of anti-idiotype (Id) mAbs to anti-GM2 mAbs, KM750 was shown to retain the parental KM697 Id. Another variant KM796 almost lost its activity in purification process in acidic condition and changes in Id were suggested. In immunofluorescence assay, KM750 was confirmed to bind to GM2-expressing tumor cell lines. The class switch hybridoma has been stably cultured with the production of the IgG3-class mAb for more than 22 months.