The E3 ubiquitin ligase NEDD4 negatively regulates HER3/ErbB3 level and signaling.

HER3/ErbB3, a member of the epidermal growth factor receptor (EGFR) family, has a pivotal role in cancer and is emerging as a therapeutic antibody target. In this study, we identified NEDD4 (neural precursor cell expressed, developmentally downregulated 4) as a novel interaction partner and ubiquitin E3 ligase of human HER3. Using molecular and biochemical approaches, we demonstrated that the C-terminal tail of HER3 interacted with the WW domains of NEDD4 and the interaction was independent of neuregulin-1. Short hairpin RNA knockdown of NEDD4 elevated HER3 levels and resulted in increased HER3 signaling and cancer cell proliferation in vitro and in vivo. A similar inverse relationship between HER3 and NEDD4 levels was observed in prostate cancer tumor tissues. More importantly, the upregulated HER3 expression by NEDD4 knockdown sensitized cancer cells for growth inhibition by an anti-HER3 antibody. Taken together, our results suggest that low NEDD4 levels may predict activation of HER3 signaling and efficacies of anti-HER3 antibody therapies.

Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis.

This report describes the isolation and partial purification of novel triterpenoid saponins [Fraction 35 (F035)] and two pure biologically active derivatives (termed avicins D and G) from Acacia victoriae, an Australian desert tree of the Leguminosae family. F035 and the avicins markedly inhibited the growth of several tumor cell lines with minimum growth inhibition in human foreskin fibroblasts, mouse fibroblasts, and immortalized breast epithelial cells at similar concentrations. F035 and the avicins induced cell cycle (G1) arrest of the human MDA-MB-453 breast cancer cell line and apoptosis of the Jurkat (T-cell leukemia) and the MDA-MB-435 breast cancer cell line. The triterpenoid saponins also partially inhibited phosphatidylinositol 3-kinase activity in Jurkat T cells in a time-dependent manner and phosphorylation in the downstream protein Akt, whereas no affect was seen on the Ras/mitogen-activated protein kinase cascade. These observations as well as other work from our laboratory demonstrating mitochondrial perturbation, chemoprevention, and inhibition of nuclear factor kappaB suggest that triterpenoid saponins from A. victoriae have potential as novel anticancer agents. Recent work linking Akt signaling with glucose metabolism, stress resistance, and longevity suggests other potential applications of these compounds.

Avicins: triterpenoid saponins from Acacia victoriae (Bentham) induce apoptosis by mitochondrial perturbation.

Anticancer agents target various subcellular components and trigger apoptosis in chemosensitive cells. We have recently reported the tumor cell growth inhibitory properties of a mixture of triterpenoid saponins obtained from an Australian desert tree (Leguminosae) Acacia victoriae (Bentham). Here we report the purification of this mixture into two biologically pure components called avicins that contain an acacic acid core with two acyclic monoterpene units connected by a quinovose sugar. We demonstrate that the mixture of triterpenoid saponins and avicins induce apoptosis in the Jurkat human T cell line by affecting the mitochondrial function. Avicin G induced cytochrome c release within 30-120 min in whole cells and within a minute in the cell-free system. Caspase inhibitors DEVD or zVAD-fmk had no effect on cytochrome c release, suggesting the direct action of avicin G on the mitochondria. Activation of caspase-3 and total cleavage of poly(ADP-ribose) polymerase (PARP) occurred between 2 and 6 h posttreatment with avicins by zVAD-fmk. Interestingly, in the treated cells no significant changes in the membrane potential preceded or accompanied cytochrome c release. A small decrease in the generation of reactive oxygen species (ROS) was measured. The study of these evolutionarily ancient compounds may represent an interesting paradigm for the application of chemical ecology and chemical biology to human health.

Emergence of cisplatin-resistant cells from the OVCAR-3 ovarian carcinoma cell line with p53 mutations, altered tumorigenicity, and increased apoptotic sensitivity to p53 gene replacement.

Resistance to chemotherapy commonly compromises the treatment of many advanced cancers. Evidence suggests a correlation between chemoresistance and more aggressive tumor growth, possibly through accumulation of additional genetic defects in drug-treated or resistant cells. To study this process in a human ovarian cancer model, we examined OVCAR-3 cells for acute sensitivity to cisplatin (cDDP) and subsequent emergence of drug-resistant clones following chronic cDDP exposure. Clonal cells (OVCAR-3/C-1) that displayed 20-fold reduced sensitivity to cisplatin but retained equivalent sensitivity to paclitaxel, as compared with the parental population, were isolated. The cDDP-resistant clone had growth kinetics similar to those of parental population, but when transplanted into the peritoneal cavity of nude mice, they acquired the ability to grow with the development of both ascites and solid tumor masses; such growth was not detectable after transplantation of the drug-sensitive parental cell line. C-1 cells had a p53 gene mutation (codon 266) that was not detected in the parental OVCAR-3 cell line, and infection of C-1 cells with p53-adenovirus (rAd-p53) caused greater apoptosis and gene transduction than that observed in the similarly infected parental population. rAd-p53 induced high levels of p21WAF1, p27Kip1, activated caspase 3 and apoptosis in C-1 cells, without causing major changes in bax or bcl-XL levels. Together, the results suggest that alterations in tumor growth and gene mutations characterize cDDP-resistance in OVCAR-3 cells, and viral replacement of one of these defective genes (p53) may provide an effective treatment for elimination of drug-resistant cells.

Phase Ia/Ib trial of anti-GD2 chimeric monoclonal antibody 14.18 (ch14.18) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in metastatic melanoma.

We performed a phase Ia/Ib trial of chimeric anti-GD2 monoclonal antibody 14.18 (ch14.18) in combination with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to determine the maximum tolerated dose as well as immunologic and biologic responses to the regimen. Sixteen patients with metastatic malignant melanoma received escalating doses of ch14.18 (15-60 mg/m2) administered intravenously for 4 h on day 1. Twenty-four hours later, subcutaneous injections of rhGM-CSF were administered daily for a total of 14 days. Significant side effects were related to ch14.18 infusion and consisted of moderate to severe abdominal and/or extremity pain, blood pressure changes, headache, nausea, diarrhea, peripheral nerve dysesthesias, myalgias, and weakness. Dose-limiting toxicity was observed at 60 mg/m2 and consisted of severe hypertension, hypotension, and atrial fibrillation in one patient each, respectively. Significant increases in white blood cell count, granulocyte count, eosinophil count, and monocyte count occurred after rhGM-CSF treatment. Significant enhancement of in vitro and in vivo monocyte and neutrophil tumoricidal activity and antibody-dependent cellular cytotoxicity along with significant elevations in C-reactive protein and neopterin were observed. Despite these immunological and biological changes, no antitumor activity was seen. In short, the combination of ch14.18 and rhGM-CSF resulted in toxicity similar to that observed with ch14.18 alone without improvement in tumor response.

Adenoviral-mediated p53 tumor suppressor gene therapy of human ovarian carcinoma.

Mutations of p53 gene are reported in 50-60% of human cancers and reintroduction of wild-type p53 can suppress cell proliferation. In this study, replication deficient recombinant adenovirus encoding wild-type p53 (ACN53) under the control of the human cytomegalovirus (CMV) promoter was constructed. A specific incorporation of the p53 gene with ACN53 reduced 3 (deleted p53 gene) cells was observed. ACN53 reduced the colony-forming ability of SK-OV-3 cells 72-216 h after single infection. A highly aggressive ovarian xenograft model was established in which animals die between 25-45 days. A localization study with the adenovirus-containing beta galactosidase reporter gene showed effective gene transfer in the tumor tissues. Ex vivo treatment of SK-OV-3 cells with ACN53 followed by injection into nude mice, increased the survival of the p53 treated mice by more than 50% compared with control animals. Gene therapy with ACN53 in intraperitoneal model of SK-OV-3 cells in two independent experiments revealed that there were some long-term survivors in the group of mice [2/5 (66 and 120 days) and [2/8 (166 and 423 days)] treated with ACN53. These findings demonstrate the potential of the p53 tumor suppressor gene therapy in human ovarian carcinoma.

Cellular resistance to the antimelanoma immunotoxin ZME-gelonin and strategies to target resistant cells.

The development of cellular resistance to immunotoxins has been demonstrated in a variety of models and can involve a number of mechanisms. For the present study, an immunotoxin was utilized composed of an anti-melanoma antibody ZME-018 recognizing a 240-kDa surface glycoprotein (gp 240) and the plant toxin gelonin. Human melanoma cells (A375-M) were grown in the presence of increasing amounts of ZME-gelonin and a clonal variant (A-375-ZR) was developed that was 100-fold resistant to ZME-gelonin compared to parental cells. Scatchard analysis showed that the A375-M parental cells had 260 X 10(3) ZME-gelonin-binding sites/cell with relatively low affinity (5 nM). In contrast, resistant A375-ZR cells demonstrated a reduced number of low-affinity sites (160 x 10(3)/cel1), but showed a small number (47 x 10(3)) of higher-affinity sites (0.8 nM). Internalization rates and degradation rates of 125I-labeled ZME-gelonin were identical in both the parental and resistant cells. A375-ZR cells were found to be more resistant to vincristine and doxorubicin than were parental cells. Both cell lines were almost equally sensitive to native gelonin, 5-fluorouracil (5-FU), cisplatin. melphalan, carmustine, interferon gamma (IFNgamma) and IFNalpha. In addition. both cell lines were equally sensitive to another gelonin antibody conjugate that binds to cell-surface, GD2 (antibody 14G2A). However, resistant cells were twice as sensitive to the cytotoxic effects of etoposide than were parental cells. Finally, a variety of agents were tested in combination with ZME-gelonin against A375-ZR cells in an attempt to identify agents to augment immunotoxin cytotoxic effects against resistant cells. The agents 5-FU, cisplatin, IFNgamma, IFNalpha, and etoposide were the most effective in augmenting the cytotoxicity of ZME-gelonin against resistant cells. These studies suggest that development of resistance to one immunotoxin does not cause development of cross-resistance to other gelonin immunotoxins. Further, specific biological response modifiers and chemotherapeutic agents may be effective in augmenting the effectiveness of immunotoxins and specifically targeting or reducing the emergence of immunotoxin-resistant cells.

An antimelanoma immunotoxin containing recombinant human tumor necrosis factor: tissue disposition, pharmacokinetic, and therapeutic studies in xenograft models.

The ability of monoclonal antibody conjugates to re-direct plant or bacterial toxins, chemotherapeutic agents and radionuclides to selected target cells has been well-documented. Recombinant human tumor necrosis factor (TNF) is a macrophage-derived, non-glycosylated (17 kDa) peptide with a broad range of biological and immunological effects including antiviral activity, cytotoxic and cytostatic effects. A conjugate of the antimelanoma antibody ZME-018 and TNF in previous studies has shown melanoma-selective cytotoxic effects in vitro. Pharmacokinetic studies of the ZME-TNF immunotoxin showed that the agent cleared from plasma biphasically with alpha- and beta-phase half-lives similar to that of ZME itself (72 min and 36 h compared to 84 min and 41 h respectively). In contrast, TNF itself was cleared rapidly from plasma with a terminal-phase half-life of only 2.7 h. The clearance rate of ZME-TNF from plasma (Clp) was almost tenfold more rapid than for ZME (1.1 versus 0.16 ml/kg x min) but was threefold slower than the clearance for TNF itself (3.4 ml/kg x min). Tissue distribution studies in nude mice bearing human melanoma xenografts showed similar tumor localization of the immunotoxin compared to the free antibody and slightly higher concentrations in liver and kidney compared to ZME itself. Treatment of nude mice bearing well-developed A375 tumors with the immunotoxin resulted in a statistically significant (P < 0.002) suppression in tumor growth rate (fivefold increase) compared to saline-treated controls, which increased 20-fold over the same period. These studies demonstrate the feasibility of this approach and suggest that TNF may represent a non-antigenic alternative to immunotoxins containing plant and bacterial toxins.

Pharmacokinetics, tissue distribution, and in vivo antitumor effects of the antimelanoma immunotoxin ZME-gelonin.

Antibody ZME-018 is directed against the gp240 glycoprotein on the surface of more than 80% of human melanoma cell lines and fresh biopsy specimens. Previous studies in our laboratory described the in vitro cytotoxicity and specificity of an immunoconjugate composed of mAb ZME-018 and the plant toxin gelonin. The present study described the in vivo pharmacokinetics and therapeutic effects of ZME-gelonin in human xenograft/nude mouse models. Pharmacokinetic studies of 125I-labeled ZME-018 and ZME-gelonin demonstrated a shorter terminal-phase plasma half-life of the immunoconjugate than native ZME (20.6 h compared to 41.3 h). The initial volume of distribution of the ZME-gelonin was also higher compared to that of ZME alone (2.85 ml compared to 1.94 ml) suggesting an enhanced distribution of the conjugate outside the vasculature. The corresponding area under the concentration/time curve for the ZME-gelonin conjugate was 40% lower than that of ZME alone (80.8 compared to 139.6 microCi.ml-1 x min). In nude mice bearing well-developed human tumor A375 melanoma xenografts, administration of 125I-labeled ZME and ZME-gelonin resulted in tumor-to-blood ratios of 1.9 +/- 0.5 and 1.5 +/- 0.6 respectively by 72 h. Compared with ZME, ZME-gelonin conjugate caused an increase in the content of radiolabel in kidney, spleen and liver. Treatment of nude mice bearing well-developed (150 mm3) s.c. A375-M xenografts with divided doses of ZME-gelonin, ZME, gelonin, or saline resulted in suppression of tumor growth in the immunotoxin group but virtually no retardation of tumor growth in the control groups. Using a murine model for a rapidly growing lethal metastatic human melanoma, treatment with ZME-gelonin resulted in a mean survival of 44 days, 213% increase in mean survival time compared with the saline treatment (14.2 +/- 2 day survival). Given these encouraging results, we are proceeding with further preclinical development of this immunotoxin.

Phase I trial of murine monoclonal antibody 14G2a administered by prolonged intravenous infusion in patients with neuroectodermal tumors.

PURPOSE: The purpose of this phase I trial was to determine the toxicity and maximum-tolerated dose (MTD) of murine monoclonal antibody (Mab) 14G2a (anti-GD2) in cancer patients. PATIENTS AND METHODS: Following tracer doses of iodine-131-labeled 14G2a to determine tumor uptake, 18 patients with refractory melanoma, neuroblastoma, or osteosarcoma received unlabeled 14G2a at total concentrations of 50, 100, and 200 mg/m2 administered as daily 24-hour infusions for 5 days. RESULTS: The overall sensitivity of external immunoscintigraphy was 64 of 74 known metastases (86%). Toxicity from prolonged infusion of 14G2a consisted of severe generalized pain, hyponatremia, fever, rash, paresthesias, weakness, and chronic refractory postural hypotension (two patients). Toxicity was less severe in pediatric patients. The MTD of Mab was 100 mg/m2. Sixteen of 18 patients developed human antimouse antibodies (HAMA) to 14G2a. Terminal-phase half-life (T1/2) of unlabeled Mab was 6.6 +/- 1.8 hours for patients receiving 50 mg/m2 and 39.5 +/- 13.3 hours at the 100-mg/m2 level. Tumor biopsies from six melanoma patients were positive for GD2 antigen, but only two of six had trace amounts of 14G2a present. Three mixed responses (two melanoma, one osteosarcoma) and two partial responses (PRs; neuroblastoma) were observed. CONCLUSION: Mab 14G2a has modest antitumor activity at the expense of significant toxicity. Dose-limiting neurologic sequelae may significantly limit phase II studies other than in pediatric patients with neuroblastoma.

Tumor necrosis factor alpha and gamma-interferon enhancement of anti-epidermal growth factor receptor monoclonal antibody binding to human melanoma cells.

Previous studies have demonstrated that the expression of tumor-associated antigens can be regulated by cytokines. The purpose of this study was to determine whether tumor necrosis factor alpha (TNF alpha) and gamma-interferon (IFN gamma) were capable of modulating epidermal growth factor receptor (EGFr) immunorecognition on a human melanoma cell line in vitro. DX-3 melanoma cells treated for 24-72 h with various concentrations of each cytokine were incubated with an anti-EGFr monoclonal antibody (Mab) (A108) that recognizes an extracellular domain of the receptor, and differences in binding were analyzed by flow cytometry and radioimmunoassay. A dose- and time-dependent enhancement in EGFr immunorecognition was measurable in TNF alpha- and IFN gamma-treated cells. Combinations of these cytokines enhanced the recognition of EGFr on DX-3 cells to a level greater than that achieved with either TNF alpha or IFN gamma alone. Scatchard analysis of receptor binding curves revealed that there was no significant change in Mab affinity between control and cytokine-treated DX-3 melanoma cells, whereas a 1.5- to 1.8-fold enhancement in the number of Mab binding sites was measurable in TNF alpha- and IFN gamma-treated cells, respectively, when compared with controls. Immune complex kinase assay of EGFr showed threefold higher tyrosine kinase activity in TNF alpha-treated cells, but no change in kinase activity was observed following IFN gamma treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Augmented binding of radiolabeled monoclonal antibodies to melanoma cells using specific antibody combinations.

Antigenic heterogeneity may limit effective cancer therapy using monoclonal antibodies (Mabs). To address this problem, combinations of two, three, or four 125I-labeled antimelanoma Mabs (NRML-05, P94, 96.5, and CL207) were incubated in vitro with three different melanoma cell lines (HS294t, A375SM, and DX3). Binding of the various Mab combinations was expressed as total cpm/10(5) cells and was compared to binding of each Mab alone. Saturating amounts (10 micrograms/ml) of two, three, or four Mabs bound to a significantly greater extent (P less than 0.05) than each individual Mab except for NRML-05. Combinations of three Mabs at a nonsaturating concentration (1.5 micrograms/ml) bound to a greater extent than single Mabs (P less than 0.05), depending on the cell line examined and the amount of antigen sites present for each Mab. Saturating or nonsaturating concentrations of unlabeled Mab 96.5 combined with 125I-labeled NRML-05 enhanced binding of the latter to HS294t by significantly modifying its affinity and by increasing the number of binding sites 3-fold. Modulation occurred only at 37 degrees C and was dependent upon protein synthesis. These data demonstrate that the effectiveness of various Mab combinations over single Mabs varies, depending on Mab concentration and the cell lines used. In addition, one Mab may significantly (P less than 0.05) enhance binding of another Mab to its antigen.

Variables influencing tumor uptake of anti-melanoma monoclonal antibodies radioiodinated using para-iodobenzoyl (PIB) conjugate.

Tumor uptake was examined with respect to antigen expression, time-dependent biodistribution, dose of Mab injected, tumor size, and tumor site (i.e., subcutaneous versus lung or liver metastases). NR-ML-05, 96.5, and P94 showed significantly greater uptake in subcutaneous tumors than CL207 and 5.1 (p less than 0.05). NR-ML-05 had a significantly higher tumor uptake at 24 hr (11.9 +/- 0.51) than at 72 hr (4.0 +/- 0.37) or 144 hr (2.7 +/- 0.84) after injection (p less than 0.001). The other four Mabs had similar tumor distribution at all three time points. The tumor uptake of four Mabs (96.5, P94, CL207. 5.1) differed with respect to in vitro versus in vivo binding to tumor, tumor type, dose of Mab, and tumor site (subcutaneous versus metastases). In contrast, NR-ML-05 demonstrated consistent uptake in tumors independent of the above parameters. These data suggest that certain host parameters can influence in vivo tumor targeting depending on characteristics of each Mab studied.

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2.

Monoclonal antibody 14G2a (anti-GD2) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD2. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagent N-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor alpha and interferon alpha and chemotherapeutic agents such as cisplatinum and N,N'-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.

Recombinant alpha-interferon enhances tumor targeting of an antimelanoma monoclonal antibody in vivo.

To determine whether recombinant human alpha-interferon (rIFN alpha A) could enhance tumor uptake of an antimelanoma monoclonal antibody (Mab) 96.5 in vivo, groups of nude mice bearing P97 antigen-positive human melanoma subcutaneous xenografts were given i.m. injections of normal saline or rIFN alpha A daily for 10 days. On day 7, mice received either 5 micrograms of 111In-labeled Mab 96.5 or irrelevant 111In-labeled subclass-matched or non-subclass-matched control Mabs. Animals were killed 72 h later and the percent injected dose per gram (%ID/g) in tumor and normal organs was determined. There was a significant (p less than 0.001) increase in 96.5 in tumors of IFN-treated mice compared to saline-treated mice and mice receiving irrelevant Mabs. There was also a significantly increased uptake of 96.5 in blood, heart, lung, kidney, and muscle of IFN-treated vs. control mice (p less than 0.05). This finding was most likely due to increased antigen shedding since significant differences in %ID/g were not observed between IFN-treated and control mice bearing antigen-negative tumors. Furthermore, P97 content in tumor and tissues of IFN-treated mice bearing melanoma xenografts was significantly higher than in mice without tumors. In summary, IFN enhanced targeting of 96.5 via an antigen-specific mechanism. These data confirm and extend previous studies in other tumor systems, and suggest that clinical trials of Mabs plus IFN might be useful in overcoming poor Mab localization that occurs as a result of antigenic heterogeneity in humans.

Functional properties and effect on growth suppression of human neuroblastoma tumors by isotype switch variants of monoclonal antiganglioside GD2 antibody 14.18.

A complete family of IgG isotype switch variant hybridomas was generated from the anti-GD2 monoclonal IgG3-producing hybridoma, 14.18, with the aid of the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a monoclonal antibodies (Mabs) produced by respective isotype switch variant hybridomas 14G1, 14G2b, or 14G2a, have binding activities for the biochemically defined GD2 antigen and GD2-expressing neuroblastoma target cell lines identical to that of IgG3 Mabs produced by the 14.18 parent cell line. This permitted us to examine the relative in vitro and in vivo cytotoxic capacities of each of the anti-GD2 antibodies for GD2-expressing neuroblastoma cells independent of antibody binding affinity or specificity. Mabs produced by 14.18, 14G2a, or 14G2b, but not 14G1, can direct efficient complement-dependent cytotoxicity against neuroblastoma tumor cells in the presence of human complement. Mabs produced by the parent 14.18 or by 14G2a are more efficient in directing antibody-dependent cell-mediated cytotoxicity than Mabs produced by 14G2b, and Mabs of 14G1 are inactive. However, despite these noted in vitro differences, antibodies produced by each member of this switch variant family suppress the growth of human neuroblastoma tumor cells in BALB/c athymic nu/nu mice. These studies suggest that a mechanism(s) other than Fc-directed complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity may account for the in vivo antitumor effects of these particular antibodies.

A human brain glycoprotein related to the mouse cell adhesion molecule L1.

We have employed monoclonal antibody 5G3, an antibody used to label human tumor cells of neural origin (Mujoo, K., Spiro, R.C., and Reisfeld, R. A. (1986) J. Biol. Chem. 261, 10299-10305), to isolate and characterize a large glycoprotein from normal adult human brain. This protein was compared to mouse L1 (Rathjen, F. G., and Schachner, M. (1984) EMBO J. 3, 1-10), a neural cell surface glycoprotein implicated predominantly in neurite-neurite interactions. On the basis of the following results the 5G3 antigen is considered to be the human homologue of mouse L1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both proteins share similar molecular masses of their carbohydrate-depleted or undepleted components. In tryptic fingerprint analyses of the iodinated L1 and 5G3 components, 65% of the resolved peptides comigrated. Comparison of NH2-terminal amino acid sequences revealed a high degree of homology between human 5G3 and mouse L1, with 11 of 15 residues being identical. Furthermore, polyclonal antibodies to human 5G3 antigen were found to be cross-reactive with mouse L1 antigen and vice versa. All components of 5G3 and L1 antigens show considerable charge heterogeneity with partial overlapping of regions in isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These findings provide a basis for studying the role of the human L1 homologue in human diseases.

Disialoganglioside GD2 on human neuroblastoma cells: target antigen for monoclonal antibody-mediated cytolysis and suppression of tumor growth.

A murine monoclonal antibody 14.18 specifically recognizes disialoganglioside GD2, the major ganglioside expressed on the surface of human neuroblastoma cells. This monoclonal antibody (Mab) is of immunoglobulin G3 isotype, has an affinity constant (KA) of 3.5 X 10(8) M-1, and reacts preferentially with tumor cells and fresh frozen tumor tissues of neuroectodermal origin in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Mab 14.18 effectively lyses a number of human neuroblastoma cell lines by two distinct mechanisms, i.e., antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. There is a good correlation between the average number of antibody-binding sites per neuroblastoma cell and the amount of cell lysis observed in complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. In addition, Mab 14.18 suppresses establishment as well as growth of progressively growing, established human neuroblastoma tumors in nude mice when injected 24 h and 9 days, respectively, after the initial s.c. inoculation of tumor cells. These data suggest that Mab 14.18 can mediate tumor cell killing in vivo and in vitro and may thereby prove useful for immunotherapy of human neuroblastoma.

Biosynthesis and expression of the disialoganglioside GD2, a relevant target antigen on small cell lung carcinoma for monoclonal antibody-mediated cytolysis.

Monoclonal antibodies (MAbs) 126 (immunoglobulin M) and 14.18 (immunoglobulin G3) react strongly with the cell surface of small cell carcinoma of the lungs (SCCL) and are unreactive with most normal tissues and other neoplasms with the notable exception of tumors derived from cells of neural crest origin. These MAbs react specifically with the oligosaccharide portion of the disialoganglioside GD2. Analysis of total gangliosides from cultured cell lines derived from SCCL indicates that GD2 is a predominant ganglioside. A comparison of the reactivities of MAbs against GD2 with those directed against gangliosides GM2 and GD3, each differing from GD2 by a single sugar residue, clearly indicates that GD2 is preferentially expressed by cultured cells derived from SCCL. Membranes isolated from these cells exhibit GD2 synthetase activity which specifically converts the precursor GD3 to GD2 in the presence of uridine diphosphate-N-acetyl galactosamine as the glycosyl donor. We present evidence that in SCCL, GD2 serves as a relevant target antigen for monoclonal antibody-mediated cytolysis. Specifically, we demonstrate that MAb 14.18 (immunoglobulin G3), can lyse small cell carcinoma of the lung targets by either complement- or antibody-dependent cellular cytotoxicity.

Characterization of a unique glycoprotein antigen expressed on the surface of human neuroblastoma cells.

In order to develop a molecular probe to delineate chemical and biological characteristics of human neuroblastoma cells, a murine monoclonal antibody (Mab 5G3) was produced that is directed to a glycoprotein, preferentially expressed on the surface of such cells. This antibody is of IgG2a isotype, has an association constant of 8 X 10(9) M-1, and reacts preferentially with human neuroblastoma cell lines and fresh frozen tissue sections in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Minimal reactivity is observed with a variety of lymphoblastoid cell lines and normal fetal and adult tissues. Mab 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa that is derived from a 200-kDa precursor, as evident from pulse-chase biosynthetic studies. Treatment with tunicamycin revealed that both molecules contain N-asparagine-linked oligosaccharides; however, only the 215-kDa species is resistant to treatment with endo-beta-N-acetylglucosaminidase H and sensitive to neuraminidase, indicating that it contains trimmed and terminally sialylated oligosaccharides of the "complex" type. In contrast, the 200-kDa precursor is sensitive to endo-beta-N-acetylglucosaminidase H and resistant to neuraminidase treatment indicating that it contains high-mannose non-processed oligosaccharides. The 215-kDa molecule is sulfated, phosphorylated at serine residues, and expressed on the cell surface. A molecule of 200 kDa is detected by Mab 5G3 in spent culture medium of human neuroblastoma cells which is neither sulfated nor phosphorylated.