ganglioside--gd2 and Melanoma

Based on the remodeling of glycosphingolipids on the human tumor cell lines with manipulation of glycosyltransferase genes, roles of sugar moieties in tumor-associated carbohydrate antigens have been analyzed. Two main topics, that is, the roles of ganglioside GD3 in human malignant melanomas and those of GD2 in small cell lung cancer (SCLC) were reported. GD3 enhances tyrosine phosphorylation of two adaptor molecules, p130Cas and paxillin, resulting in the increased cell growth and invasion in melanoma cells. GD2 also enhances the proliferation and invasion of SCLC cells. GD2 also mediates apoptosis with anti-GD2 monoclonal antibodies (mAbs) via dephosphorylation of the focal adhesion kinase. These approaches have promoted further understanding of mechanisms by which gangliosides modulate malignant properties of human cancer, and the results obtained here propose novel targets for cancer therapy.

Topics: Anoikis; Antibodies, Monoclonal; Carcinoma, Small Cell; Cell Line, Tumor; Cell Proliferation; Crk-Associated Substrate Protein; Focal Adhesion Protein-Tyrosine Kinases; Gangliosides; Glycosphingolipids; Humans; Lung Neoplasms; Melanoma; Neoplasm Invasiveness; Paxillin; Phosphorylation; Tyrosine

Anti-idiotype (Id) vaccine therapy has been tested and shown to be effective, in several animal models, for triggering the immune system to induce specific and protective immunity against bacterial, viral and parasitic infections. The administration of anti-Id antibodies as surrogate tumor-associated antigens (TAA) also represents another potential application of the concept of the Id network. Limited experience in human trials using anti-Id to stimulate immunity against tumors has shown promising results. In this "counter-point" article, we discuss our own findings showing the potential of anti-Id antibody vaccines to be novel therapeutic approaches to various human cancers and also discuss where anti-Id vaccines may perform better than traditional multiple-epitope antigen vaccines.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Breast Neoplasms; Cancer Vaccines; Carcinoembryonic Antigen; Clinical Trials as Topic; Colorectal Neoplasms; Epitopes; Evaluation Studies as Topic; Gangliosides; Glycolipids; Glycoproteins; Humans; Immunization; Leukemia-Lymphoma, Adult T-Cell; Lipid Droplets; Melanoma; Mice; Mice, Inbred C57BL; Models, Immunological; Molecular Mimicry; Neoplasms; Neoplasms, Experimental; Treatment Outcome

Topics: Animals; Antibodies, Monoclonal; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunotherapy; Melanoma; Melanoma, Experimental; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Protein Engineering; Species Specificity; Tissue Distribution; Transplantation, Heterologous

Human melanoma represents one of the most metastatic cancers in man. The capacity of melanoma cells to invade a variety of tissues and extracellular matrices is, in part, due to their repertoire of adhesion receptors. To this end, human melanoma cells express multiple integrin cell adhesion receptors among these is the vitronectin receptor, alpha v beta 3. This adhesion receptor enables melanoma cells to attach to a wide variety of extracellular matrix components containing the sequence Arg-Gly-Asp. This review will focus on the biosynthetic, biochemical and biological properties of this receptor expressed on the surface of human melanoma cells.

Topics: Amino Acid Sequence; Cell Adhesion; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Molecular Sequence Data; Neoplasm Invasiveness; Receptors, Immunologic; Receptors, Vitronectin

Anti-idiotype (Id) vaccine therapy has been tested and shown to be effective, in several animal models, for triggering the immune system to induce specific and protective immunity against bacterial, viral and parasitic infections. The administration of anti-Id antibodies as surrogate tumor-associated antigens (TAA) also represents another potential application of the concept of the Id network. Limited experience in human trials using anti-Id to stimulate immunity against tumors has shown promising results. In this "counter-point" article, we discuss our own findings showing the potential of anti-Id antibody vaccines to be novel therapeutic approaches to various human cancers and also discuss where anti-Id vaccines may perform better than traditional multiple-epitope antigen vaccines.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Breast Neoplasms; Cancer Vaccines; Carcinoembryonic Antigen; Clinical Trials as Topic; Colorectal Neoplasms; Epitopes; Evaluation Studies as Topic; Gangliosides; Glycolipids; Glycoproteins; Humans; Immunization; Leukemia-Lymphoma, Adult T-Cell; Lipid Droplets; Melanoma; Mice; Mice, Inbred C57BL; Models, Immunological; Molecular Mimicry; Neoplasms; Neoplasms, Experimental; Treatment Outcome

Immunization with GMK vaccine (G(M2) ganglioside conjugated to keyhole limpet hemocyanin mixed with QS-21 adjuvant) induces anti-G(M2) antibodies in close to 100% of patients. We found previously that anti-G(D2) antibodies could be induced in some patients using G(D2)-keyhole limpet hemocyanin + QS-21 (GDK). In this trial, we wished: (a) to determine whether immunization with both GMK and GDK vaccines could induce antibodies against both G(M2) and G(D2); and (b) to determine the optimal dose of GDK. Thirty-one patients with melanoma or sarcoma who had no evidence of disease after complete surgical resection were immunized with both GMK (30 microg of G(M2)) and GDK on weeks 1, 2, 3, 4, 12, 24, and 36. Patients were assigned to one of five GDK dose levels (3, 10, 30, 70, or 130 microg of G(D2)). Anti-G(M2) IgM or IgG were induced in 97% of patients. The dose of GDK did not affect the anti-G(M2) response, although at the highest GDK dose level, 3 of 7 patients did not make anti-G(M2) IgG. GDK was less immunogenic; overall 45% of patients developed either IgM or IgG against G(D2). At GDK doses of 30 or 70 microg, 8 of 11 patients (73%) made either IgM or IgG anti-G(D2) antibodies. We conclude that both GMK and GDK vaccines can induce antibodies against G(M2) and G(D2) in a majority of patients and are safe. The optimal dose of GDK appears to be either 30 or 70 microg when administered with GMK vaccine.

Topics: Cancer Vaccines; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; G(M2) Ganglioside; Gangliosides; Hemocyanins; Humans; Immunoblotting; Immunoglobulin G; Immunoglobulin M; Male; Melanoma; Sarcoma; Secondary Prevention; Time Factors

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.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunization, Passive; Immunotherapy, Active; Male; Melanoma; Middle Aged; Recombinant Fusion Proteins; Recombinant Proteins; Skin Neoplasms

The chimeric monoclonal anti-GD2 antibody ch14.18 is made up of the variable region of the murine anti-GD2 antibody 14.18 (or its IgG2a switch variant 14G2a) and the constant region of human IgG1k. Ch14.18 mediates antibody dependent cytotoxicity and complement dependent lysis in vitro. In a phase I trial, 13 patients with metastatic melanoma received ch14.18 as a single dose of 5-100 mg. Therapy was associated with an infusion-related abdominal/pelvic pain syndrome, which required intravenous morphine for control. The pharmacokinetics of ch14.18 best fit a two-compartment model with a T1/2 alpha of 24 +/- 1 hr and a T1/2 beta of 181 +/- 73 hr. Eight of 13 patients developed a weak-modest antibody response directed at the variable region of ch14.18. Clinical antitumor responses were not observed at the doses employed in this study. However, patients receiving greater than 45 mg of ch14.18 had antibody detectable on tumor cells analyzed by fluorescent activated cell sorter. Further modification of the therapeutic regime employing larger doses and frequent administration of ch14.18 are planned.

Topics: Adult; Aged; Antibodies, Monoclonal; Antigens, Neoplasm; Dose-Response Relationship, Drug; Flow Cytometry; Gangliosides; Humans; Infusions, Intravenous; Melanoma; Middle Aged; Time Factors

In a phase I trial, 12 patients with GD2 antigen-positive metastatic melanoma received the murine anti-GD2 monoclonal antibody 14G2a. The monoclonal antibody was administered in four doses over an 8-day period with total dose ranging from 10 to 120 mg. All patients receiving greater than 10 mg of 14G2a experienced transient abdominal/pelvic pain during the antibody infusion. Five patients had a delayed extremity pain syndrome following the third and fourth antibody infusion. Four of the five patients developed neurological toxicity, including two patients with significant although reversible motor neuropathy. Two of the patients developed hyponatremia secondary to a syndrome of inappropriate antidiuretic hormone. All 12 patients developed high levels of human anti-14G2a antibody. The plasma half-life of 14G2a was 42 +/- 6 (SD) h. One patient each had a partial response, mixed response, and stable disease, respectively. The very modest antitumor activity accompanied by dose-limiting neurological toxicity at total doses greater than 80 mg may restrict the clinical utility of murine 14G2a.

Topics: Antibodies, Monoclonal; Antibody Formation; Drug Evaluation; Female; Gangliosides; Humans; Male; Melanoma; Pain; Pain Measurement; Recurrence; Remission Induction

Exosomes (small extracellular vesicles: EVs) have attracted increasing attention from basic scientists and clinicians since they play important roles in cell-to-cell communication in various biological processes. Various features of EVs have been elucidated regarding their contents, generation and secretion mechanisms, and functions in inflammation, regeneration, and cancers. These vesicles are reported to contain proteins, RNAs, microRNAs, DNAs, and lipids. Although the roles of individual components have been rigorously studied, the presence and roles of glycans in EVs have rarely been reported. In particular, glycosphingolipids in EVs have not been investigated to date. In this study, the expression and function of a representative cancer-associated ganglioside, GD2, in malignant melanomas was investigated. Generally, cancer-associated gangliosides have been shown to enhance malignant properties and signals in cancers. Notably, EVs derived from GD2-expressing melanomas enhanced the malignant phenotypes of GD2-negative melanomas, such as cell growth, invasion, and cell adhesion, in a dose-dependent manner. The EVs also induced increased phosphorylation of signaling molecules such as EGF receptor and focal adhesion kinase. These results suggest that EVs released from cancer-associated ganglioside-expressing cells exert many functions that have been reported as a function of these gangliosides and regulate microenvironments, including total aggravation of heterogeneous cancer tissues, leading to more malignant and advanced cancer types.

Topics: Cell Line, Tumor; Extracellular Vesicles; Gangliosides; Humans; Melanoma; Tumor Microenvironment

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2-) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin β1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin β1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin β1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin β1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin β1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2- cells. All these results suggest that GD2 and integrin β1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Collagen Type I; Gangliosides; Humans; Integrin beta1; Integrins; Mass Spectrometry; Melanoma; Membrane Microdomains; Mice; Phenotype; Phosphotyrosine; Signal Transduction

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically 'cold' tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190-230 mm

Topics: Animals; Antibodies; Cancer Vaccines; Cell Line, Tumor; Female; Gangliosides; Immunotherapy; Injections, Intralesional; Interleukin-2; Kinetics; Melanoma; Mice, Inbred C57BL; Neoplasm Transplantation; Recombinant Fusion Proteins; Skin Neoplasms; Soft Tissue Neoplasms; Transplantation, Isogeneic; Tumor Burden; Vaccination

Chimeric antigen receptor (CAR)-engineered T cells have demonstrated promising clinical efficacy in patients with B cell lymphoma. However, the application of CAR-T cell therapy in the treatment of other solid tumors has been limited. We incorporated 4-1BB into the anti-GD2 CAR-T cells to test their cytotoxicity in melanoma in vitro and in vivo. Moreover, we reported the expression of ganglioside GD2 in non-Caucasian melanoma populations for the first time, thus providing a basis for future clinical research.. This study included tumor samples from 288 melanoma patients at the Peking University Cancer Hospital & Institute. Clinical data were collected. Immunohistochemical assays using antibodies against ganglioside GD2 were performed on formalin-fixed, paraffin-embedded specimens. The ability of ganglioside GD2 CAR-T cells to kill ganglioside GD2. Among the 288 samples, 49.3% of cases (142/288) demonstrated positive staining with ganglioside GD2. The median survival time in patients exhibiting ganglioside GD2 expression was significantly shorter than that in patients without ganglioside GD2 expression (31 vs. 47.1 months, P < 0.001). In the present study, CAR was constructed using a GD2-specific scFv (14.G2a), T cell receptor CD3ζ chain, and the CD137 (4-1BB) costimulatory motif. In addition, the GD2.BBζ CAR-T cells demonstrated specific lysis of ganglioside GD2-expressing melanoma cells in vitro. In two PDX models, mice that received intravenous or local intratumor injections of GD2.BBζ CAR-T cells experienced rapid tumor regression.. These data demonstrate that the rate of GD2 expression in Chinese patients is 49.3%. GD2.BBζ CAR-T cells can both efficiently lyse melanoma in a GD2-specific manner and release Th1 cytokines in an antigen-dependent manner in vitro and in vivo. Anti-GD2/4-1BB CAR-T cells represent a clinically appealing treatment strategy for Chinese melanoma patients exhibiting GD2 expression and provide a basis for future studies of the clinical application of immunotherapy for melanoma.

Topics: 4-1BB Ligand; Adult; Aged; Animals; Cell Line; China; Female; Gangliosides; Humans; Immunotherapy, Adoptive; Male; Melanoma; Mice, SCID; Middle Aged; Skin Neoplasms

Conjugation to carrier proteins is a way to improve the immunogenicity of peptides. Such is the case for peptides mimicking carbohydrate tumor-associated antigens in cancer vaccine development. The most used protein for this purpose is the keyhole limpet hemocyanin (KLH) from Megathura crenulata. Its limited bioavailability has prompted interest in finding new candidates; nevertheless, it is not known whether other hemocyanins might be equally efficient as carrier of carbohydrate peptide mimotopes to promotes anti-tumor responses. Here, we evaluated the carrier and antitumor activity of novel hemocyanins with documented immunogenicity obtained from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH), coupled through sulfo-SMCC to P10, a mimetic peptide of GD2, the major ganglioside constituent of neuroectodermal tumors, and incorporating AddaVax as an adjuvant. The humoral immune responses of mice showed that CCH-P10 and FLH-P10 conjugates elicited specific IgM and IgG antibodies against P10 mimotope, similar to those obtained with KLH-P10, which was used as a positive control. The CCH-P10 and FLH-P10 antisera, exhibited cross-reactivity with murine and human melanoma cells, like anti-CCH and anti-FLH sera suggesting a cross-reaction of CCH and FLH glycosylations with carbohydrate epitopes on the tumor cell surfaces, similar to the KLH antisera. When mice were primed with each hemocyanin-P10 and challenged with melanoma cells, better antitumor effects were observed for FLH-P10 than for CCH-P10 and, as for KLH-P10, irrespective of conjugation. These data demonstrate that CCH and FLH are useful carriers of carbohydrate mimotopes; however, the best antitumor activity of FLH preparations, indicate that is a suitable candidate for further cancer vaccines research.

Topics: Animals; Antineoplastic Agents; Dose-Response Relationship, Drug; Drug Carriers; Drug Screening Assays, Antitumor; Female; Gangliosides; Gastropoda; Hemocyanins; Immunotherapy; Melanoma; Mice; Mice, Inbred C57BL; Molecular Structure; Structure-Activity Relationship

Ganglioside GD3 is widely expressed in human malignant melanomas, and has been reported to be involved in the increased cell proliferation and invasion. In this study, we established GM3-, GM2-, GM1-, GD3-, or GD2-expressing melanoma cell lines by transfecting cDNAs of glyscosyltransferases, and effects of individual gangliosides on the cell phenotypes and signals were examined. The phenotypes of established ganglioside-expressing cells were quite different, i.e. cell growth increased as following order; GD2+, GD3+ > GM1+, GM2+, GM3+ cells. Cell invasion activity increased as GD3+ ≧ GM2+ > GM1+, GM3+, GD2+ cells. Intensity of cell adhesion to collagen I (CL-I) and spreading increased as GD2+ >> GD3+, GM1+ > GM2+, GM3+ cells. In particular, cell adhesion of GD2+ cells was markedly strong. As for cell migration velocity, GD2+ cells were slower than all other cells. The immunocytostaining revealed close localization of gangliosides and F-actin in lamellipodia. Immunoblotting of phosphorylated p130Cas and paxillin by serum treatment reveled that these phosphorylations were more increased in GD3+ cells than in GD2+ or GM3+ cells, while phosphorylation of Akt underwent similarly increased phosphorylation between GD3+ and GD2+ cells compared with GM3+ cells. While GD2 and GD3 enhanced cell growth, GD3 might also contribute in cell invasion. On the other hand, GD2 might contribute in the solid fixation of melanoma cells at metastasized sites. These results suggested that individual gangliosides exert distinct roles in the different aspects of melanomas by differentially regulating cytoskeletons and signaling molecules.

Topics: Carcinogenesis; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gangliosides; Humans; Melanoma; Skin Neoplasms

Antibody-based immunotherapy has proven efficacy for patients with high-risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single-chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T-cell-mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2-positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell-mediated therapy.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Blotting, Western; CD3 Complex; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gangliosides; Humans; Immunotherapy; Lymphocyte Activation; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Neuroblastoma; Single-Chain Antibodies; T-Lymphocytes; Tumor Cells, Cultured

Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g., neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single-chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunologic synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >10⁵-fold selectivity over normal tissues, releasing Th1 cytokines (TNFα, IFNγ, and IL2) when GD2⁺ tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells.

Topics: Animals; Antibodies, Bispecific; CD3 Complex; Cell Line, Tumor; Cell Proliferation; Gangliosides; Humans; Immunoglobulin G; Melanoma; Mice; Mice, Inbred BALB C; Mice, Knockout; Monocytes; Neuroblastoma; Single-Chain Antibodies; T-Lymphocytes; Xenograft Model Antitumor Assays

Ganglioside GD2 is expressed on plasma membranes of various types of malignant cells. One of the most promising approaches for cancer immunotherapy is the treatment with monoclonal antibodies recognizing tumor-associated markers such as ganglioside GD2. It is considered that major mechanisms of anticancer activity of anti-GD2 antibodies are complement-dependent cytotoxicity and/or antibody-mediated cellular cytotoxicity. At the same time, several studies suggested that anti-GD2 antibodies are capable of direct induction of cell death of number of tumor cell lines, but it has not been investigated in details. In this study we investigated the functional role of ganglioside GD2 in the induction of cell death of multiple tumor cell lines by using GD2-specific monoclonal antibodies.. Expression of GD2 on different tumor cell lines was analyzed by flow cytometry using anti-GD2 antibodies. By using HPTLC followed by densitometric analysis we measured the amount of ganglioside GD2 in total ganglioside fractions isolated from tumor cell lines. An MTT assay was performed to assess viability of GD2-positive and -negative tumor cell lines treated with anti-GD2 mAbs. Cross-reactivity of anti-GD2 mAbs with other gangliosides or other surface molecules was investigated by ELISA and flow cytometry. Inhibition of GD2 expression was achieved by using of inhibitor for ganglioside synthesis PDMP and/or siRNA for GM2/GD2 and GD3 synthases.. Anti-GD2 mAbs effectively induced non-classical cell death that combined features of both apoptosis and necrosis in GD2-positive tumor cells and did not affect GD2-negative tumors. Anti-GD2 mAbs directly induced cell death, which included alteration of mitochondrial membrane potential, induction of apoptotic volume decrease and cell membrane permeability. This cytotoxic effect was mediated exclusively by specific binding of anti-GD2 antibodies with ganglioside GD2 but not with other molecules. Moreover, the level of GD2 expression correlated with susceptibility of tumor cell lines to cytotoxic effect of anti-GD2 antibodies.. Results of this study demonstrate that anti-GD2 antibodies not only passively bind to the surface of tumor cells but also directly induce rapid cell death after the incubation with GD2-positive tumor cells. These results suggest a new role of GD2 as a receptor that actively transduces death signal in malignant cells.

Topics: Antibodies, Monoclonal; Apoptosis; Cell Line, Tumor; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy; Melanoma; Membrane Potential, Mitochondrial; Signal Transduction

Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model.. We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated.. Despite its low affinity of approximately 10(7) M(-1) for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC(50) of 70 ng/ml [0.47 nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4(+) and CD8(+) T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced.. Our data suggest that Surek revealed strong tumor elimination and anti-tumor immunization capabilities. The results warrant further clinical development of the human therapeutic equivalent antibody Ektomab.

Topics: Adoptive Transfer; Animals; Antibodies, Bispecific; Antibodies, Neoplasm; Antibody Specificity; Cytotoxicity, Immunologic; Dose-Response Relationship, Immunologic; Gangliosides; Humans; Immune Sera; Immunity, Humoral; Immunization; Immunoglobulin G; Melanoma; Mice; Skin Neoplasms; Survival Analysis; T-Lymphocytes; Time Factors; Treatment Outcome

Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment.

Topics: Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Binding, Competitive; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Gangliosides; Humans; Interleukin-2; Melanoma; Reproducibility of Results

Genetic engineering of human T lymphocytes to express tumor-directed chimeric antigen receptors (CAR) can produce antitumor effector cells that bypass tumor immune escape mechanisms that are due to abnormalities in protein-antigen processing and presentation. Moreover, these transgenic receptors can be directed to tumor-associated antigens that are not protein-derived, such as the ganglioside GD2, which is expressed in a high proportion of melanoma cells.. We generated chimeric T cells specific for the ganglioside GD2 by joining an extracellular antigen-binding domain derived from the GD2-specific antibody sc14.G2a to cytoplasmic signaling domains derived from the T-cell receptor zeta-chain, with the endodomains of the costimulatory molecules CD28 and OX40. We expressed this CAR in human T cells and assessed the targeting of GD2-positive melanoma tumors in vitro and in a murine xenograft.. Upon coincubation with GD2-expressing melanoma cells, CAR-GD2 T lymphocytes incorporating the CD28 and OX40 endodomains secreted significant levels of cytokines in a pattern comparable with the cytokine response obtained by engagement of the native CD3 receptor. These CAR-T cells had antimelanoma activity in vitro and in our xenograft model, increasing the survival of tumor-bearing animals.. Redirecting human T lymphocytes to the tumor-associated ganglioside GD2 generates effector cells with antimelanoma activity that should be testable in subjects with disease.

Topics: Animals; Antibodies; Gangliosides; Genetic Engineering; Humans; Immunotherapy; Melanoma; Mice; Mice, SCID; Neoplasm Metastasis; Receptors, Antigen, T-Cell; T-Lymphocytes; Xenograft Model Antitumor Assays

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Animals; Antigen Presentation; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Epitopes, T-Lymphocyte; Female; Ganglia, Spinal; Ganglioglioma; Gangliosides; Humans; Immunotherapy, Adoptive; Lymphocyte Activation; Melanoma; Mice; Mice, Inbred A; Molecular Mimicry; Neuroblastoma

Because of its restricted distribution in normal tissues and its high expression on tumors of neuroectodermal origin, GD2 ganglioside is an excellent target for active specific immunotherapy. However, GD2 usually elicits low-titered IgM and no IgG or cellular immune responses, limiting its usefulness as a vaccine for cancer patients. We have previously shown that anti-idiotypic monoclonal antibody mimics of GD2 can induce antigen-specific humoral and cellular immunity in mice, but inhibition of tumor growth by the mimics could not be detected.. Here, we isolated two peptides from phage display peptide libraries by panning with GD2-specific mAb ME361. The peptides inhibited binding of the mAb to GD2. When coupled to keyhole limpet hemocyanin (KLH) or presented as multiantigenic peptides in QS21 adjuvant, the peptides induced in mice antibodies binding specifically to GD2 and delayed-type hypersensitive lymphocytes reactive specifically with GD2-positive D142.34 mouse melanoma cells. Induction of delayed-type hypersensitivity (DTH) reaction was dependent on CD4-positive lymphocytes. The immunity elicited by the peptides significantly inhibited growth of GD2-positive melanoma cells in mice.. Our study suggests that immunization with peptides mimicking GD2 ganglioside inhibits tumor growth through antibody and/or CD4-positive T cell-mediated mechanisms. Cytolytic T lymphocytes most likely do not play a role. Our results provide the basis for structural analysis of carbohydrate mimicry by peptides.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Formation; Cancer Vaccines; Cell Line, Tumor; Female; Gangliosides; Immunity, Cellular; Immunotherapy, Active; Lymphocytes; Melanoma; Mice; Mice, Inbred C57BL; Molecular Mimicry; Molecular Sequence Data; Peptide Library; Peptides; Protein Binding; Vaccines, Subunit

This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody(sc-BsAb), and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene (NM3E2) were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b(+) plasmid from the pMD18-T easy vector by digestion with NcoI, Hind III restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 (DE3). The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni2+ -NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusion protein is SerGly4Ser. and the molecular is 53 KD.

Topics: Antibodies, Bispecific; Antibodies, Neoplasm; Base Sequence; Cell Line, Tumor; Cloning, Molecular; Escherichia coli; Gangliosides; HeLa Cells; Humans; Melanoma; Molecular Sequence Data; Receptors, IgG; Recombinant Fusion Proteins

The disialoganglioside GD2, a carbohydrate antigen, is expressed on all tumors of neuroectodermal origin, including melanoma, neuroblastoma, sarcoma and small cell lung cancer. Due to its specific expression on tumor surfaces, GD2 is an attractive target for immunotherapies. The mouse/human chimeric anti-GD2 mAb ch14.18 is already applied in melanoma and neuroblastoma trials as a passive immunotherapy. To establish an active immunotherapy alternative, we aimed to replace the poorly immunogenic ganglioside with immunogenic peptides. Previously, we used the ch14.18 antibody to select GD2 peptide mimics from a phage display library. In the present study, two mimics of the ch14.18 epitope were coupled to keyhole limpet hemocyanin and used for immunizing BALB/c mice. Induction of a specific humoral immune response towards the original antigen GD2, both purified and expressed on neuroblastoma and melanoma cells, could be demonstrated in ELISA, Western blot, and immunofluorohistochemistry. As the elicited antibodies were of the IgG isotype, the mimotope conjugates were capable of recruiting T cell help and inducing memory phenomena. In conclusion, we show that an epitope of the carbohydrate antigen GD2 can successfully be translated into immunogenic peptide mimotopes. Our immunization experiments indicate that GD2 mimotopes are suitable for active immunotherapy of GD2-expressing tumors.

Topics: Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Cross Reactions; Gangliosides; Humans; Immunoglobulin G; Melanoma; Mice; Mice, Inbred BALB C; Neuroblastoma; Vaccination

The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding Sites, Antibody; Cancer Vaccines; Cross Reactions; Female; Gangliosides; Humans; Immunoglobulin G; Immunotherapy, Active; Melanoma; Mice; Mice, Inbred BALB C; Mice, SCID; Molecular Sequence Data; Neuroblastoma; Peptide Library; Peptides; Vaccines, DNA; Xenograft Model Antitumor Assays

Matrix metalloproteinases (MMPs) are essential for tumor progression, invasion and metastases formation. Expression of these proteinases is not only restricted to the tumor cells themselves, but also is found in normal stromal cells. Moreover, immunohistochemistry suggests stromal cells as the major source. To scrutinize this hypothesis we established a slowly growing, syngeneic tumor model using the B16-melanoma cell line B78D14. In vitro analysis demonstrated that B78D14 cells secreted MMP-2, MT1-MMP, and to a lesser degree MMP-9; in addition they expressed both MT1-MMP and EMMPRIN on their surface. In subcutaneous (s.c.) tumors of these cells MMP-2 expression was predominantly present at the tumor-stroma border indicating stromal cells as primary source for this protease in vivo. Indeed, double staining experiments and in situ zymography confirmed that tumor adjacent stromal cells at the invasive front expressed MMP-2 and only at this site activated MMP-2 was detectable. Notably, in an experimental pulmonary metastases model neither tumor nor stromal cells expressed MMP-2, suggesting that the capacity of stromal cells is largely dependent on the surrounding microenvironment.

Topics: Animals; Basigin; Cell Line, Tumor; Female; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Skin Neoplasms; Stromal Cells; Tissue Inhibitor of Metalloproteinases

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

Topics: Animals; Antibodies, Monoclonal; beta-Galactosidase; Carbohydrate Metabolism; CD4-Positive T-Lymphocytes; Cell Division; Dose-Response Relationship, Drug; Gangliosides; Humans; Hybridomas; Immunoglobulin Idiotypes; Immunophenotyping; Immunotherapy; Leukocytes, Mononuclear; Lymphocytes; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Protein Binding; Rabbits; T-Lymphocytes

Melanomas, sarcomas, and neuroblastomas abundantly express the ganglioside GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2. In addition, preclinical models have demonstrated the ability of a GD2-keyhole limpet hemocyanin (KLH) conjugate vaccine to induce antibodies that eliminate micrometastases. However, vaccination of patients with GD2-KLH has previously failed to induce a consistent relevant antibody response. We test here whether the use of GD2 lactone-KLH can overcome the low immunogenicity of GD2-KLH.. Eighteen patients with melanoma were vaccinated s.c. in the adjuvant setting on weeks 0, 1, 2, 3, 10, and 24. Groups of 6 patients were entered at three dose levels (3, 10, or 30 micro g) of GD2 lactone (GD2L) in vaccines containing GD2L-KLH plus the immunological adjuvant QS-21. Blood was drawn at regular intervals to assess the antibody response.. The vaccine was well tolerated. The majority of patients in all three dose levels produced anti-GD2 antibodies detectable by ELISA assay. Specificity for GD2 was also confirmed by immune thin-layer chromatography. Although there was no statistical difference in terms of titers between the three groups, patients at the 30- micro g dose level had higher titers and longer lasting antibody responses overall by ELISA (median IgM/IgG peak titer 1:640/1:80) and generated the strongest cell surface reactivity by fluorescence-activated cell sorting (median IgM peak percentage positive cells/mean fluorescence intensity for pre- and postvaccination sera is 10%/63 and 70%/135). Patients vaccinated with the 30- micro g GD2 dose also had the most potent complement dependent cytotoxicity using human complement, with 5 of 6 patients showing strong cell surface reactivity by fluorescence-activated cell sorting and >30% cytotoxicity by chromium release with a serum dilution of 1/100.. GD2L-KLH conjugate vaccine plus adjuvant QS-21 induces antibodies against GD2 that bind to the cell surface and induce complement-dependent cytotoxicity in the majority of patients with melanoma.

Topics: Adjuvants, Immunologic; Antibody Formation; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Cancer Vaccines; Enzyme-Linked Immunosorbent Assay; Gangliosides; Hemocyanins; Humans; Immunoglobulin G; Immunoglobulin M; Lactones; Melanoma; Saponins; Skin Neoplasms; Vaccination; Vaccines, Conjugate

In this study we have documented a hybridoma secreting an unusual MAb, which expresses both IgG3 and IgG2a subclasses with a lambda-light chain. How this dual expression of isotypes was exactly brought about is not clear. To resolve this problem, it will have to wait the complete sequence analysis the heavy chain gene of MAb 9C4. Although the expression of IgG2a was about 50% that of IgG3, antibody titration studies showed the major binding affinity of MAb 9C4 to GD3-positive cells being mostly contributed by the IgG3 rather than IgG2a part of the antibody. This antibody could induce apoptosis in melanoma cells in 10-15% of cells in vitro, but the generality of this phenomenon is yet to be confirmed by the use of different cell targets and different anti-GD2 MAbs other than 9C4. Aside from the demonstrated indirect killing mechanisms of many anti-GD2 MAbs through CDC and ADCC, MAb 9C4 induction of apoptosis represents an alternative mechanism of tumor cell killing, by which direct killing of anti-GD2 antibody takes its effect. This apoptotic effect was demonstrated for the first time with an anti-ganglioside monoclonal antibody. From the therapeutic point of view, the cytolytic activity of MAb 9C4-targeted ADCC/LAK killing against GD2-positive tumor cells to be more effective than that of LAK alone and a possibility for dendritic cells to effectively acquire antigen through pulsing with MAb-induced apoptotic cells are both of great clinical importance. Further studies are warranted aiming at elucidating the molecular basis of bi-isotypic specificity and aberrant isotype switching, molecular pathway of anti-GD2 antibody-induced apoptosis, and ways to improve clinical utility of this unusual hybridoma/MAb 9C4.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Apoptosis; Cell Differentiation; Gangliosides; Humans; Hybridomas; Immunoglobulin G; Melanoma; Mice; Precipitin Tests; Tumor Cells, Cultured

Several gangliosides such as GM2, GD2, and GD3 have been thought of as target molecules for active or passive immunotherapy of human cancers because of their dominant expression on the tumor cell surface, especially in tumors of neuroectodermal origin. We established a number of mouse or rat monoclonal antibodies (mAbs) to a series of gangliosides to investigate the nature of the molecules on the cell surface. Some of those mAbs were converted to chimeric or humanized mAbs with the aim of developing immunotherapy for human cancer. It is desirable for mAbs to remain on the cell surface for a long time so that they can exert effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). We found that mAbs to GM2, GD2, and GD3 remain on the cell surface for > or =60 min after binding, while mAbs to other types of carbohydrate such as sialy Le(a) are quickly internalized. A chimeric mAb to GD3, KM871, was generated by linking cDNA sequences encoding light- and heavy-chain variable regions of mouse mAb KM641 with cDNAs encoding the constant region of human immunoglobulin gamma1 (IgG-1). KM871 bound to a variety of tumor cell lines, especially melanoma cells, including some cell lines to which R24 failed to bind. In a preclinical study, intravenous injection of KM871 markedly suppressed tumor growth and radiolabeled KM871 efficiently targeted the tumor site in a nude mouse model. This chimeric mAb is being evaluated in a phase I clinical trial in melanoma patients. The chimeric mAb KM966 and humanized mAb KM8969 to GM2 originated from a mouse IgM mAb. When human serum and human peripheral blood mononuclear cells were used as effectors in CDC and ADCC, respectively, KM966 and KM8969 killed GM2-expressing tumor cells effectively. In addition, these mAbs may induce apoptosis of a small cell lung cancer cell line cultured under conditions mimicking physiological tumor conditions.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; G(M2) Ganglioside; Gangliosides; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Recombinant Fusion Proteins; Recombinant Proteins; Tumor Cells, Cultured

Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localization of immunoliposome-entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenretinide (HPR) is an inducer of apoptosis in neuroblastoma (NB) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10-fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitro potentiation of its cytotoxic effect on melanoma cells in combination with long-circulating GD2-targeted immunoliposomes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Murine anti-GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomes by covalent coupling of Ab to the polyethylene glycol (PEG) terminus. Ab-bearing liposomes showed specific, competitive binding to and uptake by various melanoma cell lines compared with liposomes bearing non-specific isotype-matched Abs or Ab-free liposomes. Cytotoxicity was evaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non-specific adsorption of liposomes to the cells, while allowing for maximum Ab-mediated binding. When melanoma cells were incubated with 30 microM HPR entrapped in anti-GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, entrapped HPR in Ab-free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of NB cells was also inhibited by immunoliposomes with entrapped HPR.

Topics: Antibody Specificity; Antineoplastic Agents; Cell Division; Drug Carriers; Fenretinide; Gangliosides; Humans; Liposomes; Melanoma; Sensitivity and Specificity; Tumor Cells, Cultured

The expression of complement regulatory antigens C3b/C4b receptor, (CD35) membrane cofactor protein (CD46), decay accelerating factor (CD55), and homologous restriction factor 20 (CD59) was determined immunohistochemically on ten primary malignant melanomas, 16 metastatic lesions, and ten melanocytic nevi. All of the melanocytic nevi and 9/10 primary melanomas showed both expression of CD46 and CD59. In one primary melanoma lacking CD46, expression of CD35 could be detected. In metastatic melanoma, 9/16 metastases were CD46+/CD59+, two were CD46-/CD59+, one CD46+/CD59-, and four CD46-/CD59-. Additionally, CD55 could be detected in two CD46+/CD59+ metastases, and CD35 in one. Expression or lack of complement regulatory antigens did not correlate with the expression of GD2, GD3, HMB-45 or S-100. In conclusion, some cases of metastatic melanoma show loss of normal expression of complement regulatory proteins. This might have implications on the immune response or the efficacy of immune therapy in malignant melanoma.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, Neoplasm; Child; Female; Gangliosides; Humans; Immunohistochemistry; Male; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Nevus, Pigmented; S100 Proteins; Skin Neoplasms

The anti-idiotype (Id) monoclonal antibody (mAb) 1A7 immunoglobulin G1 (IgG1, kappa), raised in syngeneic mice against the murine anti-ganglioside GD2 mAb 14G2a mimics a carbohydrate epitope on GD2 and serves as a surrogate protein antigen for this disialoganglioside. Immunization of allogeneic C57BL/6 mice and rabbits with 1A7 induced anti-GD2 antibodies of IgG isotype that recognize purified GD2 by enzyme-linked immunosorbent assay (ELISA) and GD2-positive human melanoma cells (M21/P6) by fluorescence-activated cell sorter (FACS) analysis. The specificity of the antisera for GD2 was further confirmed by dot-blot analysis. These antisera also specifically lyse GD2-positive M21/P6 target cells in an antibody-dependent cellular cytotoxicity assay. Taken together, these results suggest that the anti-Id 1A7 can induce GD2-specific IgG antibodies that can recognize cell surface-associated as well as soluble disialoganglioside GD2.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gangliosides; Humans; Immunization; Immunoglobulin G; Melanoma; Mice; Mice, Inbred BALB C; Rabbits

Gangliosides GM2 [GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer] and GD2 [GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer] are cell surface tumor-associated antigens and have been demonstrated to be important markers of human malignant melanoma progression. Expression of these glycolipid antigens on melanoma tissues can be assessed by immunohistochemistry or biochemical analysis. These methodologies, however, are not logistically practical or sensitive for testing metastatic melanoma cells in blood or in tissue biopsies. In the present study, we hypothesized that the enzyme involved in GM2 and GD2 synthesis, beta 1-->4-N-acetylgalactosaminyltransferase (beta 1-->4GalNac-T), can be a useful marker for detection of occult metastatic melanoma. A reverse transcription PCR and Southern blot assay to detect beta 1-->4GalNac-T mRNA expression was developed. Beta 1-->4GalNac-T mRNA was detected in all 13 melanoma cell lines tested. Metastatic melanoma of lymph nodes and different organ sites expressed beta 1-->4GalNac-T mRNA at various levels. Detection sensitivity of the reverse transcription PCR assay was 1 ng of total RNA extracted from tumor specimens and approximately 5 melanoma cells in 20 million normal donor peripheral blood lymphocytes. In assessment of blood from 126 melanoma patients, beta 1-->4GalNac-T mRNA was more frequently found in advanced-stage melanomas and in patients showing more aggressive tumor progression. Normal donor blood samples (n = 37) were all negative for beta 1-->4GalNac-T mRNA expression. These results suggest that beta 1-->4GalNac-T mRNA is a promising molecular marker for detecting melanoma cells, characterizing antigen expression, and monitoring tumor progression.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Blotting, Southern; Carbohydrate Sequence; Disease Progression; G(M2) Ganglioside; Gangliosides; Humans; Melanoma; Molecular Sequence Data; N-Acetylgalactosaminyltransferases; Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Transcription, Genetic; Tumor Cells, Cultured

A human IgG1.k monoclonal antibody (MAb) designated GMA1 was developed by fusing pooled lymph node lymphocytes from cancer patients with the human lymphoblastoid cell line, SHFP-1. The GMA1 MAb reacted with several melanoma and neuroblastoma cell lines. Normal tissue derived from human brain and tumor-cell lines derived from colon, ovary, and breast were not reactive. FACS analysis performed using live cells demonstrated that the antibody recognizes a cell-surface antigen. Enzyme immunoassay (EIA) and thin layer chromatography (TLC) immunostaining with purified gangliosides indicated that the antibody has specificity for the major tumor associated gangliosides GD3, GM3, and GD2. GMA1 heavy and light chain genes were isolated by RT-PCR and a recombinant derivative of this human antibody was expressed in Chinese hamster ovary (CHO) cells. High-level antibody synthesis and secretion was achieved using a vector designed to maximize expression. FACS analysis and TLC immunostaining indicated recombinant GMA1 reacted with human tumor cell lines and gangliosides GD3, GM3, GD2 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Antigens, Surface; Base Sequence; G(M1) Ganglioside; Gangliosides; Humans; Hybridomas; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Melanoma; Molecular Sequence Data; Neuroblastoma; Recombinant Proteins; Tumor Cells, Cultured

In this study, we compare various methods for the detection of a tumor-associated target antigen and deposition of the bound therapeutic monoclonal antibody in patients enrolled in two separate trials, one involving the administration of two radiolabeled monoclonal antibodies and the other involving an unlabeled antibody. In the first trial, patients with TAG-72 expressing metastatic colon cancer scheduled for surgical intervention received radiolabeled murine and chimeric B72.3 antibody followed by radioimmune imaging and subsequent laparotomy. Normal and tumor tissues obtained at surgery were processed for routine histology, immunohistochemistry, radiometry, and autoradiography. Both anti-TAG-72 antibodies localized to known tumor sites as evidenced by radioimmune imaging. Resected tissue revealed a high tumor-to-normal radiolocalization ratio, and autoradiography demonstrated even deposition of the radiolabeled antibodies throughout the entire tumor deposit with sparing of surrounding normal tissue. In contrast, immunohistochemistry on the same sections revealed comparatively weak antigen expression and patchy antibody localization. In the second trial, patients with GD2 antigen expressing metastatic melanoma received the unlabeled chimeric anti-GD2 antibody C14.18. Immunologic detection of the GD2 antigen and C14.18 deposition was performed on biopsy section as well as on single cell suspension. FACS analysis of the single cell suspension proved more sensitive for the detection of bound antibody than immunohistochemistry, although both methods yielded comparable results for GD2 antigen expression. Our findings demonstrate that the optimal method for the detection of tumor-associated antigen and bound therapeutic antibody can vary depending upon the nature of the antibody (radiolabeled vs. unlabeled and murine vs. chimeric), fixation stability of the target antigen, and the type of pathologic material available for study.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Antigens, Neoplasm; Autoradiography; Clinical Trials as Topic; Colonic Neoplasms; Evaluation Studies as Topic; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Gangliosides; Glycoproteins; Humans; Melanoma; Mice

The GD2 ganglioside is a cell-surface component that appears on the surface of metastatic melanoma cells and is a marker for the progression of the disease. The ME36.1 monoclonal antibody binds to the GD2 ganglioside and has shown potential as a therapeutic antibody. ME36.1 is a possible alternative therapy to radiation, which is often ineffective in late-stage melanoma. The crystal structure of the Fab fragment of ME36.1 has been determined using molecular replacement and refined to an R factor of 20.4% at 2.8 A resolution. The model has good geometry with root-mean-square deviations of 0.008 A from ideal bond lengths and 1.7 degrees from ideal bond angles. The crystal structure of the ME36.1 Fab shows that its complementarity determining region forms a groove-shaped binding site rather than the pocket-type observed in other sugar binding Fabs. Molecular modeling has placed a four-residue sugar, representative of GD2, in the antigen binding site. The GD2 sugar moiety is stabilized by a network of hydrogen bonds that define the specificity of ME36.1 toward its antigen.

Topics: Antibodies, Monoclonal; Binding Sites, Antibody; Carbohydrate Conformation; Carbohydrate Sequence; Crystallization; Crystallography, X-Ray; Gangliosides; Hydrogen Bonding; Immunoglobulin Fab Fragments; Immunoglobulin Variable Region; Melanoma; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protein Conformation; Software

The antiganglioside GD2 monoclonal antibody 14G2a (Ab1) served as an immunogen to generate the anti-idiotype (anti-Id) 1A7 (IgG1,kappa), which mimics GD2 both antigenically and biologically. Anti-Id 1A7 induced anti-GD2 antibodies in mice and rabbits. In this preclinical study, a pair of cynomolgus monkeys, immunized with 1A7 that had been mixed with QS-21 adjuvant, produced anti-anti-Id antibodies (Ab3), which reacted with the GD2-positive melanoma cell line M21/P6 cells but not with GD2-negative LS174-T cells. The Ab3 shared Ids with mAb 14G2a (Ab1), as demonstrated by their ability to inhibit binding of 1A7 to this Ab1. The Ab3 bound specifically to purified GD2 antigen and competed with the Ab1 14G2a in binding to a GD2-positive melanoma cell line or to purified GD2, suggesting that Ab1 and Ab3 may bind to the same epitope and may behave as an Ab1-like antibody (Ab1'). The isotype of the GD2-specific antibodies was mostly IgG in nature. The specificity of the antibodies for GD2 was further confirmed by dot blot analysis. These antisera also specifically lysed GD2-positive target cells in an antibody-dependent cellular cytotoxicity assay. The induction of anti-GD2 responses in monkeys did not cause any apparent side effects, despite the fact that GD2 antigen is expressed by many normal tissues of these animals. Taken together, these results suggest that anti-Id 1A7 can induce GD2-specific antibodies in nonhuman primates and can thus serve as a potential network antigen for triggering active anti-GD2 antibodies in patients with GD2-positive neuroectodermal tumors.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody Formation; Antibody-Dependent Cell Cytotoxicity; Colonic Neoplasms; Gangliosides; Humans; Macaca fascicularis; Melanoma; Mice; Mice, Inbred BALB C; Rabbits; Tumor Cells, Cultured

Antibody-cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody-interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cgamma1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumor-specific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumor-specific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

Topics: Animals; Antibodies; Base Sequence; Cell Line; DNA Primers; ErbB Receptors; Gangliosides; Humans; Immunotherapy; Immunotoxins; Interleukin-2; Killer Cells, Lymphokine-Activated; Liver Neoplasms; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Mice, SCID; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Fusion Proteins; Skin Neoplasms; Survival Rate; Time Factors; Transplantation, Heterologous

The induction of human antimouse antibodies (HAMA) and human anti-idiotypic (anti-Id) responses in cancer patients receiving therapeutic monoclonal antibody (mAb) may limit the effectiveness of the administered mAb. This report evaluates the influence of systemic interleukin-2 (IL-2) on the anti-Id response to anti-disialoganglioside (anti-GD2) antibody given as treatment for patients with melanoma. Twenty-eight patients with melanoma received combined immunotherapy with anti-GD2 antibody and IL-2 at 1.5 x 10(6) U/m2/day given 4 days/week. The anti-GD2 antibody [murine 14.G2a mAb; dose levels of 2-5 mg/m2/day (4 patients); or human-mouse chimeric 14.18 (ch14.18) antibody; dose levels of 2-10 mg/m2/day (24 patients)] was scheduled to be given for 5 days either before, during, or after initial systemic IL-2 treatment. All four patients who received murine 14.G2a developed HAMA anti-isotype antibodies (660-1,000 ng/ml) as well as measurable anti-Id antibodies. All three patients who received initial treatment with ch14.18 alone developed a strong anti-Id antibody response after IL-2 was started 1 week later. The serum level of anti-Id antibody decreased during subsequent ch14.18 infusions, suggesting that the anti-Id antibody may be binding the administered ch14.18. In contrast, measurable anti-Id antibody was detected in only 3 of 14 patients who received IL-2 before, during, and after initial ch14.18 administration. Two of four patients receiving systemic IL-2 before and during initial ch14.18 infusions, and two of three patients receiving systemic IL-2 concurrent with initial ch14.18 infusions developed anti-Id antibodies. These data suggest that the anti-Id response to chimeric anti-GD2 antibody is influenced by the timing of systemic IL-2 in relation to antibody administration and can be suppressed by systemic treatment with IL-2 given before, during, and after the antibody administration.

Topics: Adjuvants, Immunologic; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Drug Synergism; Gangliosides; Humans; Immunoglobulin Idiotypes; Infusions, Intravenous; Interleukin-2; Melanoma; Recombinant Proteins; Skin Neoplasms

The inability of current therapy to prevent metastases arising from uveal melanoma often results in patient mortality. With the goal of developing a treatment for metastasis, gangliosides were studied as potential tumor-associated antigens. Our report describes the production of a metastatic liver variant (MH) from a human uveal melanoma cell line (SP6.5). Cells were injected into nude mouse spleens and liver metastases collected 2 months later. After 21 days of in vitro subculture, the cells were re-injected into normal nude mice spleen; 10 cycles (MH10) were performed. Gangliosides were extracted, purified, chromatographed on HPTLC plates and sprayed with a resorcinol-HCl reagent, the sialic acid spots being quantified by densitometry. Gangliosides were analyzed in each metastatic liver variant and compared with the SP6.5 s.c. tumor. The results showed a significant increase in GM3 and a significant decrease in GD3 and GD2 in the last metastatic variants obtained (MH5, MH8, MH9 and MH1O) compared with the primary s.c. tumor, SP6.5. Such evolution in the ganglioside pattern was maintained throughout the progression of the different liver variants. Our results indicate that precursor ganglioside GM3 and gangliosides GD3 and GD2 could be associated with neoplastic evolution of malignancy of human uveal melanoma in nude mice.

Topics: Animals; G(M3) Ganglioside; Gangliosides; Humans; Liver Neoplasms; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Tumor Cells, Cultured; Uveal Neoplasms

The glycosphingolipid patterns were analyzed on two clones derived from a human melanoma cell line and selected for their respectively high and low metastatic ability in immunosuppressed newborn rats. Conversely to the weakly metastatic cells which exhibited a pattern similar to that of the parental cell line, highly metastatic human melanoma cells appeared to be deficient in ganglioside biosynthesis. An accumulation of lactosylceramide was found in the latter cells, with low amounts of GM3 as the only ganglioside detected and a fourfold decreased activity of GM3 synthase (EC 2.4.99.9). After subcutaneous injection of metastatic cells in newborn rats, the cells proliferating in the tumor induced at the injection site re-expressed the four common gangliosides of melanoma: GM3, GM2, GD3 and GD2, whereas the cells growing in the lungs as metastatic nodules were deficient in ganglioside synthesis and showed an accumulation of lactosylceramide. Taken together, our results suggest that the human melanoma cells which are able to escape from the primary tumor and invade the lungs have an impaired ganglioside biosynthesis with a deficient GM3 synthase.

Topics: Animals; Animals, Newborn; Antigens, CD; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Lactosylceramides; Melanoma; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Sialyltransferases; Tumor Cells, Cultured

Human tumors originating from neuroectodermal cells such as malignant melanoma and neuroblastoma express high levels of disialogangliosides GD2 and GD3, making these antigens ideal for targeting by monoclonal antibodies (Mabs). The purpose of this study was to investigate expression and targeting of gangliosides on canine melanoma. Using immunohistochemical methods, we analyzed the expression of disialogangliosides GD2 and GD3 on canine oral malignant melanomas with murine Mabs 14.G2a and R24 that recognize GD2 and GD3 disialogangliosides, respectively, on human tumors. We also assessed the ability of Mab 14.G2a (and its mouse-human chimera, ch 14.18) to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro against a canine malignant melanoma cell line with human recombinant interleukin-2 (IL-2) activated canine peripheral blood lymphocytes (PBL), or canine neutrophil effector cells. Our data show that Mabs 14.G2a and R24 recognized fresh frozen canine oral melanoma. Mabs 14.G2a or ch 14.18, or IL-2, potentiated lysis of the canine malignant melanoma cell line by canine PBL. The killing effect observed using the combination of either Mab with IL-2 was additive. Mab 14.G2a mediated potent ADCC of canine melanoma by canine neutrophils. These studies indicate that disialogangliosides are expressed on fresh canine melanoma cells. Mabs reactive with these antigens can target and trigger tumor killing by multiple canine effector populations and IL-2 can potentiate these effects by canine lymphocytes. Thus, canine oral malignant melanoma, a spontaneously occurring, metastatic cancer in the dog, may be a relevant animal model to investigate combination immunotherapy using antitumor Mab and IL-2.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Cytotoxicity Tests, Immunologic; Disease Models, Animal; Dog Diseases; Dogs; Flow Cytometry; Gangliosides; Interleukin-2; Leukocytes; Melanoma; Mouth Neoplasms; Recombinant Proteins; Tumor Cells, Cultured

Chemotactic activity of granulocytes attracted by tumor cells loaded either with anti-ganglioside monoclonal antibodies (mAb) or with antibody-glucose oxidase conjugates (mAb-GO) was investigated. The melanoma cell line SK-Mel-28 which expresses the ganglioside GD3 at high density as well as the neuroectodermal cell line SK-N-LO which expresses GD2 were used for the experiments. In the presence of 50% human AB-serum, antibody-loaded tumor cells induced chemotactic activity on granulocytes, probably due to the generation of C3a/C5a which could be detected in serum incubated with anti-GD3 loaded SK-Mel-28 cells. Both compounds could also be detected in vivo in the plasma of patients suffering from neuroblastoma during therapy with anti-GD2 antibodies. In another set of experiments mAb-GO conjugates generating high amounts of H2O2 in the presence of glucose were bound to these tumor cells. A significant lipid peroxidation could be observed in the simultaneous presence of iron and ascorbate. The lipid peroxidation products were measured as thiobarbituric acid-reactive substances (TBARS) and were also shown to induce chemotactic effects on granulocytes.

Topics: ABO Blood-Group System; Antibodies, Monoclonal; Chemotactic Factors; Chemotaxis; Complement C3a; Complement C5a; Gangliosides; Glucose Oxidase; Granulocytes; Humans; Hydrogen Peroxide; Lipid Peroxidation; Melanoma; Neuroectodermal Tumors; Tumor Cells, Cultured

Mouse monoclonal antibodies against tumour-associated gangliosides GD2 (14.G2a) and GD3 (MB 3.6) were tested to mediate antibody-dependent cellular cytotoxicity (ADCC) with various effector cells or complement-dependent cytolysis (CDC). We also evaluated the immunomodulating potential of interferons in combination with cellular cytotoxicity. Using effector:target (E/T) ratios of 40:1, ADCC with effector cells such as granulocytes or mononuclear blood cells was not detectable against melanoma cell lines GR, SK-MEL-28 and G-361 which preferentially express GD3 and bind antibody MB 3.6. Neuroblastoma cell line SK-N-LO, which was used for comparative purposes, mainly expressed GD2 and the tumour cells were killed effectively after labelling with antibody 14.G2a. Granulocytes did not show significant killing of melanoma cells by ADCC, but neuroblastoma cells were killed very efficiently. Peripheral blood mononuclear cells (PBMC) also failed to kill melanoma cells. Interferon-beta slightly stimulated PBMC and increased killing of neuroblastoma cells, but no additive effects with ADCC were detectable. Incubation of target cells with interferons produced no significant differences in susceptibility of the target cells to interferon-activated PBMC cytotoxicity. Despite the lack of effectiveness in mediating cellular cytotoxicity, GD3 antibody MB 3.6 showed strong complement-dependent cytolysis in the presence of human plasma. There were remarkable differences in individual activity and different susceptibility of the melanoma cell lines. We assume that CDC may have more activity against melanoma cells than cytotoxicity associated with various effector cells.

Topics: Animals; Antibodies, Monoclonal; Cell Membrane; Complement Activation; Cytotoxicity, Immunologic; Gangliosides; Granulocytes; Humans; Immunotherapy; Interferon-beta; Killer Cells, Natural; Leukocytes, Mononuclear; Melanoma; Mice; Neuroblastoma; Tumor Cells, Cultured

In vivo and in vitro studies were performed to determine: (a) if human uveal melanoma cells expressed GD2 and GD3 gangliosides; (b) if anti-GD2 monoclonal antibodies would inhibit the propensity of human uveal melanoma cells to localize in the liver following intravenous injection; and (c) if anti-GD2 monoclonal antibody would reduce the spontaneous metastasis of primary intraocular melanomas in nude mice. The results showed that all three of the human uveal melanoma cell lines tested expressed GD2 and GD3 gangliosides in vitro and in vivo. The human uveal melanoma cell lines preferentially localized in the liver and entered the hepatic parenchyma following spontaneous metastasis from the eyes of nude mice. In vivo administration of anti-GD2 monoclonal antibody produced a sharp reduction in the number of uveal melanoma cells that disseminated to the liver following either intravenous injection or by spontaneous metastasis from primary intraocular melanomas. Collectively, the results demonstrate that uveal melanoma cells display a propensity to localize in the liver after entering the bloodstream; however, this localization can be significantly inhibited by in vivo administration of anti-ganglioside antibodies. The expression of GD2 and GD3 surface gangliosides on uveal melanomas and the capacity of anti-ganglioside antibodies to inhibit metastasis formation in mouse models of ocular and cutaneous melanomas raise the possibility of implementing anti-ganglioside antibodies as potential therapeutic agents for the management of uveal melanoma.

Topics: Animals; Antibodies, Monoclonal; Disease Models, Animal; Female; Flow Cytometry; Gangliosides; Humans; Immunotherapy; Liver Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Skin Neoplasms; Tumor Cells, Cultured; Uveal Neoplasms

Human tumor cells expressing ganglioside GD2 were lysed by various effector populations targeted with an anti-CD3-anti-GD2 bi-specific antibody (BAb CD3 x GD2). This antibody-heteroconjugate was prepared by chemically cross-linking the OKT-3 monoclonal antibody (MAb) reactive with CD3 antigen on T lymphocytes with the ganglioside MAb ME 361, which binds preferentially to the tumor-associated ganglioside GD2. The specificity of target-cell lysis by the cytotoxic T cells (CTL) was mediated by the specificity of the targeting antibody: GD2-negative cells were not lysed in the presence of the CD3 x GD2 BAb. A dose-dependent response was observed in a range of 10 to 10,000 ng/ml. In contrast, 2 other BAbs recognizing the tumor-associated antigens EGF-R and TKB-2 had greater potency to mediate tumor-cell lysis than the GD2 x CD3 BAb. Peripheral-blood cells (PBL) stimulated with OKT-3 MAb or with irradiated tumor cells in a mixed lymphocyte culture (MLTC) could be induced to lyse GD2-positive tumor cells in the presence of CD3 x GD2 BAb. The tumor-cell lysis could be mediated by autologous or allogeneic effector cells. NK cells had no influence on the BAb-induced cytotoxicity.

Topics: Antibodies; Antibody Specificity; CD3 Complex; Colonic Neoplasms; Cross Reactions; Gangliosides; Humans; Immunoglobulin G; Immunotherapy; Killer Cells, Natural; Melanoma; Neoplasms; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

In a phase 1 trial, patients with metastatic melanoma received the anti-GD2 murine mAb 14G2a. All patients developed human anti-14G2a antibodies including anti-Id antibodies. Peripheral blood MNCs from one such patient were fused with the murine myeloma cell line Ag8. Four human anti-14G2a secreting hybridomas were generated and the mAb product of one of the hybridomas was characterized. The human mAb 4B5 (hu-IgG, lambda) binds to the variable region of murine 14G2a (anti-Id). The 4B5 binds to the antigen-combining site of 14G2a and inhibits its binding to GD2 expressing Mel-21 cells. Rabbits were immunized with the human anti-Id 4B5. Sera from the immunized rabbits demonstrated anti-4B5 antibodies and anti-Mel-21 and anti-GD2 reactivity. Furthermore, rabbit sera competitively inhibited binding of 14G2a to Mel-21 cells. Rabbits immunized with 4B5 developed a DTH response when challenged with 4B5 antibody and Mel-21 cells. These studies demonstrate that the human anti-Id 4B5 mimics the GD2 antigen and is capable of eliciting both a humoral and cellular anti-GD2 immune response. This antibody could be potentially used as a human anti-Id vaccine in patients with malignant melanoma.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Gangliosides; Humans; Hypersensitivity, Delayed; Immunization; Immunotherapy, Active; Melanoma; Mice; Rabbits

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.

Topics: Acetyl Coenzyme A; Acetylation; Acetyltransferases; Carbohydrate Sequence; Chromatography, High Pressure Liquid; Clone Cells; Gangliosides; Gas Chromatography-Mass Spectrometry; Golgi Apparatus; Humans; Melanoma; Methylation; Molecular Sequence Data; Sialic Acids

A genetically engineered fusion protein consisting of a chimeric anti-ganglioside GD2 antibody (ch14.18) and interleukin 2 (IL2) was tested for its ability to enhance the killing of autologous GD2-expressing melanoma target cells by a tumor-infiltrating lymphocyte line (660 TIL). The fusion of IL2 to the carboxyl terminus of the immunoglobulin heavy chain did not reduce IL2 activity as measured in a standard proliferation assay using either mouse or human T-cell lines. Antigen-binding activity was greater than that of the native chimeric antibody. The ability of resting 660 TIL cells to kill their autologous GD2-positive target cells was enhanced if the target cells were first coated with the fusion protein. This stimulation of killing was greater than that of uncoated cells in the presence of equivalent or higher concentrations of free IL2. Such antibody-cytokine fusion proteins may prove useful in targeting the biological effect of IL2 and other cytokines to tumor cells and in this way stimulate their immune destruction.

Topics: Antibodies, Monoclonal; Cytotoxicity, Immunologic; Gangliosides; Humans; In Vitro Techniques; Interleukin-2; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Melanoma; Recombinant Fusion Proteins; T-Lymphocytes

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.

Topics: Antibodies, Monoclonal; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Complement System Proteins; Gangliosides; Humans; Immunologic Factors; Immunotoxins; Lysosomes; Melanoma; Monensin; Plant Proteins; Protein Synthesis Inhibitors; Ribosome Inactivating Proteins, Type 1; Tumor Cells, Cultured

GM2 ganglioside is a common cell surface constituent of human melanoma and other tumors of neuroectodermal origin, and vaccination with GM2 ganglioside results in high levels of anti-GM2 antibodies in patients with melanoma. Lymphocytes from a GM2-vaccinated patient (VS) were transformed by Epstein-Barr virus and tested for production of antibodies with reactivity for GM2-positive tumor cells. A high percentage of antibody-producing B cells was detected, but antibody reactivity was generally lost during culture expansion. Two cultures, however, remained stable for antibody productivity and one was used to develop a stable hybrid line with mouse myeloma. The monoclonal antibody (designated 3-207) derived from patient VS has dual specificity for GM2 and GD2, despite the fact that only GM2 antibody could be detected in the patient's serum. Monoclonal antibody 3-207 shows high-titered reactivity with a range of melanoma, astrocytoma, neuroblastoma, and leukemia cell lines, cells with prominent cell surface expression of GM2 and GD2. The cell surface reactivity of monoclonal antibody 3-207 was not abolished by treatment of target cells with neuraminidase, as the enzyme converted GD2 to GM2, which was still detected by monoclonal antibody 3-207.

Topics: Animals; Antibodies, Monoclonal; BCG Vaccine; Cell Fusion; Cell Line; Cell Transformation, Viral; Chromatography, Thin Layer; G(M2) Ganglioside; Gangliosides; Herpesvirus 4, Human; Humans; Melanoma; Mice; Neuraminidase; Tumor Cells, Cultured; Vaccines

Recombinant techniques allow one to engineer an antibody molecule and, in this way, manipulate its properties and functions. We engineered a chimeric human/mouse antibody to the tumor-associated antigen ganglioside GD2, with the aim of decreasing its serum half-life, maintaining its full antigen-binding capacity, and deleting its effector functions, thus making it a potentially useful reagent for the radioimaging of tumors. To this end, the constant region of the human gamma 1 chain was mutated by deleting the second domain (CH2). Here we show that the CH2-deleted antibody (ch14.18-delta CH2) was cleared from the blood of athymic (nu/nu) mice bearing human melanoma tumors with the same kinetics as human IgG F(ab')2. At a beta t1/2 of 12 hr, 0.9% of the injected dose of 125I-labeled ch14.18-delta CH2 was found per milliliter of blood 24 hr after i.v. injection. In biodistribution experiments, 125I-labeled ch14.18-delta CH2 targeted specifically to melanoma xenografts, achieving optimal tumor-to-tissue ratios 12-16 hr after i.v. injection. ch14.18-delta CH2 was localized to the melanoma tumors more rapidly and with better localization ratios than the intact chimeric antibody ch14.18. Sixteen hours after i.v. injection, the tumor-to-blood and tumor-to-liver ratios of ch14.18-delta CH2 were 5 and 12, respectively, while optimal localization ratios obtained for ch14.18 were 1 and 5, respectively, but 96 hr after injection. A reagent such as ch14.18-delta CH2 should be useful for radioimmunodetection of human tumors because of reduced immunogenicity, increased targeting specificity, and rapid clearance from circulation.

Topics: Animals; Cell Line; Chimera; Chromosome Deletion; Fluorescent Antibody Technique; Gangliosides; Humans; Immunoglobulin Fab Fragments; Immunoglobulin G; Melanoma; Metabolic Clearance Rate; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

The relationship between the cellular internalization of an anti-ganglioside GD2 monoclonal antibody (14.G2a) and the toxic effect of its ricin A-chain immunotoxin (14.G2a-RA) was examined on GD2-bearing M21 human melanoma and T293 small cell lung carcinoma cell lines. The capacity for ligand uptake was determined by examining the parameters that contribute to this constant, including the number of cell-surface binding sites and the internalization rate constant (ke). The maximum uptake of 14.G2a is 11-fold greater for M21 than for T293 cells, due to a 2.7-fold difference in binding sites and a 4-fold difference in the rate of antibody internalization. The capacity for ligand uptake correlates with the cytotoxic activity of the 14.G2a-RA immunotoxin against these two cell lines. Furthermore, we were able to demonstrate that the consequence of internalization of 14.G2a-RA is the intracellular release of undegraded ricin A-chain from the antibody. These studies indicate that the rate of internalization is a quantitative parameter that plays a key role in predicting the cytotoxic potency of this immunotoxin.

Topics: Antibodies, Monoclonal; Biological Transport; Carcinoma, Small Cell; Cell Survival; DNA Replication; Gangliosides; Humans; Immunotoxins; Kinetics; Lung Neoplasms; Melanoma; Ricin; Tumor Cells, Cultured

Previous studies in vitro have shown that monoclonal antibodies (MAbs) against gangliosides GD3 and GD2 potentiate lymphocyte responses to a variety of stimuli. The purpose of the present study was to determine by immunohistological techniques whether GD3 and GD2 was expressed on lymphoid cells in vivo around melanoma cells. Studies on metastases in lymph nodes indicated that the lymphoid infiltrate around the margins of the metastases was predominantly CD4+ T cells, which were shown by dual labelling techniques to express mainly GD2 and to a lesser extent GD3. CD4+GD3+ T cells were detected more frequently in cortical regions of the lymph nodes. CD8+ T cells were less numerous than CD4+ T cells and expressed both GD3 and GD2. Expression of GD2 was also prominent on CD4+ T cells, B lymphocytes and dendritic reticular cells in germinal centres, whereas GD3 was mainly expressed on T cells in the margins of the follicles. In contrast to the predominance of CD4+ T cells in lymph nodes, CD4+ and CD8+ T cells were in approximately equal proportions about primary melanoma and metastases in skin. GD2 was largely undetectable on lymphocytes at these sites. In contrast, GD3 was detected on both CD8+ and CD4+ lymphocytes but not on B lymphocytes. The absence of GD2 on CD4+ T cells in skin suggested the latter were a different subpopulation to those in lymph nodes. There appeared to be no clear correlation, however, with subsets of CD4 T cells defined by the 2H4 and Leu 8 MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies, Monoclonal; Dendritic Cells; Gangliosides; Humans; Immunoenzyme Techniques; Lymph Nodes; Lymphatic Metastasis; Lymphocytes; Melanoma; Skin Neoplasms

Gangliosides appear to be important target molecules for immunological effector mechanisms on neuro-ectodermal tumors. Therefore in vitro studies were performed to examine whether ganglioside GD3, which is highly expressed on the cell surface of cultured human melanoma cells, is being shed into the culture medium. Measurable quantities of gangliosides GM3 and in particular GD3 were shed by the melanoma cells we have tested as detected on thin-layer chromatograms (TLC) stained with orcinol. Ganglioside GD3 was also evidenced by immunostaining with anti-GD3 MAb and by ELISA. The concentration of GD3 in the supernatant of human melanoma cells depended on the ganglioside pattern of the cell line. Cells containing high levels of GD3 shed large amounts, cells with low levels shed no detectable GD3. Ganglioside GD3 was detectable in sera, but no major quantitative differences were observed in sera of patients with GD3-positive tumors and normal controls. This points to a local accumulation of ganglioside GD3 at the tumor site.

Topics: G(M3) Ganglioside; Gangliosides; Humans; Melanoma; Tumor Cells, Cultured

Levels of GD2, GD3, and 9-O-acetyl GD3 were monitored in sera of patients with melanoma and healthy adults with two monoclonal antibodies that specifically detect these gangliosides. By direct measurement of radioactivity in the immunolabeled chromatogram, GD2 could be detected in normal sera at 2 ng/mL. Serum levels of GD2 and GD3 were increased approximately sixfold and fivefold, respectively, in patients with disseminated melanoma, compared with those of healthy adults. The acetylated derivative of GD3, which is highly specific for melanoma cells, was not detected in serum. This sensitive assay allows the quantitation of tumor-associated gangliosides that are circulating in sera of melanoma patients.

Topics: Adult; Antibodies, Monoclonal; Autoradiography; Biomarkers, Tumor; Chromatography, Thin Layer; Gangliosides; Humans; Immunoglobulin Fab Fragments; Immunoglobulin G; Immunohistochemistry; Iodine Radioisotopes; Melanoma

Expression of the gangliosides GM3, GD3 and GD2 was studied in tissue sections from 19 naevi, 29 primary and 83 metastatic melanoma using the ABC immunoperoxidase technique. GM3 was not detected in normal skin whereas GD2 was detected on the basal and stratum spinosum of the epidermis and on peripheral nerves in the dermis. GD3 was expressed on melanocytes but not on most other components of normal skin. However, GD3 was strongly expressed on epidermis adjacent to naevi and primary melanoma whereas GD2, in contrast to that in normal skin, was not expressed on the epidermis adjacent to 26/29 primary melanoma. All naevi were positive for GM3 and GD3 except that GM3 was not detected on junctional components of naevi. GD2 was not expressed on naevi except in areas showing neuroid differentiation. Studies on melanoma revealed that approximately 60% of primary and 75% of metastatic melanoma expressed GM3 to a varying extent. With 2 exceptions, all primary and metastatic melanomas expressed GD3 although there was variable expression within most of the individual tumours. GD2 was detected in only approximately 25% of primary and 50% of metastatic melanomas. Both GD2 and GD3 were detected on lymphocytes surrounding melanoma. The higher expression of GD2 on metastases compared to primary melanomas was consistent with the view that GD2 expression was associated with increased metastatic potential. However, the low proportion of metastases expressing GD2 and the absence of any correlation with thickness of the primary tumour suggested that GD2 expression was not a reliable marker of metastatic potential. No differences could be detected in ganglioside expression on metastases in skin or lymph nodes. These results appear to have implications for the use of MAbs against gangliosides in therapy of melanoma and in the study of melanocytic differentiation.

Topics: Antigens; G(M3) Ganglioside; Gangliosides; Humans; In Vitro Techniques; Melanoma; Nevus; Skin; Skin Neoplasms

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.

Topics: Antibodies, Monoclonal; Binding Sites; Carbohydrate Sequence; Epitopes; Gangliosides; Humans; Killer Cells, Natural; Melanoma

The murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, activates human complement, is active in antibody-dependent cell-mediated cytotoxicity (ADCC), and can target specifically to human neuroblastoma in patients with metastatic disease. In a phase I study, 3F8 was administered intravenously (IV) to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma at doses of 5, 20, 50, and 100 mg/m2. Serum 3F8 levels achieved were proportional to the dose of 3F8 infused. However, serum antimouse antibody levels did not increase with the amount of 3F8 administered. Toxicities included pain, hypertension, urticaria, and complement depletion. All acute side effects were controllable with symptomatic therapy. No long-term side effects were detected in patients observed for more than 14 months. None of the 17 patients received any antitumor therapy postantibody treatment. Antitumor responses occurred in seven of 17 patients. These ranged from complete clinical remissions to mixed responses. The murine monoclonal antibody (MoAb) 3F8 has clinical utility for the diagnosis and therapy of neuroblastoma and melanoma.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Child; Child, Preschool; Drug Evaluation; Female; Gangliosides; Humans; Infant; Male; Melanoma; Middle Aged; Neuroblastoma

A monoclonal antibody is described that specifically detects the ganglioside antigens GD2 and GD3, binding preferentially to GD2, in melanoma. Antibody specificity was demonstrated with solid-phase radioimmunoassay and enzyme-linked immunosorbent assay as well as by immunostaining on thin-layer chromatography plates using structurally characterized gangliosides. Binding of both the IgG3 antibody and its IgG2a switch variant were assayed on live cells by cytofluorography and by immunoperoxidase staining on frozen tissue sections. The binding patterns correlated with antitumor activity in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays with human effector cells and complement in an 111In-release assay using cell lines derived from the same individual. The significant level of killing in all tumor cells tested that express GD2, GD3, or both, suggests the importance of multiple specificity towards tumor antigens, i.e., binding of a monoclonal antibody to two or more tumor-associated antigens.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Complement System Proteins; Cytotoxicity, Immunologic; Enzyme-Linked Immunosorbent Assay; Gangliosides; Humans; Melanoma; Mice; Mice, Inbred BALB C

Human melanoma cells express relatively large amounts of the disialogangliosides GD3 and GD2 on their surface whereas neuroblastoma cells express GD2 as a major ganglioside. Monoclonal antibodies (Mabs) directed specifically to the carbohydrate moiety of GD3 and GD2 inhibit melanoma and neuroblastoma cell attachment to various substrate adhesive proteins, e.g. collagen, vitronectin, laminin, fibronectin, and a heptapeptide, glycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-prolyl-L-cysteine, which constitutes the cell attachment site of fibronectin. Cells that are preattached to a fibronectin substrate can also be induced to detach and round up in the presence of purified anti-ganglioside Mab. Moreover, when melanoma cells that contain both GD2 and GD3 are incubated with Mabs directed to both of these molecules an additive inhibition is observed. The specificity of this inhibition is demonstrated since Mabs of various isotypes directed to either protein or carbohydrate epitopes on a number of other major melanoma or neuroblastoma cell surface antigens have no effect on cell attachment. A study of the kinetics involved in this inhibition indicates that significant effects occur during the first 5 min of cell attachment, suggesting an important role for GD2 and GD3 in the initial events of cell-substrate interactions. The role of gangliosides in cell attachment apparently does not directly involve a strong interaction with fibronectin since we could not observe any binding of radiolabeled fibronectin or fragments of the molecule known to contain the cell attachment site to melanoma gangliosides separated on thin-layer chromatograms. An alternative explanation would be that gangliosides may play a role in the electrostatic requirements for cell-substrate interactions. In this regard, controlled periodate oxidation of terminal, unsubstituted sialic acid residues on the cell surface not only specifically destroys the antigenic epitopes on GD2 and GD3 recognized by specific Mabs but also inhibits melanoma cell and neuroblastoma cell attachment. In fact, the periodate-induced ganglioside oxidation and the inhibition of cell attachment are equally dose dependent. These data suggest that cell-substratum interactions may depend in part on the electrostatic environment provided by terminal sialic acid residues of cell surface gangliosides and possibly other anionic glycoconjugates.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antigens, Surface; Cell Adhesion; Cell Line; Extracellular Matrix; Fibronectins; Gangliosides; Humans; Laminin; Lung Neoplasms; Melanoma; Neuroblastoma; Periodic Acid

Human melanoma cells (M21) actively attach and spread on a fibronectin substrate. Indirect immunofluorescence assays with specific monoclonal antibodies directed to the disialoganglioside GD2, the major ganglioside expressed on M21 melanoma cells, indicate that during the cell attachment process this molecule redistributes into microprocesses that make direct contact with the fibronectin substrate. Scanning and transmission immunoelectron microscopic studies with anti-GD2 monoclonal antibodies and immuno-gold staining demonstrate that GD2 preferentially localizes into substrate-associated microprocesses that emanate from the plasma membrane of the M21 cells. Staining with monoclonal antibodies directed to other melanoma surface antigens fails to demonstrate a similar distribution pattern on these cells. Direct evidence is provided that GD2 is involved in M21 cell attachment to fibronectin, since treatment of these cells with anti-GD2 monoclonal antibodies causes cell rounding and detachment from a fibronectin substrate. Moreover, scanning electron microscopy demonstrates that this loss of attachment of fibronectin is characterized by a perturbation of the cell attachment-promoting microprocesses that in the presence of these antibodies lose contact with the fibronectin substrate.

Topics: Antibodies, Monoclonal; Antibody Specificity; Cell Adhesion; Cell Membrane; Edetic Acid; Extracellular Matrix; Fibronectins; Gangliosides; Humans; Melanoma; Microscopy, Electron, Scanning

The cell membranes of human melanoma express a tumor-associated ganglioside, GD2. We previously established a human melanoma cell line, M14-A, that metastasizes to the lung, liver, skin, lymph nodes, and abdominal organs of nude mice in addition to forming ascites and pleural effusions. We also reported the successful in vitro production of human IgM monoclonal antibody to GD2. In the present study, we evaluated the GD2 expression of human melanoma cells at the primary and metastatic sites and their reactivity to human monoclonal anti-GD2 antibody in vivo. GD2 was expressed strongly on the melanoma cells from both primary and metastatic sites, except for cells from pleural effusions and ascites. When M14-A-bearing nude mice received systemic injections of the human monoclonal antibody, the anti-GD2 titer in the sera was reduced markedly at 2 hours, whereas the reduction was minimal in sera from tumor-free mice and mice bearing GD2-negative human M24 cells. The immune adherence test confirmed that antibody was fixed on cells of primary subcutaneous M14-A tumors and on their metastases to liver, lung, abdominal organs and skin. These results suggest that this large molecule protein can penetrate the blood-tumor barrier and bind immunologically to antigen-positive melanoma cells in vivo.

Topics: Animals; Antibodies, Monoclonal; Complement System Proteins; Female; Fluorescent Antibody Technique; Gangliosides; Guinea Pigs; Humans; Immune Adherence Reaction; Immunoglobulin M; Male; Melanoma; Mice; Mice, Inbred Strains; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Rosette Formation; Transplantation, Heterologous

Patients with stage II melanoma were vaccinated with vaccinia virus-induced melanoma cell lysates (VMCL). The vaccine contained viable vaccinia virus, membranous fragments and no intact nuclei. A number of antigens defined by monoclonal antibodies were detected in the vaccine including the ganglioside GD3 and DR antigens. Administration of the vaccine was associated with depression of natural killer cell activity against melanoma and K562 target cells in the first 3-6 months of treatment. Leucocyte dependent antibody (LDA) activity against melanoma cells was induced or increased in titre in approximately half of the patients studied. Continued vaccination was associated in a number of patients with a decrease in LDA titres. Studies on a small sample of patients revealed that this was associated with the development of serum factors which inhibited LDA activity. LDA activity appeared directed to non-MHC antigens on melanoma cells which were of at least two specificities. One specificity which was shared with antigens on a number of non-melanoma carcinoma cells was removed by absorption on fetal brain and may be similar to oncofetal antigens described by other workers. Reactivity against melanocytes was induced in some patients and may underline the development of vitiligo in several patients. These results suggest that vaccines prepared from VMCL may be a favourable method for increasing immune responses against melanoma.

Topics: Antibodies, Neoplasm; Antibody Specificity; Antigens, Viral; Combined Modality Therapy; Cytotoxicity, Immunologic; Female; Gangliosides; Histocompatibility Antigens Class II; HLA Antigens; HLA-DR Antigens; Humans; Immunization; Killer Cells, Natural; Leukocytes; Male; Melanoma; Vaccinia virus; Viral Vaccines

In this study we used human monoclonal antibody (Hu-mAb) L72 as an intratumoral injection of cutaneous metastasis of melanoma to study its anti-tumor effects in human patients. Hu-mAb L72 was developed by transforming peripheral blood lymphocytes from a melanoma patient in vitro with the Epstein-Barr virus, forming a human lymphoblastoid cell line that produces 2-5 micrograms of IgM per ml. This IgM Hu-mAb was shown to react specifically with ganglioside GD2 and have a strong cytotoxic effect on human melanoma cells in the presence of complement. Patients with cutaneous metastatic melanoma were given intralesional injections on a daily or weekly injection schedule. Regression was seen in all tumors except in those of two patients whose tumors were shown to have low antigenicity. Histopathological data showed tumor degeneration, fibrosis, free melanin, and some degree of lymphocyte or macrophage infiltration. One patient with melanoma satellitosis treated with Hu-mAb showed complete regression with no sign of recurrence 20 months after the initial treatment. With the exception of mild erythema, no side effects were observed in any patient.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Female; Gangliosides; Humans; Immunoglobulins; Male; Melanoma; Middle Aged; Skin Neoplasms

Gangliosides from cell cultures established from melanocytic lesions, representing different stages of melanoma tumor progression, were analyzed by chemical and immunological means on thin-layer chromatograms. The GD2 ganglioside and N-acetylgalactosaminyl transferase, which catalyzes the biosynthesis of GD2 from its precursor GD3, were detected in cultures established from advanced primary and metastatic melanomas, but not in cultures of normal melanocytes. Immunohistochemical studies on tissue sections from all progression stages confirmed GD2 expression only in these advanced lesions. A distinct biochemical event thus coincides with the onset of faster growth and acquisition of metastatic competence in human melanoma tumor progression.

Topics: Autoradiography; Cells, Cultured; Chromatography, Thin Layer; Gangliosides; Histocytochemistry; Humans; Immunoenzyme Techniques; Melanoma

Human IgM kappa antibody to a membrane antigen of human tumors of neuroectodermal origin (melanoma, glioma and neuroblastoma) has been detected in the spent culture fluid of an Epstein-Barr virus (EBV)-transformed human B-lymphoblastoid cell line, L72. The chemical nature of the antigen was identified as ganglioside GD2. The antibody was purified by precipitation of L72 culture fluid with ammonia sulfate and hypotonic buffer followed by ultracentrifugation and Sephacryl S-300 super gel filtration. Approximately 27 mg of pure human IgM was obtained from 101 of spent medium. Total IgM and antibody activity recovery efficiency was 60% and 75%, respectively. The monoclonal character of the immunoglobulin produced by the L72 cell line was determined by agarose isoelectric focusing and immunofixation techniques. 1 mg of the purified IgM possessed an antibody titer endpoint to a GD2-positive melanoma cell line of 1:10,000 as assayed by immune adherence and 1:100 titer by complement-dependent cytotoxicity in vitro. The effect of pure anti-GD2 on suppression of melanoma growth in vivo was tested using a nude mouse model. Three-week-old CD-1 nude mice bearing 2-3 mm M14-A subcutaneous melanoma nodules were treated intraperitoneally with anti-GD2 and rabbit complement. Tumor growth was retarded for 25 days when compared to that of control mice receiving non-specific human IgM and complement. On Day 15, treated tumors were 80% smaller than control tumors. These result indicated that the pure human monoclonal antibody to GD2 may have potential for cancer therapy.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Cell Line; Cell Transformation, Viral; Complement System Proteins; Cytotoxicity, Immunologic; Gangliosides; Herpesvirus 4, Human; Humans; Immunoglobulin M; Melanoma; Mice; Mice, Nude

The predominant gangliosides produced by two cultured human melanoma cell lines are GD3 and/or GD2. These gangliosides were found to be cell associated and present in substratum-attached material after cell removal by EDTA. Monoclonal antibodies directed to GD2 and GD3 specified the cell-surface distribution of these gangliosides and localized them in focal adhesion plaques at the interface of cells and their substratum. These attachment sites did not represent indiscriminant membrane fragments remaining after removal of cells with EDTA, because neither melanoma-associated proteoglycan nor class I histocompatibility antigens were detected by their respective antibodies. Our data suggest that the disialogangliosides GD2 and GD3 may be involved in the interaction between human melanoma cells and solid substrata.

Topics: Cell Adhesion; Cell Line; Cell Membrane; Chromatography, Thin Layer; Fluorescent Antibody Technique; Gangliosides; Humans; Melanoma; Membrane Lipids

A human IgM monoclonal antibody was produced in vitro against OFA-I-2, a human tumor membrane antigen. This antigen is expressed on tumors of neuroectodermal origin, and has been identified as the ganglioside GD2. This study examines the anti-tumor effect of the monoclonal antibody against a GD2-positive human melanoma cell line, M14, inoculated subcutaneously into athymic CD-1 nude mice. Tumor-free survival was prolonged markedly when the monoclonal antibody and M14 cells were inoculated simultaneously. When antibody and complement were also injected into established tumor nodules, M14 tumor growth was suppressed. However, intraperitoneal injection of the antibody did not alter the growth of the subcutaneously inoculated M14 cells. The antibody has no effect on the growth of a GD2-negative melanoma cell line, M24. These results indicate that the human monoclonal antibody to GD2 may be useful for the suppression of GD2-positive tumor cells in cancer patients if the tumor can be directly exposed to the antibody and complement.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Antigens, Surface; Cell Line; Complement System Proteins; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunization, Passive; Immunoglobulin M; In Vitro Techniques; Male; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

Human IgM kappa monoclonal antibody (anti-GD2) to a tumor antigen, ganglioside GD2, has been produced by Epstein-Barr virus-transformed human B lymphoblasts. In the present study, we demonstrated the anti-tumor effects of passively administered anti-GD2 against an ascites-form human melanoma cell line, M14-A, transplanted into athymic nude mice. M14-A expresses GD2 in vivo. Cells (5 X 10(5) were inoculated intraperitoneally (i.p.) or subcutaneously (s.c.) into CD-1 nude mice that had received i.p. injections of 200 micrograms anti-GD2 and rabbit complement. Significant tumor-free intervals were observed in the treated mice (P less than 0.005) for i.p. tumors and P less than 0.025 for s.c. tumors). M14-A formed well-vascularized s.c. tumors if injected into young nude mice. Three-wk-old CD-1 nude mice bearing 2-3 mm M14-A s.c. tumor nodules were treated i.p. with anti-GD2 and rabbit complement. Tumor growth was delayed for 25 days. On day 15, treated tumors were 20% the size of control tumors. Because most biopsied or autopsied melanomas express GD2, and because patients with melanoma produce autoantibodies to GD2, the results in this study may provide important information for future passive immunization with human monoclonal antibody and for active specific immunization with GD2 antigen.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Antigens, Surface; Cell Line; Gangliosides; Humans; Immunization, Passive; Immunoglobulin M; Melanoma; Mice; Mice, Inbred Strains; Mice, Nude