g(m2)-ganglioside and Melanoma

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Autoantibodies; Autoantigens; Autoimmune Diseases of the Nervous System; Brain Chemistry; Carbohydrate Sequence; Epitopes; G(M1) Ganglioside; G(M2) Ganglioside; Galactosylceramides; Gangliosides; Globosides; Glycolipids; Glycosphingolipids; Humans; Immune Tolerance; Immunoglobulin G; Immunoglobulin M; Melanoma; Melanoma, Experimental; Mice; Molecular Sequence Data; Molecular Structure; N-Acetylneuraminic Acid; Oligosaccharides; Paraneoplastic Syndromes, Nervous System

Gangliosides are neuraminic acid-containing glycosphingolipids that are anchored into the cell membrane lipid bilayer by lipophilic ceramide chains. They are overexpressed on tissues of neuroectodermal origin, and particularly in tumors such as melanomas, sarcomas, neuroblastomas, astrocytomas, and small cell lung cancers. Both active and passive immunotherapy trials have identified gangliosides as uniquely effective targets for antibody mediated melanoma immunotherapy. Induction of antibodies against GM2 by vaccination has correlated with an improved prognosis in American Joint Committee on Cancer (AJCC) stage III melanoma patients and vaccines containing GM2 chemically conjugated to keyhole limpet hemocyanin (KLH; GM2-KLH) plus the immunologic adjuvant QS-21 have proven to be consistently immunogenic. Phase III trials with this vaccine are ongoing in patients with melanoma in the United States, Canada, Europe, Australia, and New Zealand. GD2, fucosylated GMI, and GD3-KLH conjugates plus QS-21 are also consistently immunogenic, inducing IgM and IgG antibodies in the majority of patients. Polyvalent ganglioside-KLH conjugate plus QS-21 vaccines should be available in early 1999 for testing in phase II and III clinical trials.

Topics: Adjuvants, Immunologic; Antibody Formation; Antigens, Neoplasm; Antigens, Surface; Cancer Vaccines; Clinical Trials as Topic; Epitopes; G(M2) Ganglioside; Gangliosides; Hemocyanins; Humans; Immunotherapy; Melanoma; Saponins; Vaccines, Conjugate

Topics: Adjuvants, Immunologic; Antineoplastic Agents; Cancer Vaccines; Chemotherapy, Adjuvant; Clinical Trials as Topic; Disease-Free Survival; Dose-Response Relationship, Immunologic; Drug Combinations; Female; G(M2) Ganglioside; Humans; Interferon Type I; Interferon-gamma; Interleukin-2; Male; Melanoma; Multicenter Studies as Topic; Neoplasm Staging; Recombinant Proteins; Skin Neoplasms; Vaccines, Conjugate

The factors involved in constructing vaccines for clinical testing are shown in Figure 3. The definition of melanoma antigens which are likely to be immunogenic is greatly facilitated by identification of reactive antibodies in sera or human hybridoma supernatants. It is also possible that antigens defined by murine monoclonal antibodies or extraction of melanoma specimens will be proven immunogenic with appropriately constructed vaccines, but this is unlikely unless immunohistologic testing and extraction of normal tissues show lack of expression on normal cells. With the use of purified antigens it has been possible to immunize with more antigen and to present the antigen in a more immunogenic fashion. On the other hand, the use of whole tumor cells or cell fractions provides the possibility of obtaining immune responses against antigens not previously known to be immunogenic, especially if linked with the production of human monoclonal antibodies. Experimental models for vaccine construction play a critical role in the selection of optimal adjuvants or vehicles for antigen presentation and for comparing different approaches to anti suppressor cell treatment. (Formula: see text). The ability to consistently induce high titer antibody responses against a single antigen, the ganglioside GM2, represents a foot in the door of active specific immunotherapy against malignant melanoma in man. When we are able to immunize as well against 2 or 3 additional melanoma antigens, the door will be open for testing the hypothesis that active specific immunotherapy can play a role in preventing recurrence or treating measurable cancer in man.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Clinical Trials as Topic; Cyclophosphamide; G(M2) Ganglioside; Humans; Immunity, Active; Immunization; Immunotherapy; Melanoma; Mice; Neoplasms; Neoplasms, Experimental

We evaluated Eastern Cooperative Group phase II and III trials E2696 and E1694 to assess the incidence and prognostic significance of autoimmunity induced by adjuvant high-dose interferon-α2b (HDI). In E2696, patients with resectable high-risk melanoma were randomized to receive vaccination with GM2-KLH/QS-1 (GMK) plus concurrent HDI, GMK plus sequential HDI, or GMK alone. E1694 randomized patients to either HDI or GMK. Sera from 103 patients in E2696 and 691 patients in E1694 banked at baseline and up to three subsequent time points were tested by ELISA for the development of five autoantibodies. In E2696, autoantibodies were induced in 16 patients (23.2%; n=69) receiving HDI and GMK and two patients (5.9%; n=34) receiving GMK alone (P=0.031). Of 691 patients in E1694, 67 (19.1%) who received HDI (n=350) developed autoantibodies, but only 16 patients (4.7%) developed autoantibodies in the vaccine group (n=341; P<0.001). Almost all induced autoantibodies were detected at ≥12 weeks after the initiation of therapy. A 1-year landmark analysis among resected stage III patients treated with HDI in E1694 showed a trend toward a survival advantage associated with HDI-induced autoimmunity (hazard ratio=0.80; 95% confidence interval: 0.50-1.98; P=0.33). Therefore, adjuvant HDI therapy is associated with the induction of autoimmunity that should be further investigated prospectively as a surrogate marker of adjuvant therapeutic benefit. This potential biomarker develops over the course of up to 1 year, and cannot be used to alter the course of therapy. Studies of the genetic determinants of this response may better discriminate patients more likely to benefit from HDI immunomodulatory therapy.

Topics: Autoantibodies; Autoimmunity; Cancer Vaccines; Chemotherapy, Adjuvant; Disease-Free Survival; Enzyme-Linked Immunosorbent Assay; G(M2) Ganglioside; Hemocyanins; Humans; Interferon-alpha; Melanoma; Prognosis; Skin Neoplasms

The GM2 ganglioside is an antigen expressed in the majority of melanomas. The GM2-KLH/QS-21 vaccine induces high immunoglobulin M (IgM) and IgG antibody responses. The EORTC 18961 trial compared the efficacy of GM2-KLH/QS-21 vaccination versus observation.. A total of 1,314 patients with a primary tumor > 1.50 mm in thickness (T3-4N0M0; American Joint Committee on Cancer stage II) were randomly assigned to GM2-KLH/QS-21 vaccination (n = 657) or observation (n = 657). Treatment consisted of subcutaneous injections once per week from week 1 to 4, then every 3 months for the first 2 years and every 6 months during the third year. Primary end point was relapse-free survival (RFS). Secondary end points were distant metastasis-free survival (DMFS) and overall survival (OS). Analyses were by intent to treat.. After a median follow-up of 1.8 years, the trial was stopped at the second interim analysis for futility regarding RFS (hazard ratio [HR], 1.00; P = .99) and detrimental outcome regarding OS (HR, 1.66; P = .02). After a median follow-up of 4.2 years, we had recorded 400 relapses, nine deaths without relapse, a total of 236 deaths. At 4 years, the vaccination arm showed a decreased RFS rate of 1.2% (HR, 1.03; 95% CI, 0.84 to 1.25) and OS rate of 2.1% (HR, 1.16; 95% CI, 0.90 to 1.51). Toxicity was acceptable, with 4.6% of patients ending study participation because of toxicity.. GM2-KLH/QS-21 vaccination does not improve outcome for patients with stage II melanoma.

Topics: Adjuvants, Immunologic; Adult; Aged; Aged, 80 and over; Cancer Vaccines; Disease-Free Survival; Female; G(M2) Ganglioside; Hemocyanins; Humans; Kaplan-Meier Estimate; Lymph Nodes; Lymphatic Metastasis; Male; Melanoma; Middle Aged; Neoplasm Staging; Saponins; Skin Neoplasms; Watchful Waiting

High-dose interferon alfa-2b (IFNalpha2b) is the only established adjuvant therapy of resectable high-risk melanoma. GM2-KLH/QS-21 (GMK) is a chemically defined vaccine that is one of the best developed of a range of vaccine candidates for melanoma. A single-institution phase III trial conducted at Memorial Hospital served as the impetus for an intergroup adjuvant E1694/S9512/C509801 trial, which recently completed enrollment of 880 patients. To build on the apparent benefit of IFNalpha2b in resectable high-risk American Joint Committee on Cancer (AJCC) stage IIB or III melanoma, this phase II study was designed to evaluate the combination of GMK and IFNalpha2b. The E2696 trial was undertaken to evaluate the toxicity and other effects of the established adjuvant high-dose IFNalpha2b regimen in relation to immune responses to GMK and to evaluate the potential clinical and immunologic effects of the combined therapies.. This trial enrolled 107 patients with resectable high- or very high-risk melanoma (AJCC stages IIB, III, and IV).. The results demonstrate that IFNalpha2b does not significantly inhibit immunoglobulin M or G serologic responses to the vaccine and that the combination of high-dose IFNalpha2b and GMK is well tolerated in this patient population.. Cox analysis of the results of the combination with IFNalpha2b show improvement in the relapse-free survival of patients with very high-risk melanoma (including those with resectable M1 disease).

Topics: Adult; Antibody Formation; Antineoplastic Agents; Cancer Vaccines; Chemotherapy, Adjuvant; Combined Modality Therapy; Disease-Free Survival; Female; G(M2) Ganglioside; Humans; Interferon alpha-2; Interferon-alpha; Male; Melanoma; Middle Aged; Neoplasm Recurrence, Local; Recombinant Proteins; Risk Factors; Skin Neoplasms

Vaccine alternatives to high-dose interferon alfa-2b therapy (HDI), the current standard adjuvant therapy for high-risk melanoma, are of interest because of toxicity associated with HDI. The GM2 ganglioside is a well-defined melanoma antigen, and anti-GM2 antibodies have been associated with improved prognosis. We conducted a prospective, randomized, intergroup trial to evaluate the efficacy of HDI for 1 year versus vaccination with GM2 conjugated to keyhole limpet hemocyanin and administered with QS-21 (GMK) for 96 weeks (weekly x 4 then every 12 weeks x 8).. Eligible patients had resected stage IIB/III melanoma. Patients were stratified by sex and number of positive nodes. Primary end points were relapse-free survival (RFS) and overall survival (OS).. Eight hundred eighty patients were randomized (440 per treatment group); 774 patients were eligible for efficacy analysis. The trial was closed after interim analysis indicated inferiority of GMK compared with HDI. For eligible patients, HDI provided a statistically significant RFS benefit (hazard ratio [HR] = 1.47, P = .0015) and OS benefit (HR = 1.52, P = .009) for GMK versus HDI. Similar benefit was observed in the intent-to-treat analysis (RFS HR = 1.49; OS HR = 1.38). HDI was associated with a treatment benefit in all subsets of patients with zero to > or = four positive nodes, but the greatest benefit was observed in the node-negative subset (RFS HR = 2.07; OS HR = 2.71 [eligible population]). Antibody responses to GM2 (ie, titers > or = 1:80) at days 29, 85, 365, and 720 were associated with a trend toward improved RFS and OS (P2 = .068 at day 29).. This trial demonstrated a significant treatment benefit of HDI versus GMK in terms of RFS and OS in melanoma patients at high risk of recurrence.

Topics: Adult; Aged; Cancer Vaccines; Disease-Free Survival; Female; G(M2) Ganglioside; Hemocyanins; Humans; Immunoglobulin G; Immunoglobulin M; Interferon alpha-2; Interferon-alpha; Male; Melanoma; Middle Aged; Neoplasm Staging; Prospective Studies; Recombinant Proteins; Saponins; Vaccination; Vaccines, Conjugate

In a previous randomized Phase III trial (P. O. Livingston et al, J. Clin. Oncol., 12: 1036-1044, 1994), we demonstrated that immunization with GM2 and bacille Calmette-Guerin reduced the risk of relapse in stage III melanoma patients who were free of disease after surgical resection and who had no preexisting anti-GM2 antibodies. That vaccine formulation induced IgM anti-GM2 antibodies in 74% but induced IgG anti-GM2 antibodies in only 10% of the patients. To optimize the immune response against GM2, a reformulated vaccine was produced conjugating GM2 to keyhole limpet hemocyanin (KLH) and using the adjuvant QS21 (GM2-KLH/QS21). In pilot studies, 70 microg of vaccine induced IgG anti-GM2 antibodies in 76% of the patients. We wished to define the lowest vaccine dose that induced consistent, high-titer IgM and IgG antibodies against GM2. Fifty-two melanoma patients who were free of disease after resection but at high risk for relapse were immunized with GM2-KLH/QS21 vaccine at GM2 doses of 1, 3, 10, 30, or 70 ILg on weeks 1, 2, 3, 4, 12, 24, and 36. Serum collected at frequent and defined intervals was tested for anti-GM2 antibodies. Overall, 88% of the patients developed IgM anti-GM2 antibodies; 71% also developed IgG anti-GM2 antibodies. GM2-KLH doses of 3-70 microg seemed to be equivalent in terms of peak titers and induction of anti-GM2 antibodies. At the 30-microg dose level, 50% of the patients developed complement fixing anti-GM2 antibodies detectable at a serum dilution of 1:10. We conclude that the GM2-KLH/QS21 formulation is more immunogenic than our previous formulation and that 3 microg is the lowest dose that induces consistent, high-titer IgM and IgG antibodies against GM2.

Topics: Adult; Aged; Animals; Antibody Formation; Cancer Vaccines; Cattle; Dose-Response Relationship, Drug; Fatigue; Female; Fever; G(M2) Ganglioside; Humans; Immunization; Immunoglobulin G; Immunoglobulin M; Inflammation; Male; Melanoma; Middle Aged; Neoplasm Staging; Pain; Pruritus; Time Factors; Tumor Cells, Cultured; Vaccines, Conjugate

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

The cell surface gangliosides GM2, GD2, and GD3 are often overexpressed in malignant melanoma. We have shown previously that immunization of melanoma patients with GM2 and Bacillus Calmette-Guérin induced an IgM antibody response in most patients and that patients with high titer GM2 antibodies showed increased survival. As is commonly seen with carbohydrate antigens (which are T independent), the IgM response was short lived, and an IgG response was rarely observed. To increase immunogenicity, we conjugated GM2 covalently with keyhole limpet hemocyanin (KLH). GM2-KLH vaccine was given to melanoma patients alone or with one of the three adjuvants: Bacillus Calmette-Guérin, DETOX, or QS-21. The most effective vaccine was GM2-KLH with QS-21. It induced a much higher titer, a longer-lasting IgM GM2 antibody response, and a consistent IgG response (isotype IgG1 and IgG3). It also induced the highest titer anti-KLH response. The results suggest that the conjugate GM2-KLH plus QS-21 vaccine elicited significant T-cell help. Because there was no serious toxicity, this vaccine approach is attractive for augmenting the immunogenicity of other gangliosides, such as GD2 and GD3, and to determine the effects of ganglioside antibodies on the course of melanoma. In addition, the finding that QS-21 significantly increased the immunogenicity of GM2-KLH suggests that it may do the same for other conjugate vaccines, many of which are currently used without adjuvant.

Topics: Adjuvants, Immunologic; Antibody Formation; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; G(M2) Ganglioside; Hemocyanins; Humans; Immunoglobulin G; Immunoglobulin M; Melanoma; Vaccines

Increasing doses of saponin fraction QS-21 were administered as immunological adjuvant in a Phase 1 trial with a constant dose of the melanoma ganglioside GM2 covalently attached to keyhole limpet haemocyanin (KLH). Twenty-eight patients with AJCC Stage III or IV melanoma who were free from disease after surgery were treated with six vaccinations administered subcutaneously over a 5-month period. Local and systemic reactions were QS-21 dose-related. Doses of < or = 100 micrograms induced mild local tenderness and inflammation at vaccination sites lasting 2-4 days and occasional brief low-grade fever and malaise, but no significant incapacitation. The 200 micrograms dose induced low-grade fever and malaise after 30% of vaccinations and local reactions as large as 20 cm in diameter were seen in all patients, resulting in discomfort with usage of the injected extremity for 5-10 days. The titres of IgM and IgG antibodies against GM2, and IgG antibodies against KLH, were highest at the 100 and 200 micrograms QS-21 doses. No antibodies against QS-21 were detected. This trial identifies the 100 micrograms dose of QS-21 as the optimal well tolerated dose for induction of antibodies against both the melanoma ganglioside/GM2 and the protein KLH in melanoma patients.

Topics: Adjuvants, Immunologic; Antibodies, Neoplasm; G(M2) Ganglioside; Hemocyanins; Humans; Hypersensitivity, Delayed; Melanoma; Saponins

To perform a double-blind randomized trial with American Joint Commission on Cancer (AJCC) stage III melanoma patients for the following reasons: (1) to confirm our previous finding that patients with antibodies against the melanoma differentiation antigen GM2 have an improved prognosis, and (2) to demonstrate clinical benefit from GM2 antibody induction.. One hundred twenty-two patients with AJCC stage III melanoma who were free of disease after surgery were randomized: 58 to receive treatment with the GM2/BCG vaccine, and 64 to receive treatment with bacille Calmette-Guèrin (BCG) alone. All patients were pretreated with low-dose cyclophosphamide (Cy).. GM2 antibody was detected in 50 of 58 patients treated with GM2/BCG and seven of 64 patients treated with BCG alone. With a minimum follow-up period of 51 months, there was a highly significant increase in the disease-free interval (P = .004) and a 17% increase in overall survival (P = .02) in these 57 antibody-positive patients, confirming our earlier experience. Exclusion of all patients with preexisting GM2 antibodies (one in the GM2/BCG group and five in the BCG group) from statistical analysis resulted in a 23% increase in disease-free interval (P = .02) and a 14% increase in overall survival (P = .15) at 51 months for patients treated with the GM2/BCG vaccine. However, when all patients in the two treatment groups were compared as randomized, these increases were 18% for disease-free interval and 11% for survival in the GM2/BCG treatment group, with neither result showing statistical significance.. (1) Vaccination with GM2/BCG induced immunoglobulin M (IgM) antibodies in most patients. (2) GM2 antibody production was associated with a prolonged disease-free interval and survival. (3) Comparison of the two arms of this trial as randomized fails to show a statistically significant improvement in disease-free interval or survival for patients treated with GM2/BCG vaccines.

Topics: Adolescent; Adult; Aged; Antibody Formation; Antigens, Differentiation; Antigens, Neoplasm; BCG Vaccine; Chemotherapy, Adjuvant; Double-Blind Method; Female; G(M2) Ganglioside; Humans; Immunoglobulin G; Immunoglobulin M; Male; Melanoma; Middle Aged; Survival Analysis

The factors involved in constructing vaccines for clinical testing are shown in Figure 3. The definition of melanoma antigens which are likely to be immunogenic is greatly facilitated by identification of reactive antibodies in sera or human hybridoma supernatants. It is also possible that antigens defined by murine monoclonal antibodies or extraction of melanoma specimens will be proven immunogenic with appropriately constructed vaccines, but this is unlikely unless immunohistologic testing and extraction of normal tissues show lack of expression on normal cells. With the use of purified antigens it has been possible to immunize with more antigen and to present the antigen in a more immunogenic fashion. On the other hand, the use of whole tumor cells or cell fractions provides the possibility of obtaining immune responses against antigens not previously known to be immunogenic, especially if linked with the production of human monoclonal antibodies. Experimental models for vaccine construction play a critical role in the selection of optimal adjuvants or vehicles for antigen presentation and for comparing different approaches to anti suppressor cell treatment. (Formula: see text). The ability to consistently induce high titer antibody responses against a single antigen, the ganglioside GM2, represents a foot in the door of active specific immunotherapy against malignant melanoma in man. When we are able to immunize as well against 2 or 3 additional melanoma antigens, the door will be open for testing the hypothesis that active specific immunotherapy can play a role in preventing recurrence or treating measurable cancer in man.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Clinical Trials as Topic; Cyclophosphamide; G(M2) Ganglioside; Humans; Immunity, Active; Immunization; Immunotherapy; Melanoma; Mice; Neoplasms; Neoplasms, Experimental

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; G(M2) Ganglioside; Heterografts; Humans; Male; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; N-Acetylgalactosaminyltransferases; Neovascularization, Pathologic; Neuroblastoma; RNA-Seq; Skin Neoplasms; Transfection; Tumor Burden

The GM2 ganglioside represents an important target for specific anticancer immunotherapy. We designed and synthesized a neoglycopeptide immunogen displaying one or two copies of the GM2 tetrasaccharidic moiety. These glycopeptides were prepared using the Huisgen cycloaddition, which enables the efficient ligation of the alkyne-functionalized biosynthesized GM2 with an azido CD4(+) T cell epitope peptide. It is worth noting that the GM2 can be produced on a gram scale in bacteria, which can be advantageous for a scale-up of the process. We show here for the first time that a fully synthetic glycopeptide, which is based on a ganglioside carbohydrate moiety, can induce human tumor cell-specific antibodies after immunization in mice. Interestingly, the monovalent, but not the divalent, form of GM2 peptide construct induced antimelanoma antibodies. Unlike traditional vaccines, this vaccine is a pure chemically-defined entity, a key quality for consistent studies and safe clinical evaluation. Therefore, such carbohydrate-peptide conjugate represents a promising cancer vaccine strategy for active immunotherapy targeting gangliosides.

Topics: Animals; Antibodies; Antibody Specificity; Carbohydrate Sequence; G(M2) Ganglioside; Humans; Jurkat Cells; Melanoma; Mice; Mice, Inbred BALB C; Molecular Sequence Data

Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Apoptosis; Carcinoma, Small Cell; Cricetinae; Epitopes; Female; G(M2) Ganglioside; Humans; Immunization, Passive; Jurkat Cells; Lung Neoplasms; Melanoma; Mice; Mice, Inbred AKR; Mice, SCID

This article proposes a new semiparametric Bayesian hierarchical model for the joint modeling of longitudinal and survival data. We relax the distributional assumptions for the longitudinal model using Dirichlet process priors on the parameters defining the longitudinal model. The resulting posterior distribution of the longitudinal parameters is free of parametric constraints, resulting in more robust estimates. This type of approach is becoming increasingly essential in many applications, such as HIV and cancer vaccine trials, where patients' responses are highly diverse and may not be easily modeled with known distributions. An example will be presented from a clinical trial of a cancer vaccine where the survival outcome is time to recurrence of a tumor. Immunologic measures believed to be predictive of tumor recurrence were taken repeatedly during follow-up. We will present an analysis of this data using our new semiparametric Bayesian hierarchical joint modeling methodology to determine the association of these longitudinal immunologic measures with time to tumor recurrence.

Topics: Bayes Theorem; Cancer Vaccines; Clinical Trials as Topic; G(M2) Ganglioside; Humans; Immunotherapy; Longitudinal Studies; Melanoma; Models, Biological; Models, Statistical; Neoplasm Recurrence, Local; Survival Analysis

Dendritic cell (DC) development and function is critical in the initiation phase of any antigen-specific immune response against tumours. Impaired function of DC is one explanation as to how tumours escape immunosurveillance. In the presence of various soluble tumour-related factors DC precursors lose their ability to differentiate into mature DC and to activate T cells. Gangliosides are glycosphingolipids shed by tumours of neuroectodermal origin such as melanoma and neuroblastoma. In this investigation we address the question of whether gangliosides suppress the development and function of monocyte-derived DC in vitro. In the presence of gangliosides, the monocytic DC precursors showed increased adherence, cell spreading and a reduced number of dendrites. The expression of MHC class II molecules, co-stimulatory molecules and the GM-CSF receptor (CD116) on the ganglioside-treated DC was significantly reduced. Furthermore, the function of ganglioside-treated DC was impaired as observed in endocytosis, chemotactic and T cell proliferation assays. In contrast to monocytic DC precursors, mature DC were unaffected even when higher doses of gangliosides were added to the culture. With regard to their carbohydrate structure, five different gangliosides (GM2, GM3, GD2, GD3, GT1b), which are typically shed by melanoma and neuroblastoma, were tested for their ability to suppress DC development and function. Suppression was induced by GM2, but not by the other gangliosides. These data suggest that certain gangliosides impair DC precursors, implying a possible mechanism for tumour escape.

Topics: Cell Adhesion; Cell Differentiation; Cells, Cultured; Chemotaxis, Leukocyte; Dendritic Cells; Endocytosis; Flow Cytometry; G(M2) Ganglioside; Gangliosides; HLA-D Antigens; Humans; Immunophenotyping; Lymphocyte Culture Test, Mixed; Melanoma; Monocytes; Neuroblastoma; Neuroectodermal Tumors; Tumor Escape

The incidence of melanoma is estimated to be growing at the second fastest rate among all cancers in the United States. The progression of the melanocyte to a malignant melanoma involves various sequential steps: development of benign naevocellular naevus, preneoplastic dysplastic naevus, primary melanoma, and metastatic melanoma. Despite these clearly defined stages, very little is known about the molecular events leading to melanoma progression. We established a human congenital naevus cell line (UISO-CMN-1). UISO-CMN-1 cells were confirmed to have melanocytic origin by S100 immunoreactivity and the presence of melanin granules and melanosomes. UISO-CMN-1 cells, even though they showed structural and numerical abnormalities in karyotype, were non-tumorigenic when transplanted into athymic mice. However, following frequent exposure to ultraviolet C radiation, UISO-CMN-1 cells acquired tumorigenic potential. Transformation of UISO-CMN-1 cells into tumorigenic cells was accompanied by induction of ganglioside-2 expression without any significant changes in cellular ganglioside-3. These transformed and non-transformed UISO-CMN-1 cell lines can serve as excellent research tools for studying the molecular changes associated with melanoma development and progression, and for identifying agents that might prevent development of malignant melanoma.

Topics: Animals; Cell Line; Cell Line, Transformed; Female; G(M2) Ganglioside; G(M3) Ganglioside; Humans; Immunohistochemistry; Infant; Karyotyping; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Nevus; Transformation, Genetic; Ultraviolet Rays

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 cells express ganglioside antigens GM3, GD3, GM2, and GD2 on their surface. This study examined whether immunization with a melanoma cell vaccine induced anti-ganglioside antibody responses in melanoma patients and whether these responses were correlated with survival. Sixty-six patients who had received melanoma cell vaccine immunotherapy after surgical removal of regional metastatic melanoma were identified. Cryopreserved serum samples from these patients were used in an enzyme-linked immunsorbent assay to determine the IgM antibody levels to GM2, GD2, GM3, and GD3 prior to melanoma cell vaccine treatment and 4 wk after the first melanoma cell vaccine immunization. All antibody levels significantly increased by week 4 (p < 0.001 for all four antibodies) and all increases were significantly associated with survival (anti-GD2, p < 0.001; anti-GM2, p = 0.001; anti-GD3, p < 0.001; anti-GM3, p < 0.001). Anti-tumor activity of these antibodies was proved using five representative antibody-positive sera in a complement-dependent cytotoxicity assay with cultured melanoma cell lines. These studies suggest that GM2, GD2, GM2, and GD3 expressed by melanoma cells can induce specific IgM antibodies and that high levels of these antibodies might have a beneficial impact on survival.

Topics: Antibodies, Anti-Idiotypic; Antibody Formation; Cancer Vaccines; Cytotoxicity, Immunologic; G(M2) Ganglioside; Humans; Immunoglobulin M; Melanoma; Survival Rate

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

Natural IgM antibodies against the melanoma cell-surface ganglioside GM2, and IgM antibodies induced by vaccination with GM2 adherent to bacillus Calmette-Guerin, have been correlated with increased disease-free and overall survival in melanoma patients in previous phase I and II clinical trials. A vaccine containing GM2 covalently attached to keyhole limpet hemocyanin (KLH) plus the immunological adjuvant QS-21 now induces higher-titer, longer-lasting IgM antibodies against GM2 and has recently entered phase III clinical trials. For the first time this new vaccine also induces IgG antibodies against GM2 in the majority of immunized patients. With regard to immunity against bacteria, IgM antibodies have been described to be 1000-fold more effective than IgG antibodies at opsonification, complement-mediated cytotoxicity and protection from bacterial challenge. Though IgG antibodies have the theoretical advantage of being able to mediate antibody-directed cell-mediated cytotoxicity (ADCC), they may inhibit complement mediated IgM effector mechanisms against melanoma cells. Our goal was to confirm the functional characteristics of the anti-GM2 IgM and IgG antibodies induced by vaccination and to determine the impact that IgG antibodies might have on IgM antibody reactivity with GM2-positive tumor cells. Post-immunization sera from seven immunized patients were separated by size-exclusion chromatography into IgM and IgG fractions and a variety of serological assays were performed with the individual fractions and their combinations. Assays identifying specific IgM or IgG reactivity demonstrated partial inhibition by the opposite fraction. However, when the endpoint was complement-mediated lysis or overall antibody binding, which may more faithfully predict in vivo complement-mediated opsonification and lysis, the combinations of IgM and IgG fractions consistently demonstrated higher reactivity than either fraction alone. In addition, ADCC was induced in all seven patients. The results were the same whether the sera were obtained after 2 months or 2 years of immunizations. These findings suggest that IgG antibodies induced by the GM2-KLH plus QS-21 vaccine will not inhibit and should further augment the clinical impact of induced IgM antibodies.

Topics: Antibodies, Neoplasm; Cancer Vaccines; G(M2) Ganglioside; Haptens; Hemocyanins; Humans; Immunoglobulin G; Immunoglobulin M; Melanoma

After treatment of melanomas with anti-GD2 monoclonal antibody (MAb) (14G2a), some patients develop sensorimotor demyelinating polyneuropathy with and without the syndrome of inappropriate antidiuretic hormone (SIADH). To clarify what causes the neurotoxicity of anti-GD2 MAb, we investigated the immunohistochemical localization of GD2 in the human nervous system. Anti-GD2 MAb (14G2a) reacted with the myelin sheaths in the peripheral nerves as well as with the pituicyte cytoplasm in the posterior lobe of the pituitary gland. We assume that the binding of anti-GD2 MAb to peripheral nerve myelin and the pituicytes in the posterior pituitary causes sensorimotor demyelinating neuropathy and SIADH.

Topics: Animals; Antibodies, Monoclonal; Biopsy; Central Nervous System; Demyelinating Diseases; G(M2) Ganglioside; Humans; Inappropriate ADH Syndrome; Melanoma; Mice; Myelin Sheath; Neurotoxins; Oculomotor Nerve; Peripheral Nervous System Diseases; Pituitary Gland; Sural Nerve; Tibial Nerve; Trochlear Nerve

A human B-lymphoblastoid cell clone, L55-81, that produces human monoclonal antibody (MAb) to ganglioside G(M2) was established from peripheral blood B lymphocytes of a melanoma patient. L55-81 secretes IgMkappa light chain antibody in a serum-free medium. G(M2) specificity of the antibody was tested by immune adherence assay, TLC immunostaining, and ELISA. Anti-G(M2) antibody was shown to have the ability to kill the G(M2)-rich human melanoma cell line M14 in the presence of human or rabbit complement. A purified L55-81 MAb (>99.5% purity in protein concentration) was biotinylated and tested for its reactivity to various histological-type biopsied tumor and normal tissues in an avidin-biotin detection system. L55-81 MAb (20 microg/ml) reacted with several types of tumor tissues such as melanoma (7 of 10), colon carcinoma (4 of 5), ovary carcinoma (4 of 5), breast carcinoma (1 of 5), kidney carcinoma (1 of 5), and prostate carcinoma (1 of 5). None of the normal tissues derived from 24 different organs and adjacent normal tissues surrounding the cancerous tissues were stained. Production of the antibody in a serum-free medium, the cytotoxic potential with human complement, the inability to react to normal tissues, and the ability to target antigen-specific target cells make L55-81 a potential therapeutic agent for the treatment of cancers expressing ganglioside G(M2).

Topics: Antibodies, Monoclonal; Antibody Specificity; B-Lymphocytes; Cell Line, Transformed; Epitopes; G(M2) Ganglioside; Humans; Immunotherapy; Melanoma; Tumor Cells, Cultured

Previous studies (Y. Nagata, S. Yamashiro, J. Yodoi, K. O. Lloyd, H. Shiku, and K. Furukawa (1992) J. Biol. Chem. 267, 12082-12089) had isolated a putative cDNA coding for beta 1,4 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and demonstrated the presence of GM2 and/or GD2 gangliosides in melanoma cell lines stably transfected with this gene. We have now measured the levels of glycosyltransferase activities and mRNA levels in five transfected cell lines in comparison with their parent cell lines (mouse melanoma B16 and human melanoma MeWo). Membrane preparations from the transfected cell lines demonstrated de novo synthesis of GM2 or GD2 in in vitro assays using GM3 or GD3, respectively, as ganglioside acceptors. The enzyme levels, however, varied considerably among the different transfectants, as did the mRNA levels for the beta 1,4 Gal-NAc-transferase. The effect of the transfected gene on levels of preexisting glycosyltransferases involved in ganglioside biosynthesis was also measured and the ganglioside composition of the cell lines was determined. The level of beta 1,4 GalNAc-transferase expressed in the different cell lines was found to dramatically influence the overall ganglioside composition of the cell. In the transfected cell line with the highest levels of the transferase, for example, biosynthesis was almost completely redirected away from the b pathway to the a pathway with the resulting expression of only GM2. These data from this family of related cell lines clearly demonstrate the primary roles that relative glycosyltransferase levels play in determining the ganglioside composition of cells.

Topics: Animals; Cell Membrane; G(M2) Ganglioside; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; N-Acetylgalactosaminyltransferases; Transfection; Tumor Cells, Cultured

Differential cell- and immuno-biological properties of two murine melanoma B16 variants, B16-F1 and F10, were investigated. Studies focused on the expression of proto-oncogene c-fos, sensitivities to LAK cells and/or IL-2, and modulation of the expression of ganglioside components after treatment with IL-2. Proto-oncogene c-fos was found to be highly expressed in F10 lines by an in situ hybridization technique and also in F10 lung metastatic nests by immunofluorescent staining with anti-c-fos antibody. F1 melanomas were more sensitive to local injection of IL-2. F10 melanomas hardly responded to IL-2 treatment, but successive injections of a combination of LAK cells and IL-2 did cause prolongation of survival rates, even of F10 melanoma-burdened mice. A major component of gangliosides of both F1 and F10 melanomas was GM3. Production of GM3 in F10 melanomas treated with IL-2 for 4 days increased, and, if the treatment was continued for 7 days, minor components of gangliosides, such as GM2, GM1, and GD1a, appeared only in F1 melanomas, while the increase of production of GM3 disappeared in both melanomas. These experimental results may provide clues for additional mechanisms which allow these two murine melanoma variants to show different implantation and metastasis rates.

Topics: Animals; Female; Fluorescent Antibody Technique; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Gene Expression Regulation, Neoplastic; Genes, fos; In Situ Hybridization; Interleukin-2; Killer Cells, Lymphokine-Activated; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Survival Rate; Tumor Cells, Cultured

Gangliosides, such as GM2, GD2, GD3, and 9-O-acetyl GD3, are receiving attention as targets for antibody-based and vaccine-based therapies of melanoma. GM2 appears to be a particularly immunogenic ganglioside in humans, as indicated by the presence of naturally occurring IgM anti-GM2 antibodies in approximately 5% of humans and the fact that immunization with irradiated GM2-expressing melanoma cells or purified GM2 adherent to bacillus Calmette-Guérin elicits GM2 antibodies of low to moderate titers in a high proportion of vaccinated patients. To develop vaccines that consistently induce high titers of IgM as well as IgG anti-GM2 antibodies, vaccines containing GM2 conjugated to keyhole limpet hemocyanin as the carrier protein and QS-21 as the adjuvant have been constructed. The serological response of vaccinated patients was monitored by ELISA using purified GM2 ganglioside for IgM and IgG anti-GM2 antibodies and for GM2 cell surface-reactive antibodies by immune adherence assays and cytotoxic tests (IgM antibodies) and mixed hemadsorption assays (IgG antibodies). The majority of vaccinated patients developed IgM and IgG antibodies detectable by ELISA. In most cases, the results of IgM ELISA correlated with assays for cell surface-reactive IgM antibodies. This was not true for IgG anti-GM2 antibodies, where strong discrepancies were seen between high titers in ELISA and little or no reactivity in mixed hemadsorption tests for cell surface-reactive antibodies. These IgG antibodies (and the less frequent IgM antibodies that show similar discrepancies) may be directed against GM2 determinants that are buried, hidden, or not present on GM2-expressing target cells. With regard to a major objective of ganglioside vaccines--i.e., generation of cytotoxic antibodies--the GM2-keyhole limpet hemocyanin/QS-21 vaccine is clearly superior to the previously tested GM2/bacillus Calmette-Guérin vaccine. However, variability in patient response and lack of persistence of high-titered IgM cytotoxic antibodies in many patients are problems that remain to be solved.

Topics: Adjuvants, Immunologic; Animals; Brain; Cats; Cattle; Cyclophosphamide; Enzyme-Linked Immunosorbent Assay; G(M2) Ganglioside; Hemocyanins; Immunoglobulin G; Immunoglobulin M; Melanoma; Monitoring, Immunologic; Neoplasm Staging; Tay-Sachs Disease; Time Factors; Vaccines, Synthetic

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

GM2 is usually significantly elevated in human melanoma cells. The lysosomal hydrolase beta-hexosaminidase A (Hex A), is an isoenzyme required for terminal GalNAc hydrolysis from intact GM2 to GM3. The objective of the present studies is to determine if the elevated levels of gangliosides, particularly GM2, correlate with the activity of Hex A in cultured melanoma cells. The Hex A activity in 13 melanoma cell lines ranged from 26%-88.3% There was a inverse correlation between GM2 nmole/g and Hex A activity (r = -0.48; p less than 0.003). An inverse correlation (p less than 0.03) occurred between GD2 nmole/g and Hex A activity. There was an inverse correlation (r = -0.53; p less than 0.05 and r = -0.51; p less than 0.05, respectively) between the ratio of substrate/product (GM2/GM3) and (GD2/GD3) and Hex A activity. These results indicate that the level of GM2 in melanoma is inversely correlated with the level of activity of Hex A.

Topics: Analysis of Variance; beta-N-Acetylhexosaminidases; Carbohydrate Sequence; G(M2) Ganglioside; Hexosaminidase A; Humans; Lysosomes; Melanoma; Molecular Sequence Data; 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

In order to investigate GM2 expression in gliomas, the GM2-positive human glioma cell line (HGL) D-54 MG, which contains 0.6 nmol GM2/mg protein, representing 77% of the total monosialoganglioside fraction, was used as an immunogen for the production of anti-GM2 monoclonal antibodies. For ganglioside designations, see IUPAC-IUB (Eur. J. Biochem., 79: 11-21, 1977) and Svennerholm (J. Neurochem., 10: 613-623, 1963). Five IgM monoclonal antibodies (DMAb-1 through DMAb-5) specifically recognizing the GalNAc beta1-4(NeuAc alpha 2-3)Gal-terminal epitope common to GM2 and GalNAC-GD1a are reported. The antibodies did not react with GM1, GM3, GD2, GD3, GD1a, GD1b, and GQ1b. Purified anti-GM2 MAbs were used to define the expression of the "GM2" terminal epitope by cultured human malignant and normal cells by radioimmunoassay and membrane immunofluorescence. Among neuroectodermal tissue-derived cell lines, DMAb-3, at an optimal concentration of 5 micrograms/ml, showed high reactivity (radioimmunoassay binding ratios greater than 20) with 9 of 19 HGLs, 3 of 5 medulloblastoma, 4 of 5 neuroblastoma, and 1 of 3 melanoma lines. Moderate reactivity (binding ratio, 10-20) was exhibited by 3 HGL, 2 medulloblastoma, and 1 neuroblastoma lines and low reactivity (binding ratio, 3-10) by 5 HGL lines; no reactivity was detected with 2 HGL and 2 melanoma lines. Densitometric evaluation of monosialoganglioside extracts from human glioma and medulloblastoma cell lines in conjunction with immunostaining on thin-layer chromatograms showed that GM2 represents the major monosialoganglioside in 8 of 10 HGL and in 3 of 4 Med lines. In these lines the amount of GM2 ranged from less than 0.1 to 0.6 nmol/mg protein. These results indicate that GM2 represents a proportionally increased ganglioside of most glioma, medulloblastoma, and neuroblastoma cells in vitro.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Antigens, Neoplasm; Dose-Response Relationship, Immunologic; Epitopes; G(M2) Ganglioside; Gangliosides; Glioma; Humans; Medulloblastoma; Melanoma; Neuroblastoma

Topics: Aged; Antibodies, Monoclonal; Anus Neoplasms; Facial Neoplasms; Female; G(M2) Ganglioside; Gangliosides; Humans; Immunoglobulin M; Melanoma; Rectal Neoplasms

The ganglioside GM2 is a differentiation antigen expressed on the cell surface of human malignant melanomas and other cancers of neuroectodermal origin. We have previously reported that immunization with purified GM2 combined with Bacillus Calmette-Guérin as adjuvant and pretreatment with low-dose cyclophosphamide induced production of antibodies against GM2 in five of six patients. We have now extended our study and analyzed the induced antibodies against GM2 were not detected. After immunization, high-titer IgM antibodies were induced in 17 of 24 patients, and high-titer IgG antibodies in eight cases. Additional treatment of 12 patients with cimetidine, a histamine H2 receptor antagonist reported to have antisuppressor cell activity, had no effect on GM2 antibody titers. Antibodies against asialo-GM2 were present in all patients, and antibodies against GM1 were present in 33% of patients, before and after immunization. Antibodies induced by immunization were specific for GM2, though some reacted predominantly with N-acetylneuraminic acid GM2 (GM2), and some reacted with GM2 and N-glycolylneuraminic acid GM2(NeuGcGM2). The pattern of reactivity with GM2 is consistent with the response to T-cell-independent antigens: both IgM and IgG antibody responses against GM2 were short lived; peak titers seen after initial and secondary vaccinations were similar; and delayed-type hypersensitivity responses to skin test challenges with GM2 were not detected in any patients. However, the IgG response typed as predominantly IgG1 and IgG3, not IgG2 as might be expected for carbohydrate antigens (which are generally T-cell-independent antigens). Because IgG1 and IgG3 antibody responses are usually to T-cell-dependent antigens, the humoral immune response elicited by GM2 vaccination has both T-cell-dependent and T-cell-independent characteristics. These IgM and IgG responses against this neuroectodermal differentiation antigen expressed on melanoma cells have been induced without evidence of neurological or other toxicity.

Topics: Antibodies; Antibody Formation; BCG Vaccine; Enzyme-Linked Immunosorbent Assay; G(M2) Ganglioside; Gangliosides; Humans; Immunization; Immunoglobulin G; Immunoglobulin M; Melanoma

Ganglioside GM2 is expressed on cell surface membranes of a variety of human malignant cells and has been demonstrated to be immunogenic in humans. We have assessed the role of the antigen GM2 on melanoma cells as a recognition structure for lymphokine-activated killer (LAK) cells. LAK cells were generated by stimulation of non-adherent peripheral blood lymphocytes (PBL) from human donors with recombinant interleukin-2 (IL-2). The selection of target cells was based on GM2 content and included 11 human melanoma cell lines and 2 human leukemia lines. Using a single-cell binding assay, LAK cell binding to target lines expressing high levels of GM2 was significantly greater than to those expressing minimum GM2. This cell-binding was specifically inhibited by addition of purified GM2 but not by other gangliosides. LAK-melanoma cell-binding was also specifically inhibited by anti-GM2 monoclonal antibody (MAb). For further analysis LAK cell lysis of melanoma target cells expressing various amounts of GM2 was assessed. A significant correlation occurred with GM2 expression and LAK cell lysis (p less than 0.025; r = 0.623). Three other gangliosides commonly expressed on human melanoma, GM3, GD3 and GD2, had no correlation with LAK cell lysis. These studies suggest that GM2 on melanoma cells is a marker for LAK cell sensitivity, as well as indicate that GM2 is a potential target recognition structure for human LAK cells.

Topics: Antibodies, Monoclonal; Cell Line; Cell Membrane; Cytotoxicity, Immunologic; G(M2) Ganglioside; Gangliosides; Humans; Killer Cells, Natural; Lymphocyte Activation; Lymphokines; Melanoma; Membrane Lipids; Tumor Cells, Cultured

GM2, GD2, and GD3 gangliosides are expressed on the surface of human melanoma cells and represent potential targets for immunological control of melanoma growth by monoclonal antibodies and active immunization. The immunogenicity of GM2 was investigated by analyzing the humoral immune response of melanoma patients to vaccination with cell lines selected for high GM2 expression and with vaccines containing purified GM2. The whole-cell vaccine and vaccines containing purified GM2 and bacillus Calmette-Guérin (BCG) elicited GM2 antibody in a high proportion of patients, particularly in GM2/BCG-vaccinated patients pretreated with cyclophosphamide and given a GM2/BCG booster immunization. Vaccines containing purified GM2 and Salmonella minnesota R595 as the adjuvant were also effective, but only in patients pretreated with cyclophosphamide. GM2 antibodies in vaccinated patients were of the IgM class and were cytotoxic for GM2-positive targets in the presence of human complement.

Topics: Animals; Antibody Formation; Cell Line; G(M2) Ganglioside; Gangliosides; Humans; Melanoma; Melanoma, Experimental; Mice; Vaccines

A monospecific antibody produced in vitro by a B-lymphoblastoid cell line transformed with Epstein-Barr virus has been shown to recognize a membrane antigen (OFA-I-1) on human tumors and fetal brain. This study identifies the chemical nature of OFA-I-1. The glycolipid fraction of antigen-rich spent medium of an OFA-I-1-positive melanoma cell line, M14, was extracted by chloroform/methanol/water, 4:8:3 (vol/vol), and was separated into fractions of neutral glycolipids and gangliosides by DEAE-Sephadex followed by base treatment and Bio-sil A column elution. OFA-I-1 antigens were found exclusively in the ganglioside fraction when assayed with monospecific anti-OFA-I-1 by an immune adherence inhibition test. The results obtained from thin-layer chromatography of the antigenic M14 ganglioside and sequential glycosidase digestion suggested that the antigen was a ganglioside GM2, GalNAc beta 1 leads to 4(NeuAc alpha 2 leads to 3)Gal beta 1 leads to 4Glc leads to Cer. These results were further supported by testing various authentic gangliosides and neutral glycolipids for OFA-I-1 antigenicity. Only GM2 showed positive reactivity.

Topics: Adult; Antigens, Neoplasm; Brain; Carbohydrates; Cell Line; Epitopes; Female; Fetus; G(M2) Ganglioside; Gangliosides; Glycolipids; Humans; Liver; Melanoma; Pregnancy

AH antigen, initially defined by an antibody present in a melanoma patient, is a cell surface antigen found on approximately 65% of melanoma cell lines. Absorption analysis indicates that AH is a differentiation antigen marking normal and malignant cells of neuroectodermal origin. The AH determinant has been found to be related to GD2 ganglioside.

Topics: Antigens; Antigens, Neoplasm; Autoantigens; Binding, Competitive; Cell Line; G(M2) Ganglioside; Gangliosides; Humans; Melanoma; Phenotype