Polyvalent melanoma vaccine improves survival of patients with metastatic melanoma.
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Antibodies, Neoplasm↗
Biomedical subjects
D S Hoon
Publications and source records attributed to D S Hoon.
Augmentation of T-cell response with a melanoma cell vaccine expressing specific HLA-A antigens.
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Antigens, Neoplasm↗
A preclinical model to assess the antigenicity of an HLA-A2 melanoma cell vaccine.
BACKGROUND: The authors have demonstrated that immunization with melanoma whole-cell vaccine (MCV) augments T-cell responses to melanoma and that cytotoxic T-cells (CTL) recognize allogeneic melanoma-bearing shared HLA-A antigens. A preclinical model was developed to assess CTL activation in vitro using melanoma lines as stimulators. HLA-A2 expression is predominant in melanoma patients and plays a role in HLA class I restricted CTL killing of melanomas. The authors hypothesized that a MCV consisting of allogeneic HLA-A2 melanomas may be as good as autologous melanoma MCV for HLA-A2 patients. METHODS: CTL were generated from peripheral blood lymphocytes of patients with HLA-A2 melanoma by stimulation with autologous melanoma, allogeneic melanoma (HLA-A2 or non-HLA-A2), or allogeneic MCV (mixed HLA-A2 and non-HLA-A2 melanomas). RESULTS: HLA-A2 MCV and autologous melanoma were similar and significantly better stimulators than the others. Specificity also was supported by CTL killing and mixed lymphocyte tumor reaction assays. CONCLUSIONS: These studies provide important information for the studying immunization of patients with HLA-A2 melanoma with an allogeneic HLA-A2 MCV in a Phase I clinical trial.
Antigens, Neoplasm↗
Transfection of interleukin 2 gene into human melanoma cells augments cellular immune response.
A preclinical model was used to determine if transfection of the interleukin 2 (IL-2) gene into human melanoma cells would augment the response of autologous and allogeneic peripheral blood lymphocytes (PBLs) from melanoma patients. IL-2 gene was transfected into three human melanoma cell lines; secretion of IL-2 from stable transfected cells was confirmed by enzyme-linked immunosorbent assay. The PBL response to these melanoma cells was then examined in a mixed-lymphocyte tumor reaction using PBLs from eight melanoma patients. The PBL response to autologous (P < 0.01) or human leukocyte antigen A cross-reactive (P < 0.05) transfected melanoma cells was significantly higher than it was to nontransfected melanoma cells. These data suggest that IL-2 gene transfection may be an important strategic approach to enhancing specific immune responses induced by a polyvalent melanoma cell vaccine.
Base Sequence↗
Interleukin-4 plus tumor necrosis factor alpha augments the antigenicity of melanoma cells.
Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor alpha (TNF) or interferon gamma (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3+CD4+ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.
Antigens, CD↗
Aberrant expression of gangliosides in human renal cell carcinomas.
Aberrant and elevated ganglioside expression has been observed in neoplasms, and has been shown to be an important marker of tumor progression. We therefore studied the gangliosides of renal cell carcinoma (RCC) by analyzing gangliosides from 18 RCC biopsies, 10 RCC lines and 5 normal kidney biopsies. A comparison of tumor with normal tissue revealed a significant difference in individual ganglioside expression in which the former consistently expressed eight major gangliosides, GM3, GM2, GM1, GD3, GD1A, GD2, GD1B and GT1B, according to the nomenclature of Svennerholm. There was a notable significant mean increase in the expression of GM2, GM1 and GD1A and a significant decrease in the expression of GD3 in tumor tissue compared with normal kidney tissue. Compared with tumor biopsy tissue, RCC cell lines showed a significant decrease in the expression of GM3, but a significant increase in GM2, GM1 and GD2. There was a marked increase in a pathway gangliosides (GM2, GM1, and GD1a) in RCC biopsies and cell lines compared with normal kidney. These studies indicating that RCC have markedly aberrant ganglioside expression similar to neural origin tumors may relate to the activation of a ganglioside pathway enzymes. Gangliosides expressed on RCC tumors may be important markers of tumor progression and target antigens for immunotherapy.
Biomarkers, Tumor↗
Growth inhibition and modulation of cell markers of melanoma by S-allyl cysteine.
A sulfur-containing amino acid compound, S-allyl cysteine (SAC), derived from garlic extract inhibited proliferation of nine human and murine melanoma cell line in a dose-dependent manner (1.2-10 mM) assessed by a [3H]thymidine incorporation assay. Three control human lymphoblastoid cell lines were not inhibited by SAC concentrations < 5 mM. Four human melanoma cell lines in a soft-agar assay also showed dose-dependent inhibition of colony formation by SAC. Melanin content was increased up to 95% compared to the same untreated cell lines in these four human melanoma and two B16 murine melanoma sublines. Expression of cell surface gangliosides, cellular-differentiation and transformation markers, decreased after SAC treatment. Significant morphological changes including 'flattening and/or dendritic-like elongations' were also observed. Thus SAC inhibited cellular growth and proliferation and modulated major cell differentiation markers of melanoma.
Animals↗
Interleukin 4 receptor expression and growth inhibition of gastric carcinoma cells by interleukin 4.
The expression of the interleukin 4 (IL-4) receptor (IL-4R) and effects of human recombinant IL-4 on human gastric carcinoma cell lines were studied. We demonstrated that IL-4 inhibited the growth of gastric carcinoma cells in a dose dependent manner (0.1-100 units/ml) in a [3H]thymidine incorporation proliferation assay. The gastric carcinoma cells varied in sensitivity to treatment with low dose IL-4. Treatment of cells with IL-4 altered the morphology of the cells to a "flattened" morphological shape resembling differentiation. The IL-4-mediated growth inhibition was significantly abrogated by neutralization of IL-4 with specific anti-IL-4 antibody. IL-4R expression on the cell surface was determined by assessing biotin-labeled IL-4 binding to cells using flow cytometry. IL-4R expression ranged from 5 to 85% of total cell population in the gastric carcinoma cell lines assessed. There was a positive correlation between the sensitivity to IL-4-mediated growth inhibition and IL-4R expression. By Northern blot analysis, we demonstrated that mRNA of IL-4R was expressed in the gastric carcinoma cells. Using in situ hybridization, we confirmed that IL-4R mRNA was expressed in the gastric carcinoma cell at the single cell level. By using a sensitive polymerase chain reaction technique, we demonstrated that gastric carcinoma cells expressed IL-4 mRNA, suggesting a possible autocrine loop. These studies indicate that IL-4 can significantly modulate gastric carcinoma cells that possess IL-4R. IL-4R on gastric carcinoma cells may be a potential therapeutic target site for IL-4-directed therapy.
Base Sequence↗
Cytotoxic T cell lines recognize autologous and allogeneic melanomas with shared or cross-reactive HLA-A.
Cytotoxic T lymphocytes (CTL), CD3+, alpha/beta T-cell-receptor-positive, are important effector cells with specific immunity in melanoma patients. The establishment and expansion in vitro of CTL of a specific phenotype to tumor cells strongly depends on the method of activation and sensitization with tumor cells. We generated CD3+ CTL lines to melanoma by co-culturing peripheral blood lymphocytes with autologous irradiated melanoma cells and repetitive stimulation with high-dose interleukin-4 in a "cocktail" culture medium. CTL lines were investigated for their specificity to kill autologous and allogeneic melanoma. Histocompatibility locus antigen (HLA) class I (A, B) molecules are important restrictive recognition antigens for CTL. Although these antigens are highly polymorphic, they can share a similar immunogenic molecular epitope(s) and can be immunologically cross-reactive. The CTL lines generated were found to kill not only autologous melanoma, but also allogeneic melanomas having class I HLA-A antigens shared or "cross-reactive" with autologous HLA-A. These CTL lines were poor killers of melanomas bearing non-shared or non-cross-reactive HLA-A. Cold-target inhibition assays demonstrated this CTL cross-reactivity to allogeneic melanoma specificity. Epstein-Barr-virus-transformed autologous and allogeneic B lymphoblastoid cell lines failed to block autologous melanoma killing, indicating that CTL were not recognizing major histocompatibility complex antigens, serum proteins or culture medium products as the primary target antigen. HLA-A2 was the major shared HLA-A antigen recognized by CTL lines on the melanoma lines studied. CTL lines also recognized shared HLA-A11 and A24 on allogeneic melanoma. There were no CTL lines showing restriction to HLA-B. These results suggest that common tumor-associated antigens are present on melanomas and are recognized in association with distinct HLA-A epitopes by CTL.
Antigens, Neoplasm↗
Induction of CD4+ cytotoxic T cells by sensitization with allogeneic melanomas bearing shared or cross-reactive HLA-A.
Human melanoma is an immunogenic neoplasm whereby enhancement of specific cell-mediated immunity can alter tumor progression. HLA-A2-restricted CTL have been demonstrated to kill allogeneic HLA-A2-matched melanoma. We investigated the ability of allogeneic melanoma cells sharing HLA-A antigens to sensitize melanoma patients' lymphocytes to induce HLA-A-restricted CTL to autologous melanoma. PBL from melanoma patients were cocultured with autologous melanoma cells in defined "cocktail medium" to generate melanoma-specific HLA-A-restricted CTL lines. CTL generated by sensitization with allogeneic melanoma bearing shared HLA-A2, A11, A24, or "cross-reactive" HLA-A antigens could kill almost as many autologous melanoma cells as CTL sensitized with autologous melanoma. There are HLA-A antigens that are immunogenically cross-reactive because they share determinant epitopes. CTL were not activated NK or LAK cells. The HLA restriction and melanoma cell specificity of the CTL were demonstrated by cold target inhibition with autologous and allogeneic melanoma and B lymphoblasts. Anti-CD3 and anti-HLA AB inhibited CTL killing of melanoma. The CTL were predominantly CD3+CD4+ TCR alpha/beta+. These studies demonstrate that melanomas being shared or cross-reactive HLA-A can be used for in vitro generation of HLA-restricted CTL that recognize melanoma-associated antigens. The findings have very important implications in human tumor immunotherapy.
CD4 Antigens↗
Prolongation of survival in metastatic melanoma after active specific immunotherapy with a new polyvalent melanoma vaccine.
A new polyvalent melanoma cell vaccine (MCV) was administered to 136 stage IIIA and IV (American Joint Committee on Cancer) melanoma patients. Induction of cell-mediated and humoral immune responses to common melanoma-associated antigens present on autologous melanoma cells was observed in patients receiving the new MCV. This was accompanied by increased activation of tumor-infiltrating lymphocytes. Survival correlated significantly with delayed cutaneous hypersensitivity (p = 0.0066) and antibody responses to MCV (p = 0.0117). Of 40 patients with evaluable disease, nine (23%) had regressions (three complete). From our historical database of 126 stage IIIA and 1275 stage IV melanoma patients, there were no significant changes in the natural history of metastatic melanoma during the past 20 years. Univariate and multivariate analyses demonstrated prognostic significance for site of metastases (p = 0.0001) and immunotherapy with the new MCV (p = 0.0001). Overall our new MCV increased the median and 5-year survival of stage IIIA melanoma patients with regional soft tissue metastases twofold (p = 0.00024), and stage IV patients threefold (p = 0.0001) compared with previous immunotherapy and other treatments.
Adult↗
Ganglioside GM2 levels in human melanoma cells: inverse correlation with lysosomal beta-hexosaminidase A activity.
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.
Analysis of Variance↗
Interleukin 4 alone and with gamma-interferon or alpha-tumor necrosis factor inhibits cell growth and modulates cell surface antigens on human renal cell carcinomas.
Immune cytokines have been shown to play important roles in regulating immune cell functions as well as neoplastic cells. Interleukin-4 (IL4), primarily known as a B-cell growth factor, can also activate and differentiate other immune cells. This cytokine has recently been shown to have immunotherapeutic benefit in tumor-bearing hosts. The present study assessed the effect on human renal cell carcinoma cell lines of recombinant IL4 alone and in combination with recombinant gamma-interferon (IFN) or recombinant alpha-tumor necrosis factor (TNF). IL4 inhibited cell growth of all lines at 250-500 units/ml in a differential manner. Expression of IL4 receptors was demonstrated on renal cell carcinomas. Overall, IFN (500 units/ml) alone inhibited cell growth; however, TNF (500 units/ml) was not as strong an inhibitor. When IL4 was combined with IFN or TNF there was a significant augmentation of cell growth inhibition and modulation of cell morphology of the cell lines. Tumor-associated ganglioside antigens (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, GalNAc beta 1-4 (NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer, and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer) HLA class I, HLA-DR, and beta 2-microglobulin on the cell surface of renal cancer lines were assessed by flow cytometry and radiometric binding assay. IL4 alone or in combination with other cytokines modulated HLA class I and HLA-DR expression. IL4 and IFN consistently enhanced NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer expression on individual cell lines. The study demonstrated that IL4 alone or in combination with other cytokines can significantly inhibit growth, and modulate the expression of surface major histocompatibility and tumor-associated antigens of renal cell carcinomas.
Carcinoma, Renal Cell↗
Modulation of human melanoma cells by interleukin-4 and in combination with gamma-interferon or alpha-tumor necrosis factor.
Immune cytokines have been shown to play important roles in regulating immune cell functions. Interleukin-4 (IL4), originally described as a B-cell growth factor, is known to activate and differentiate other immune cells. IL4 has been given as an immunotherapeutic to tumor-bearing hosts. In this report, we set out to determine whether IL4 can directly modulate growth and expression of surface antigens on human melanoma cells. The effect of recombinant IL4 alone and in combination with recombinant gamma-interferon (IFN) or recombinant alpha-tumor necrosis factor (TNF) on melanoma cell lines was examined. IL4 significantly inhibited cell growth of all cell lines examined at 100-500 units/ml; but a dose-dependent differential response to individual cell lines occurred. The effect of IL4 was augmented by combination with IFN or TNF. Melanoma-associated ganglioside antigens (GM3, GD3, GM2, GD2) and human leukocyte antigen class I and DR on the cell surface of melanoma cells were assessed by flow cytometry and/or a radiometric binding assay. IL4, IFN, or TNF alone enhanced human leukocyte antigen class I, DR, and beta 2-microglobulin antigen expression. IL4 alone and in combination with IFN or TNF increased the GM3/GD3 ratio expression. GD2 was enhanced significantly by IL4, IFN, and TNF. Pretreatment of melanoma with IL4 or with other cytokines prior to stimulation with peripheral blood lymphocytes significantly enhanced mixed lymphocyte tumor reaction activity as compared with non-treated melanoma used as stimulators. These studies demonstrate that IL4 alone or in combination with IFN and TNF can modulate melanoma growth activity and surface antigen expression to a more differentiated and immunogenic phenotype.
Antigens, Neoplasm↗
Anti-idiotype monoclonal antibody carrying the internal image of ganglioside GM3.
Murine anti-idiotype monoclonal antibodies were generated against a human IgM monoclonal antibody (L612) that recognizes ganglioside GM3 on human melanoma. Hybridomas secreting antibodies that bound specifically to L612 were selected by enzyme-linked immunosorbent assay using L612 and three negative control human IgMs, including monoclonal anti-GM2 and anti-GD2 antibodies, as well as purified serum IgM, as antigen sources. GM3-binding inhibition and cell-binding inhibition assays were used to identify seven anti-idiotype monoclonal antibodies that recognized determinants located within the antigen-combining sites of L612. To determine whether these anti-idiotype monoclonal antibodies possessed the internal image of the original antigen, we immunized syngeneic BALB/c mice with one of the anti-idiotype monoclonal antibodies, 4C10, coupled with keyhole limpet hemocyanin. Sera from the immunized mice reacted strongly with an antigen-positive M12 melanoma cell line and with purified GM3. Because L612 detects and kills melanoma tumor cells in vitro and in vivo in the presence of complement without affecting normal tissues, anti-idiotype monoclonal antibodies carrying the internal image of GM3 may be an effective tool for active specific immunotherapy in patients with melanoma.
Animals↗
Suppressor cell activity in a randomized trial of patients receiving active specific immunotherapy with melanoma cell vaccine and low dosages of cyclophosphamide.
Previous studies have shown that melanoma patients develop an immune response to cell surface melanoma-associated antigens. The presence of this antibody response to cell surface antigens has been correlated with a better clinical outcome when melanoma patients are treated with an allogeneic melanoma cell vaccine (MCV) as an active immunotherapy protocol. It was hypothesized that the inability to consistently induce or enhance existing immune responses to melanoma-associated antigens was related to the downregulation by suppressor cells. Patients received treatments of MCV 3 times in a 4-week interval and then every fourth week. The biological response modifier cyclophosphamide (CYP) is an immunomodulator of suppressor T-cell function. In this study we set out to determine whether CYP given prior to MCV could reduce suppressor cell activity during vaccination. In a randomized trial stage II and III melanoma patients (n = 41) were given MCV alone or in conjunction with CYP at dosages of 300, 150, or 75 mg/m2. CYP was given 3 days prior to each MCV treatment. Suppressor cell activity in patients was monitored by a concanavalin A suppressor assay using peripheral blood lymphocytes from serial phlebotomies during a 12-week period of treatment. In each trial group there were patients who had major reduction in suppressor cell activity (greater than 50%). Overall, the greatest reduction in suppressor cell activity occurred in patients receiving 300 mg/m2 CYP compared to the other CYP dosages or MCV alone. For the first two treatments at all CYP dosages there was a greater number of patients showing reduced suppressor cell activity compared to later treatments. In a comparison of patients receiving MCV alone to MCV + CYP 300 mg/m2 phenotypic analysis of lymphocyte subsets showed significant (P = 0.03) reduction in the CD8+CD11B+ (suppressor) cells of the latter group. These studies suggest that CYP can be used at low dosages in conjunction with MCV to reduce suppressor cell activity.
Adult↗
Generation of lymphokine-activated killer cell activity by low-dose recombinant interleukin-2 and tumor cells.
The generation of lymphokine-activated killer (LAK) cells in vitro has been reported to require 100-1000 units of recombinant interleukin-2 (IL2). In this study we investigated the generation of human LAK cells with low-dose IL2 (1-10 U) in combination with human tumor cell lines. A significant LAK activity was generated within 3- to 5-days culture of PBL. Among six human tumor cell lines tested, the K562 cell line had the greatest stimulating activity, and the degree of cytotoxicity was comparative to that of PBL stimulated with higher doses of IL2 alone. The origin of this LAK activity was primarily the E(-) rosetting cell population. Cocultures of E- cells with 1 U/ml IL2 plus K562 had significantly higher cytotoxicity (P less than 0.05) compared to using E+ cells. Phenotypic analysis indicated that 1 U/ml IL2 plus K562 cell stimulation enhanced CD56+ and CD16+ cells. These studies suggest that very low dosages of IL2 with stimulator tumor cells can generate LAK activity comparable to that generated with high dosages of IL2 alone.
Antigens, CD↗
Ganglioside GM2 expression on human melanoma cells correlates with sensitivity to lymphokine-activated killer cells.
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.
Antibodies, Monoclonal↗