Generation of melanoma-specific, cytotoxic CD4(+) T helper 2 cells: requirement of both HLA-DR15 and Fas antigens on melanomas for their lysis by Th2 cells.

Recognition of melanoma antigens by HLA class-II-restricted CD4(+) T lymphocytes has been investigated. Two cytotoxic CD4(+) T cell lines were established by stimulating PBLs from a melanoma patient with either parental or IFN-gamma-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-gamma, or TNF-beta, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-gamma-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-gamma-modified melanoma cell lines shared melanoma antigen(s) presented in the context of HLA-DR15. HLA-DR15(+) nonmelanoma cells were resistant targets indicating that the shared antigen(s) is melanoma associated. Parental autologous and HLA-DR-matched allogeneic melanoma cell lines, displaying low levels of HLA-DR antigens, induced Th2 proliferation and cytokine release, but were insensitive to lysis prior to upregulation of HLA-DR and Fas antigens by IFN-gamma. Cytolysis was inhibited by anti-HLA-DR and by anti-Fas antibodies, suggesting that the cytolysis is mediated via the Fas pathway. While small amounts of HLA-DR15 molecules on melanoma cells are sufficient for Th2 proliferation and cytokine release, higher amounts of HLA-DR15 and the expression of Fas are required for CD4(+)-mediated lysis.

MCA106 fibrosarcoma cells transduced with granulocyte/macrophage colony-stimulating factor are not superior to the wild-type cells in suppressing the growth of hepatic metastases.

BACKGROUND AND OBJECTIVES: Vaccination with cytokine gene-modified tumor cells augments the immune response against established tumors and protects against tumor challenges. In this study, we investigated the vaccine potential of GM-CSF-transduced MCA106 fibrosarcoma (MCA-GMCSF) cells in the C57BL/6 (B6) murine hepatic metastasis model. METHODS: Experimental mice received one to three weekly vaccines (subcutaneous/intramuscular, s.c./i.m.) of irradiated, parental, or GM-CSF-transduced MCA106 tumor cells. One week after the last immunization, hepatic metastases were established through the intrasplenic injection of live MCA106 parental (MCA106P) tumor cells. The animals were then sacrificed 3-4 weeks after surgery for evaluation of hepatic tumor burden. RESULTS: Based on in vivo experiments, both GM-CSF-modified and parental MCA106 tumor cell vaccines induced strong protection against hepatic tumor growth with grossly visible tumors rarely identified. This protection was evident even at a single vaccine dose of as low as 1x10(5) irradiated cells. Unimmunized control mice, on the other hand, consistently developed substantial hepatic tumors. Cytotoxicity assays on splenocytes (cultured in vitro for 4-5 days) showed that both groups of vaccinated mice developed strong tumor-specific cytotoxic T-lymphocyte (CTL) responses. Immunohistochemical analysis of injection sites showed infiltration of dendritic cells (DCs) and macrophages into subcutaneously injected MCA-GMCSF cells. Mostly macrophages, however, were seen at the injection site of MCA106P cells. Furthermore, the MCA106P cells expressed high levels of MHC class I antigens and the level of expression was not significantly altered by transduction with the GM-CSF gene. The high expression of MHC class I antigens probably contributed to the strong immunogenicity of the MCA106P cell vaccine. CONCLUSIONS: This study demonstrates that MCA106 parental cells are as effective as the GM-CSF-transduced cells in suppressing the growth of hepatic metastases. The cellular immune responses induced by these two vaccines, however, are probably mediated by different subsets of host effector cells. These results have important implications for the use of GM-CSF-transduced cell vaccines in the immunotherapy of tumors that have the propensity to metastasize through the lymphatic channels and the circulatory system.

Pulsing of dendritic cells with cell lysates from either B16 melanoma or MCA-106 fibrosarcoma yields equally effective vaccines against B16 tumors in mice.

BACKGROUND AND OBJECTIVES: Dendritic cells (DC) pulsed in vitro with a variety of antigens have proved effective in producing specific antitumor effects in vivo. Experimental evidence from other laboratories has confirmed that shared antigens can be encountered in histologically distinct tumors. In our experiments, we set out to evaluate the immunotherapeutic potential of vaccines consisting of DC pulsed with MCA-106 fibrosarcoma or B16 melanoma cell lysates and to determine whether a cross-reactivity exists between the two tumors. METHODS: DC were prepared from the bone marrow of C57BL/6 (B6) mice by culturing progenitor cells in murine granulocyte-macrophage colony-stimulating factor (GM-CSF). They were separated into three equal groups and were either pulsed with B16 melanoma cell lysates (BDC), pulsed with tumor extract from the syngeneic fibrosarcoma MCA-106 (MDC), or left unpulsed (UDC). DC were then used to immunize three groups of mice, with all mice receiving two weekly intravenous (IV) doses of 1 x 10(6) DC from their respective preparations on days -14 and -7. A fourth group of control mice were left untreated. On day 0, all mice were challenged with subcutaneous injections of 1 x 10(5) B16 and 1 x 10(5) MCA tumor cells, administered in the left and right thighs, respectively. After the inoculations, the mice were monitored closely with respect to tumor growth and survival. RESULTS: The MDC mice developed specific cellular immunity directed against not only MCA-106 tumor cells, but also against B16 melanoma, as measured through chromium-release assays of splenocyte preparations, while remaining ineffective at killing both L929 fibroblasts and CT26 tumor cells. By day 30 after tumor inoculations, control mice manifested the largest B16 tumor volumes at a mean of 2185 mm3, followed by the UDC, MDC, and BDC groups at 92 mm3 (P=0.00008), 3 mm3 (P=0.000002), and 2 mm3 (P=0.00004), respectively. The survival data mirrored this pattern, with control animals displaying the shortest mean survival time (37.1+/-4.0 days), followed by UDC (44.8+/-6.6), MDC (56.2 +/-14.7), and BDC (56.4+/-18.3) animals. No significant differences were noted between MCA-106 and B16 cell lysate-pulsed DC vaccines with respect to their abilities to inhibit B16 tumor growth and to prolong survival. These findings were confirmed using a B16 pulmonary metastasis model. Likewise, vaccination with interferon-gamma gene-modified MCA-106 tumor cells was shown to be effective at protecting against a subsequent subcutaneous B16 tumor challenge in 3 of 4 mice observed. CONCLUSIONS: These results demonstrate that immunization with antigen-pulsed DC confers cellular immunity, retards tumor growth, and prolongs the survival of tumor-challenged mice. The ability of MCA-106 cell lysate-pulsed DC vaccines to inhibit the growth of subcutaneous B16 tumors also suggests the presence of shared tumor-associated antigens between these two histologically distinct tumors.

Human dendritic cells, pulsed with either melanoma tumor cell lysates or the gp100 peptide(280-288), induce pairs of T-cell cultures with similar phenotype and lytic activity.

Dendritic cells (DCs) pulsed with unfractionated tumor cell lysates or defined tumor peptides provide potent vaccines which elicit strong antitumor immunity. In this study, we generated DCs from the 2-h adherent progenitor cells obtained from the peripheral blood of melanoma patients. These DCs were able to capture biotinylated melanoma tumor cell lysates. We examined the efficacy of immunogens composed of DCs loaded either with the melanoma peptide gp100 [amino acids 280-288 (DC/gp100)] or with lysates from melanoma tumor cells (DC/lysates) in inducing cytotoxic T-cells from autologous PBLs of HLA-A2 melanoma patients. After four to five weekly stimulations of bulk PBLs with DC/gp100 or DC/lysates, the cultures were enriched with CD3+ T-cells and exhibited one of three phenotypic and functional patterns: (1) Predominant expression of CD8+ and MHC class I-restricted CTLs which displayed strong lytic activity against melanoma cells and T2 cells loaded with the gp100 peptide, (2) mixed CD4+/CD8+ phenotype and weak lytic activity, or (3) nonlytic and predominantly CD4+ cultures. Interestingly, T-cell cultures from each patient exhibited similar phenotypes and lytic activities whether the stimulant was DC/gp100 or DC/cell lysates. Our study demonstrates that DCs pulsed with soluble melanoma peptides or cell lysates are capable of inducing CD8+ CTLs from autologous PBLs of some, but not all, melanoma patients. The function and phenotype of the generated T-cell cultures are governed by DCs since both antigens (the gp100 peptide and melanoma lysates), when presented by a given DC preparation, induced similar T-cell cultures. In summary, it may be difficult to predict the nature of the cellular responses elicited by DC/tumor antigen vaccines from patient to patient.

Vaccination with dendritic cells inhibits the growth of hepatic metastases in B6 mice.

Dendritic cells (DC) are specialized antigen-presenting cells that can activate naive and mature T-cells, induce cellular immunity, and stimulate strong antitumor reactions in vivo. This study was undertaken to examine the function of DC vaccines in suppressing the growth of hepatic metastases in C57BL/6 mice. Experimental mice received two i.v. doses of 1 x 10(6) bone marrow-derived DC, either unpulsed or pulsed with MCA-106 fibrosarcoma cell lysates, on days -14 and -7. Controls were injected with HBSS. Hepatic metastases were established on day 0 through intrasplenic injections of 1 x 10(5) MCA-106 tumor cells. Animals were sacrificed on day 21 and their livers were excised to assess tumor burden. Splenocytes from DC-treated groups were cytotoxic against MCA-106 cells, but not against the L929 and CT26 (syngeneic fibroblast and colon tumor, respectively) cell lines. All control mice developed grossly evident hepatic metastases, while 62 and 44% of the mice receiving MCA-106 cell lysate-pulsed DC and unpulsed DC vaccines, respectively, were completely free of tumor. Mean hepatic mass for the controls, including tumor, was almost double that for treated animals. Antibody depletion of either CD4+ or CD8+ lymphocytes abrogated the protective effect of the vaccine. This study demonstrates that immunization with DC confers cellular immunity, with both CD4+ and CD8+ T-cells playing a significant role, and impedes the subsequent establishment and growth of hepatic metastases in mice. The antitumor capabilities of DC justify their use in immunotherapeutic vaccines against human cancers.

A Phase I clinical trial of immunotherapy with interferon-gamma gene-modified autologous melanoma cells: monitoring the humoral immune response.

BACKGROUND: Tumor cells transduced with cytokine genes provide immunogenic vaccines for cancer immunotherapy. METHODS: A Phase I clinical trial was conducted for the specific active immunization of melanoma patients with interferon-gamma (IFN-gamma) gene-modified autologous melanoma tumor cells. Short term melanoma cultures were transduced retrovirally with the gene for human IFN-gamma. The genetically modified melanoma cells secreted biologically active IFN-gamma and showed enhanced expression of major histocompatibility complex class I and class II surface antigens. These cells were inactivated by irradiation (50 gray) and were cryopreserved for the vaccine. Twenty melanoma patients were enrolled in this clinical trial. The immunizations were administered in escalating doses once every 2 weeks for 3 months. The first and second injections consisted of 2 million cells, followed by 6 million for the third and fourth injections, and then 18 million for the fifth and sixth injections. The humoral immune responses of the patients were assessed by enzyme-linked immunoadsorbent assay, radioimmunoassay, and radioimmunoprecipitation. RESULTS: Thirteen of the 20 patients completed the immunization protocol. Eight of these 13 patients showed a humoral immunoglobulin (Ig)G response against autologous and allogeneic melanoma cells. The other five patients either had no detectable antimelanoma antibodies or showed a weak IgG response that did not rise significantly above the preimmune level. All the sera contained low or undetectable levels of antimelanoma IgM antibodies. The IgG response increased progressively in titer during the course of immunization. The positive sera showed preferentially strong binding to melanoma cell lines and some cross-reactivity to nonmelanoma tumors. A 75-80 kD antigen on melanoma cells was immunoprecipitated by postimmune sera of 3 of the responding patients. Preimmune sera from these three patients and sera from other patients immunized with a standard nontransduced melanoma cell vaccine failed to precipitate this antigen. Two patients with significant increases in serum IgG had clinical tumor regression, and two additional patients with low serum IgG response had transient shrinkage of nodular disease during therapy. CONCLUSIONS: These data suggest that gene therapy with IFN-gamma-transduced melanoma cells is safe and worthy of further investigation in patients with less advanced stage malignant melanoma. The ability to monitor changes in the humoral responses of the immunized patients has been demonstrated.

Eradication of melanoma pulmonary metastases by immunotherapy with tumor cells engineered to secrete interleukin-2 or gamma interferon.

This study was undertaken to investigate the effectiveness of interleukin-2 (IL-2) and gamma interferon (gammaIFN)-modified B16 melanoma cells in the immunotherapy of established melanoma pulmonary metastases. The genes for IL-2 and gammaIFN were introduced retrovirally into B16 melanoma cells. Transduction with the gammaIFN, but not the IL-2, gene caused significant increases in the expression of major histocompatibility complex (MHC) antigens on B16-gammaIFN cells. The in vivo tumor-forming capacity of both IL-2- and gammaIFN-transduced B16 cells was drastically reduced when the cells were inoculated subcutaneously (SC) in syngeneic C57BL/6 mice. After intravenous (IV) inoculation, most of the B16-gammaIFN cells were rejected, but B16-IL-2 cells were relatively tumorigenic and formed pulmonary metastases. C57BL/6 mice bearing 4-day established parental B16 lung metastases were treated with B16 parental (B16P) unmodified cells, IL-2- or gammaIFN-modified B16 cells, or a combination of both transduced cells. Treatment consisted of a weekly intraperitoneal (IP) injection of one million irradiated (10,000 rad) tumor cells alone or in combination with exogenous IL-2 for a total of three to four injections. Immunotherapy with B16 parental or B16-IL-2 secreting cells caused a moderate reduction in the number of lung metastases. However, mice treated with gammaIFN-secreting B16 cells showed a significant reduction or complete elimination of lung metastases. There was no additive effect for combining both IL-2- and gammaIFN-modified tumor cells in the immunotherapy. Exogenous IL-2 (50,000-100,000 U/day for 3 days) caused a significant enhancement of the immunotherapeutic benefit of the vaccines. Moreover, mice treated with gammaIFN-modified B16 cells survived longer than the other groups. Twenty-five percent of these mice were tumor free and remained alive for an observation period of 4 months. The in vitro cytolytic activity of splenocytes in chromium release assays did not correlate in every case with the in vivo antitumor effect of the treatment. Our findings have implications for the use of cytokine-modified cells for immunotherapy and for evaluating the therapeutic benefit of this novel treatment.

Modulation of specific active immunization against murine melanoma using recombinant cytokines.

Specific active immunization with tumour cells and IL-1beta or IL-2 was examined in a murine model. Mice were treated with irradiated B16 melanoma, IL-1beta or IL-2 only, or with B16 plus cytokines prior to i.v. challenge with viable B16. Lung metastases were recorded after 28 days. Treatment with cytokine alone was not protective. Treatment with B16 alone afforded moderate protection. Treatment with B16 in combination with either cytokine resulted in a significant level of B16 specific protection which was dependent on the dose of cytokine used. Multiple immunizations with B16 provided limited protection which was significantly improved with IL-2. Immunization with B16 in combination with both cytokines at doses that alone failed to enhance immunity resulted in significant protection, suggesting that the two cytokines act at least additively. These studies demonstrate the significant benefit of specific active immunization with tumour cells in combination with low doses of IL-1beta or IL2.

Effect of irradiation on cytokine production, MHC antigen expression, and vaccine potential of interleukin-2 and interferon-gamma gene-modified melanoma cells.

Recent studies have shown that tumor cells transduced with interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) genes stimulated a potent and specific antitumor immunity in experimental animals. For use as a human vaccine, tumor cells must be inactivated by irradiation to ensure the arrest of their growth. This study was undertaken to examine the effects of irradiation (10,000 rad) on the growth characteristics and vaccine potential of IL-2 and IFN-gamma-modified human melanomas and B16 murine melanoma. Irradiation caused cessation of cell growth and gradual reduction of cell number. Irradiated melanoma cells displayed 1.5 to 10-fold increases in the surface expression of MHC class I and/or class II antigens. The increases in MHC antigens persisted for 7-14 days postirradiation and then declined thereafter. Furthermore, IL-2- and IFN-gamma-transduced melanoma cells showed enhanced expression of the cytokine mRNA and increased cytokine secretion after irradiation. The effect of irradiation on the vaccine potential of the transduced cells was examined in C57BL/ 6 mice by prophylactic immunization and immunotherapy, and in nude mice by mixed transplantation assays. The irradiated, cytokine-transduced B16 cell vaccine was as or more effective than the unirradiated vaccine. These irradiated vaccines protected the animals against a challenging tumorigenic dose of B16 parental cells and suppressed the growth of 4-day-established B16 lung metastases. The ability of the irradiated IL-2-transduced human melanomas to inhibit the growth of admixed parental melanoma cells was retained but was less efficacious than unirradiated cells. The results suggest that irradiation does not abrogate the vaccine potential of IL-2- and IFN-gamma-transduced melanomas. These findings have implications for designing specific active immunotherapy protocols utilizing cytokine gene-modified tumor cells.

Recognition and lysis of human melanoma by a CD3+, CD4+, CD8- T-cell clone restricted by HLA-A2.

The in vitro cytotoxic response to human melanoma is characterized by CD3+ CD8+ T-cells which recognize shared peptide antigens presented in the context of HLA class-I-encoded gene products. We report here studies of a CD3+, CD4+, CD8-, HLA-A2-restricted, melanoma-specific cytotoxic T-cell clone derived by limiting dilution from a T-cell line induced in PBLs from a melanoma patient following in vitro stimulation with an HLA-A2-matched melanoma cell line. The CD4+ cytotoxic T-cell clone is lytic only for melanomas which share the HLA-A2 allele, and the cytotoxicity is blocked by antibody to the T-cell receptor and by antibody to HLA class I. The clone proliferates only following stimulation with HLA-A2-matched melanoma tumor cells. The data suggest that cytotoxic CD4+ T-cells may play a significant role in immunity to melanoma, and HLA class-I-restricted recognition of melanoma may not necessarily require the CD8 molecule on the lytic T-cell.

Dual recognition of a human cytotoxic T-cell clone for melanoma antigens.

It is well known that tumor-specific CTLs have a crucial role in the elimination of tumors and that different CTL populations recognize tumor antigens in MHC-restricted and MHC-unrestricted manners. We have established two alpha beta CTL clones that recognize melanoma antigens in both human lymphocyte antigen (HLA)-A2-restricted and HLA-unrestricted manners. Flow cytometry analysis showed that these CTL clones carry CD3, CD8, and alpha beta T-cell receptor (TCR) and express low levels of CD56. In contrast, these CTL clones do not express CD16, indicating that they do not contain natural killer cells. TCR analysis of these CTL clones using an anchored PCR method revealed that each clone carries a single alpha beta TCR. Both CTL clones contained the same Valpha and Vbeta gene segments although they carried different Jalpha and Jbeta gene segments. Taken together, these results confirm that CTL clones that carry a single alpha beta TCR recognize melanoma antigens in both HLA-A2-restricted and HLA-unrestricted manners. It is strongly suggested that the dual recognition of these CTL clones for the melanoma antigens is mediated by TCRs. The novel mechanism for antitumor immunity by these CTLs may be important in the effective elimination of tumors in vivo.

Immunological memory induced by genetically transduced tumor cells.

BACKGROUND: Recent studies have demonstrated the usefulness of gene-modified tumor cells for immunotherapy. Using the tumorigenic murine fibrosarcoma, MCA 106, we investigated the effects of localized interferon-gamma (IFNg) secretion on tumorigenicity and on long-term memory. METHODS: The murine IFNg (MuIFNg) gene was introduced into tumor cells. High and low IFNg-secreting clones were isolated. C57BL/6 mice were injected subcutaneously (s.c.) with either parental (P), high or low IFNg-secreting (H- or L-IFNg) cells, and tumor growth was assessed weekly. Spleens were harvested on different days postinjection (p.i.) to assess in vitro cytolytic activity. In parallel, tissues from injection sites were stained with macrophage-, CD4-, and CD8-detecting antibodies. Mice were injected s.c. with H-IFNg MCA106 tumor. After 150 days the animals were rechallenged s.c. with MCA106P in one leg and with irrelevant syngeneic tumor in the other. RESULTS: Both P- and L-IFNg cells had similar growth, whereas the H-IFNg cells never grew. Only splenocytes from the H-IFNg animals showed in vitro CTL activity persisting until day 30 p.i. Histological data revealed a macrophage and CD4+ infiltrate much earlier in the H-IFNg group compared with the P group. Only the irrelevant, syngeneic tumor grew in animals previously injected with H-IFNg cells, whereas both P and irrelevant syngeneic tumors grew in controls. CONCLUSIONS: Transduction of MCA106 cells with the MuIFNg gene diminished in vivo tumorigenicity in proportion to the amount of IFNg secreted. Immunization with H-IFNg cells elicited a host response characterized by macrophages and CD4+ cells. Long-term tumor-specific memory was seen after immunization with H-IFNg cells.

Immunotherapy of Human Melanoma With Gene-Modified Tumor Cell Vaccines.

The incidence of melanoma in the United States is increasing at a faster rate than that of any other cancer. The prognosis for metastatic disease is poor, and more effective treatments for disseminated disease are needed. Since melanoma is one of the more immunogenic tumors, strategies have focussed on immune recognition. In vitro studies suggest that potent tumor-specific cytotoxic T cells can be induced against human melanoma. Melanoma specific T-cell activation depends on appropriate presentation to the immune system of recently defined melanoma-associated antigens presented in the context of self-HLA gene products. Full T-cell activation requires the co- stimulation by B7-CD28 interactions at the T-cell surface and the elaboration of immune cytokines to promote T-cell growth. Data from animal models of tumor-specific immunization with tumor cells engineered to express immune cytokines or the B7 co-stimulatory molecule suggest that gene therapy for human melanoma may be an effective means to treat disseminated disease.

Transduction of human melanoma cells with interleukin-2 gene reduces tumorigenicity and enhances host antitumor immunity: a nude mouse model.

Human melanoma tumor cells were genetically modified in vitro by transferring the interleukin-2 (IL-2) gene via a retroviral vector into established or fresh tumor cells. In addition, human melanoma cells were transduced in vivo by the direct injection of the IL-2/retroviral vector into melanoma xenografts in nude mice. The gene-modified melanoma cells expressed the IL-2 cytokine gene and secreted biologically active IL-2. Transduction of melanoma cells with the IL-2 gene did not affect the antigenic profile of the cells, but caused a strong abrogation of their tumorigenicity. One million parental cells formed subcutaneous tumors in nude mice. In contrast, various doses of up to 20 x 10(6) IL-2-transduced cells failed to form tumor in the mice. Coinjection of IL-2-producing cells with parental cells inhibited tumor formation even when highly tumorigenic doses of parental cells were used. Histochemical analysis of the injection sites of IL-2-modified cells showed an influx of host immune cells, predominantly macrophages, as early as the third day after inoculation. Neutrophils, mast cells, and eosinophils were also seen in the inflammatory exudate. Eventually, transduced cells showed signs of degeneration and necrosis and ultimately died in 4 weeks. Macrophages were seen in parental tumor sites only during the first few days after injection, and then parental tumors exhibited fast, progressive growth. The study suggests that melanoma cells transduced with the IL-2 cytokine gene may provide an effective vaccine for melanoma patients, whereas the in vivo transduction of tumors with cytokine genes is feasible and may represent a novel approach for the immunotherapy of cancer patients.

Transduction of human melanoma cells with the gamma interferon gene enhances cellular immunity.

Human tumor cells transduced with the gamma interferon (gamma IFN) gene are currently used in specific active immunotherapy protocols to enhance the antitumor immune responses of cancer patients. This in vitro study was undertaken to examine the initial events in the cellular immune response that may occur following the administration of the gamma IFN-transduced cell vaccine. Human melanoma tumor cell lines were transduced with a MoMLV-based retroviral vector carrying the human gamma IFN gene. The transduced cells expressed the cytokine gene, secreted biologically active gamma IFN, and exhibited enhanced expression of MHC class I and class II (HLA-DR), and ICAM-1 surface antigens. The gamma IFN-transduced and corresponding parental melanoma cells were used for the induction of short-term lymphocyte cultures. Peripheral blood lymphocytes or lymph node cells from 20 melanoma patients were stimulated for 5 to 15 days with autologous or MHC class I-matched allogeneic parental or gamma IFN-transduced melanoma cells. Seven of the 20 lymphocyte cultures showed substantial increases in lytic activity following stimulation with the transduced melanoma cells in comparison to control lymphocyte cultures stimulated with unmodified parental melanoma. The cytolytic activity stimulated with gamma IFN-modified melanomas was mediated partly by MHC-restricted cytotoxic T lymphocytes and partly by NK cells. Lymphocyte cultures that displayed increases in cytotoxicity after stimulation with the gamma IFN-transduced melanoma cells also exhibited enhanced expression or induction of one or more of the following lymphokines: IL-4, IL-1 alpha, IL-1 beta, gamma IFN, and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrovirus-mediated gene transfer of the human gamma-IFN gene: a therapy for cancer.

A retroviral vector-mediated gene transfer system was used to introduce m gamma-IFN and h gamma-IFN genes into mouse and human tumor cells, respectively. Murine tumor cell lines and primary human melanoma tumor cells were successfully transduced with gamma-IFN vector, and these transduced cells secreted measurable levels of biologically active m gamma-IFN and h gamma-IFN, respectively. Both murine and human tumor cell lines that expressed gamma-IFN exhibited increased surface expression of HLA class I antigens when tested by Western blot and FACS analysis. gamma-IFN--transduced human melanoma cells were more active in stimulating tumor-specific cytolytic activity of CTLs from melanoma patients in vitro. m gamma-IFN--transduced tumor cells were substantially less tumorigenic than the corresponding parent tumor cell lines in immune-competent mice. In addition, injection of m gamma-IFN--transduced tumor cells resulted in activation of tumor-specific CTL in vivo. We plan to use gamma-IFN--transduced autologous tumor cells to boost host immune responses as a potential therapy for human melanoma.

Cell surface reactive human monoclonal antibody directed to human melanoma-associated gangliosides.

An IgM human monoclonal antibody (human MAb) was generated by fusing lymph node cells isolated from a surgical specimen of malignant melanoma with the heteromyeloma cell line SHMD-33. The antibody, designated 7c11.e8, reacted with surface antigens on human melanoma cells as shown by live cell immunofluorescence and absorption assays. The MAb 7c11.e8 reacted with DSI, SPG, GM4, GM3 and GD3 in enzyme-linked immunosorbent assays (ELISA), and did not react with GD2, GM1, GM2, GD1a, GD1b, GT1b and a number of neutral glycosphingolipids. The main binding epitope for the MAb was, therefore, the terminal N-acetylneuraminic acid 2-3 Gal linked by a beta 1-1 bond to the ceramide, or a beta 1-4 bond to glucose or glucosamine. As shown by immunohistochemical assays, 7c11.e8 antigen was expressed on all melanoma tumour tissues, and on a few samples of colon carcinoma, normal colon, skin, spinal cord, kidney and liver. However, other normal organs such as breast, lung, small intestine, stomach and lymph nodes did not react with the MAb. In the presence of human serum the antibody initiated a strong lysis of melanoma tumour cells in complement-dependent cellular cytotoxicity (CDCC) assays. This study demonstrates that it is possible to isolate human monoclonal antibodies directed to cell surface antigens using viable cell assays in the screening protocol. The preferential binding of 7c11.e8 to melanoma tissues and the reactivity with two of the major melanoma gangliosides (GM3 and GD3) suggest that 7c11.e8 may provide a useful reagent for diagnosis and therapy of malignant melanoma.

Inhibition of the growth of human melanoma metastases in nude mice by melanoma-specific murine monoclonal antibody.

The administration of anti-melanoma murine monoclonal antibody (MAB) 16.C8 (IgG2a) to nude mice bearing established human melanoma lung or liver metastases resulted in a significant inhibition of tumour growth. A total dose of 2 mg of affinity purified 16.C8 caused complete inhibition of tumour growth in 89 and 100% of animals in the liver and lung model, respectively. In contrast, a significant tumour growth was found in most control animals which received an irrelevant IgG2a MAB or 2% human serum albumin in Hanks Balanced Salt Solution (HBSS). The MAB was most effective when treatment was started on day 1 or 4 following tumour inoculation. When the 16.C8 MAB treatment was delayed 7 or 14 days, 33 and 67% of 16.C8 treated animals, respectively, developed tumours. The MAB-mediated anti-tumour activity appeared to be dose dependent, and the effect of a suboptimal dose was potentiated by the concomitant administration of recombinant interleukin 2 (rIL-2). Recombinant IL-2 alone in a similar dose did not elicit comparable anti-tumour activity. Moreover, the MAB 16.C8 inhibited tumour growth in irradiated animals which may suggest the involvement of host-radioresistant cellular elements in the 16.C8 antibody-mediated anti-tumour activities in nude mice. These results suggest that MAB 16.C8 alone or combined with rIL-2 may prove useful in the immunotherapy of metastatic melanoma.

Generation of human IgG, IgA, and IgM anti-melanoma monoclonal antibodies utilizing lymphocytes of an actively immunized melanoma patient.

Active specific immunotherapy with irradiated allogeneic melanoma cells has been shown to enhance the humoral immune response in melanoma patients. An increased titer of melanoma-binding antibodies was demonstrated in sera of immunized patients. Lymph node cells and splenocytes isolated from an actively immunized melanoma patient were fused with the human-murine heteromyeloma cell lines SHMD-33, SPM4-0, and SBC-H20. A group of human anti-melanoma monoclonal antibodies (MABs) were generated from the SHMD-33 fusion. Isolated MABs (one IgG2, one IgA, and two IgM) have been stable in cultures for more than 12 months and have produced human immunoglobulins at 0.2-0.9 Ug/ml/day. As shown by solid phase radioimmunoassays, the MABs react with autologous tumor cells and allogeneic melanoma tumors, including the cell line that was used for immunotherapy. In immunocytochemical assays, all four MABs react with a number of melanoma tumor cell lines. The IgG2 and IgA MABs stained preferentially melanoma tumor cells. In contrast, the IgM MABs cross-reacted with a broad panel of tumor cells from colon, prostate, pancreas, lung, and other human tumors. The MABs appear to be directed to intracellular rather than membrane-associated antigens as shown by immunofluorescence assays on live and permeabilized cells. The IgG2 antibody recognizes a 70 kDa antigen in melanoma cell lysates by Western immunoblotting. The target antigens for the other MABs have not yet been defined. Stability in culture and strong binding to melanoma tumor cells provide the basis for evaluating the potential of these human MABs. The IgG2 MAB, in particular, may prove useful for diagnostic and therapeutic applications in humans.(ABSTRACT TRUNCATED AT 250 WORDS)