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-γ-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-γ, or TNF-β, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-γ-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-γ-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-γ. 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.

Genetic modification of tumor cells with the gene for the B7.1 or with the genes for cytokines results in increased tumor cell immunogenicity. in the work reported here, immunization of naive animals with either B7.1 or gamma-IFN gene-modified MCA 106 tumor cells effectively protects the host from subsequent challenge with parental tumor. The same treatment fails to induce regression of established tumors, although tumor-specific CTL are generated in the tumor-bearing animals. In contrast, a large tumor burden of the MCA106 fibrosarcoma can be successfully eliminated by treatment with MCA 106 tumor cells cotransduced with the B7.1 and gamma-IFN genes. Antitumor immunity induced by the cotransductants is primarily dependent on CD8(+) T cells and partly on CD4(+) T cells and NK cells, and the enhanced therapeutic effect may be attributed to the in vivo increase of CTL precursors following treatment. The gamma-IFN and B7.1 genes must be expressed on the same tumor cell for optimal therapeutic effect. Our results suggest that tumor vaccines with a potent immunoprotective effect do not necessarily have therapeutic potential and that weakly immunogenic tumors may be rendered highly immunogenic by cotransfection with the genes for B7.1 and gamma-IFN.

Dendritic cells (DCs) are potent inducers of cytotoxic T lymphocytes (CTLs) when pulsed with an antigenic peptide or tumor lysate. In this report, we have used liposome‐mediated gene transfer to examine the ability of plasmid DNA encoding the human melanoma‐associated antigen gp100 to elicit CD8+ and CD4+ T‐cell responses. We also compared the efficacy between gp100 gene–modified DCs and naked DNA (pCDNA3/gp100)–based vaccines at inducing anti‐tumor immunity. DCs were generated from murine bone marrow and transfected in vitro with plasmid DNA containing the gp100 gene. These gp100‐modified DCs (DC/gps) were used to stimulate syngeneic naive spleen T cells in vitro or to immunize mice in vivo. Antigen‐specific, MHC‐restricted CTLs were generated when DC/gps were used to prime T cells both in vitro and in vivo. Thus, these CTLs were cytolytic for gp100‐transfected syngeneic (H‐2b) tumor MCA106 (MCA/gp) and vaccinia‐pMel17/gp100‐infected syngeneic B16 and MCA106, but not parental tumor MCA106 and B16, or gp100‐transfected allogeneic tumor P815 (H‐2d). Immunization with DC/gp protected mice from subsequent challenge with MCA/gp but not parental MCA106. Antibody‐mediated T‐cell subset depletion experiments demonstrate that induction of CTLs in vivo is dependent on both CD4+ and CD8+ T cells. Furthermore, DC/gp immunization elicits an antigen‐specific CD4+ T‐cell response, suggesting that DC/gps present MHC class II epitopes to CD4+ T cells. In addition, our data show that gene‐modified, DC‐based vaccines are more effective than the naked DNA‐based vaccines at eliciting anti‐tumor immunity in both prophylactic and therapeutic models. These results suggest that the use of DCs transfected with plasmid DNA containing a gene for TAA may be superior to peptide‐pulsed DCs and naked DNA‐based vaccines for immunotherapy and could provide an alternative strategy for tumor vaccine design. Int. J. Cancer 83:532–540, 1999. © 1999 Wiley‐Liss, Inc.

Genetic modification of tumor cells with the gene for the B7.1 or with the genes for cytokines results in increased tumor cell immunogenicity. In the work reported here, immunization of naive animals with either B7.1 or γ-IFN gene-modified MCA106 tumor cells effectively protects the host from subsequent challenge with parental tumor. The same treatment fails to induce regression of established tumors, although tumor-specific CTL are generated in the tumor-bearing animals. In contrast, a large tumor burden of the MCA106 fibrosarcoma can be successfully eliminated by treatment with MCA106 tumor cells cotransduced with the B7.1 and γ-IFN genes. Antitumor immunity induced by the cotransductants is primarily dependent on CD8 + T cells and partly on CD4 + T cells and NK cells, and the enhanced therapeutic effect may be attributed to the in vivo increase of CTL precursors following treatment. The γ-IFN and B7.1 genes must be expressed on the same tumor cell for optimal therapeutic effect. Our results suggest that tumor vaccines with a potent immunoprotective effect do not necessarily have therapeutic potential and that weakly immunogenic tumors may be rendered highly immunogenic by cotransfection with the genes for B7.1 and γ-IFN.

Expression of B7.1 costimulatory molecules on tumor cells has been shown to elicit antitumor immunity in mice. In the present study, we have developed a human B7.1 retroviral vector system to effectively transduce human melanoma cell lines and investigated the potential role of B7.1 in the generation of tumor-specific CTLs from peripheral blood lymphocytes (PBLs) in vitro. We have demonstrated that B7.1-modified melanoma cells are able to induce primary CTL activity from autologous, human lymphocyte antigen (HLA) class I-matched allogeneic PBLs and purified CD8+ T cells in the absence of exogenous cytokines. CTLs generated by B7.1 are tumor specific and HLA class I restricted, and CD8+ T cells are primarily responsible for this specific cytotoxicity. Furthermore, CTLs generated from HLA class I-matched PBLs by B7.1 are cytolytic to tumor cells autologous to the stimulated PBLs. These data suggest that B7.1-modified tumor cells can be used as a potent tumor vaccine for both autologous and HLA class I-matched allogeneic patients.

Dendritic cells (DC) are highly efficient antigen-presenting cells able to capture, process, and present antigens to naive and primed T-cells. In this study, we have investigated the ability of DC, derived from murine bone marrow and pulsed with tumor cell extracts, to induce regression of preexisting tumors. In an experimental model of B16 melanoma in B6 mice, a significant reduction in metastatic nodules in the lungs was observed in tumor-bearing animals treated with either DC alone or DC pulsed with tumor extracts. Kinetic studies demonstrate that the efficacy of these tumor vaccines is inversely related to tumor burden. In this model, tumor-specific cytotoxic T-cells (CTL) could also be induced in vitro from spleen cells derived from tumor-bearing animals treated with DC pulsed with tumor extracts. Untreated mice had no CTL. Furthermore, DC alone elicited tumor-specific CTL responses in tumor-bearing mice, but not in naive mice. Immune cell depletion experiments show that the therapeutic effects of DC are primarily mediated by CD8+ T-cells, while CD4+ T-cells and NK cells are involved in DC-mediated antitumor immunity to a limited extent. These results illustrate the potential use of DC and DC pulsed with tumor extracts as potent therapeutic reagents for cancer and provide a rationale for using DC in vivo to eliminate disseminated tumors or residual tumor deposits following surgery.

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.

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-a-transduced human melanomas to inhibit the growth of admired 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. (C) 1996 Academic Press, Inc.

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.

Thein vitrocytotoxic 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 followingin vitrostimulation 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.

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.

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.

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)

Human Gene TherapyVol. 5, No. 6 News and CommentsA Phase I Trial of Human Gamma Interferon Transduced Autologous Tumor Cells in Patients with Disseminated Malignant Melanoma. Duke University Medical Center, Durham, North CarolinaINVESTIGATORS Hilliard F. Seigler, Timothy L. Darrow, Zeinab Abdel-Wahab, Ram Gangavalli, and Jack BarberINVESTIGATORS Hilliard F. Seigler, Timothy L. Darrow, Zeinab Abdel-Wahab, Ram Gangavalli, and Jack BarberPublished Online:19 Mar 2008https://doi.org/10.1089/hum.1994.5.6-761AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByThe evolving role of gene-based treatment in surgery4 November 2005 | British Journal of Surgery, Vol. 92, No. 12Vaccines in cancer: GVAX®, a GM-CSF gene vaccine9 January 2014 | Expert Review of Vaccines, Vol. 4, No. 3Granulocyte-Macrophage Colony-Stimulating Factor Gene–Transfected Autologous Tumor Cell Vaccine: Focus on Non–Small-Cell Lung CancerClinical Lung Cancer, Vol. 5, No. 3Interferon-Gamma or Granulocyte-Macrophage Colony-Stimulating Factor Administered as Adjuvants With a Vaccine of Irradiated Autologous Tumor Cells From Short-Term Cell Line Cultures: A Randomized Phase 2 Trial of the Cancer Biotherapy Research GroupJournal of Immunotherapy, Vol. 26, No. 4Are Vaccinations for Prostate Cancer Realistic?Cytokines and Immune Response in the Tumor MicroenvironmentJournal of Immunotherapy, Vol. 24, No. 5HLA class I expression on human cancer cellsHuman Immunology, Vol. 61, No. 2Immune Modulation as Cancer Treatment Using Gene Therapy28 January 2018 | Baylor University Medical Center Proceedings, Vol. 12, No. 4Potential use of T cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancerPathology & Oncology Research, Vol. 5, No. 1Cancer VaccinesJournal of Clinical Oncology, Vol. 17, No. 3GENE THERAPY FOR MELANOMA IN HUMANSHematology/Oncology Clinics of North America, Vol. 12, No. 3Human 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 ActivityCellular Immunology, Vol. 186, No. 1Clinical Approaches to Cancer Gene TherapyThe effector functions of macrophages transfected with interferon-gamma gene mediated by recombinant adenovirusChinese Journal of Cancer Research, Vol. 9, No. 4A phase I clinical trial of immunotherapy with interferon-? gene-modified autologous melanoma cellsCancer, Vol. 80, No. 3Rapid Production of Interleukin-2-Secreting Tumor Cells by Herpes Simplex Virus-Mediated Gene Transfer: Implications for Autologous Vaccine Production20 March 2008 | Human Gene Therapy, Vol. 7, No. 18Immunotherapy of Human Melanoma with Gene-Modified Tumor Cell Vaccines30 August 2017 | Cancer Control, Vol. 2, No. 5Cytokines and clinical gene therapyEuropean Journal of Immunology, Vol. 25, No. 4 Volume 5Issue 6Jun 1994 InformationCopyright 1994, Mary Ann Liebert, Inc.To cite this article:INVESTIGATORS Hilliard F. Seigler, Timothy L. Darrow, Zeinab Abdel-Wahab, Ram Gangavalli, and Jack Barber.A Phase I Trial of Human Gamma Interferon Transduced Autologous Tumor Cells in Patients with Disseminated Malignant Melanoma. Duke University Medical Center, Durham, North Carolina.Human Gene Therapy.Jun 1994.761-777.http://doi.org/10.1089/hum.1994.5.6-761Published in Volume: 5 Issue 6: March 19, 2008PDF download

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.

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 glycosphingollplds. The main binding epitope for the MAb was, therefore, the terminal N-acetylneuramlnlc acid 2–3 Gal linked by a β1–1 bond to the ceramlde, or a β-4 bond to glucose or glucosamine. As shown by immunohlstochemlcal 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.

McCallister, T; Burrascano, M; Chong, K; Howard, B; Gangavalli, R; Darrow, T; Vervaert, C; Wahab, Z; Jolly, D J; Seigler, H F; Barber, J R Author Information