Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high-affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3.

To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.

Murine dendritic cells transfected with human GP100 elicit both antigen-specific CD8(+) and CD4(+) T-cell responses and are more effective than DNA vaccines at generating anti-tumor immunity.

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-2(b)) 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-2(d)). 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.

Tumor cells cotransduced with B7.1 and gamma-IFN induce effective rejection of established parental tumor.

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 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 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.

Immunotherapeutic potential of tumor antigen-pulsed and unpulsed dendritic cells generated from murine bone marrow.

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.

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.

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.

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)

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.

Human xenograft-nude mouse model of adoptive immunotherapy with human melanoma-specific cytotoxic T-cells.

We investigated the efficacy of human melanoma-specific cytotoxic T-cells (CTLs) in treating experimental human melanoma metastases in a nude mouse model of adoptive immunotherapy. Hepatic metastases were generated by the intrasplenic injection of 1.5 x 10(6) human melanoma cells. Animals were then randomized to receive saline, interleukin-2 only, or CTLs and interleukin-2. CTLs were effective when administered 3 or 7 days after generation of hepatic metastases, with 96 and 88% of animals disease-free, respectively, when examined at one month. Interleukin-2 alone was not effective. In addition, CTLs were effective when as few as 2.5 x 10(6) T-cells were adoptively transferred. Only 33% of the animals were tumor-free when CTLs were administered on day 10, and CTLs were not effective when given at day 14. Human CTLs that were not cytotoxic for the tumor line used in vivo, when tested in a 51Cr assay, were also not effective in the model of immunotherapy. This suggests that the tumor-specific CTLs maintain their specificity in vivo, and eliminates a nonspecific inflammation directed against the human CTLs as a possible cause of the antitumor effect. These studies lay the foundation for clinical trials of CTLs in the adoptive immunotherapy of patients with metastatic melanoma.

Cell binding and tumor inhibiting functions of a new antihuman melanoma murine monoclonal antibody.

Murine, antihuman melanoma cell monoclonal antibody (mAb) 16.C8 was generated by fusing the murine myeloma cell line P3X63/Ag8.653 with splenocytes from a nude mouse bearing a human melanoma xenograft, after reconstitution with splenocytes from syngeneic immunocompetent BALB/c mice. The antibody reacted strongly with fresh human melanoma cells and exhibited preferential reactivity with established human melanoma and neuroectodermal tumor cell lines. Electrophoresis and Western blotting experiments indicated that 16.C8 is directed against a sialoglycoprotein antigen with a molecular weight of 110-120 kDa. mAb 16.C8 mediated lysis of melanoma cells in vitro in antibody-dependent cellular cytotoxicity assays using human mononuclear effector cells isolated from normal volunteers or malignant melanoma patients. In addition, the administration of mAb 16.C8 to nude mice bearing established human melanoma lung and liver metastases resulted in significant inhibition of tumor growth as shown by gross and histologic examination. In contrast, animals treated with Hanks' balanced salt solution or nonspecific immunoglobulin exhibited a large tumor burden. These results suggest that mAb 16.C8 may be of value in treatment of metastatic melanoma in humans.

Treatment of human melanoma hepatic metastases in nude mice with human cytotoxic T lymphocytes.

We investigated the effects of human melanoma-specific cytotoxic T lymphocytes in treating experimental human melanoma hepatic metastases in a nude mouse model of adoptive immunotherapy. Hepatic metastases were generated by intrasplenic injection of 1.5 x 10(6) human melanoma cells. Three days after injection, animals received salt solution and interleukin 2 or interleukin 2 and cytotoxic T lymphocytes. Twenty-four of 25 control animals had developed multiple tumor nodules in the liver; 11 of 13 animals receiving only interleukin 2 also had significant tumor burdens. In striking contrast, 17 of 18 animals receiving cytotoxic T lymphocytes and interleukin 2 had no gross or histologic evidence of tumors. The remaining animal had a 2-mm nodule. Human tumor-specific cytotoxic T lymphocytes are effective in vivo in a model of adoptive immunotherapy and may prove useful in adoptive immunotherapy of humans with metastatic melanoma.

Inhibition of the growth of human melanoma xenografts in nude mice by human tumor-specific cytotoxic T-cells.

Melanoma-specific T-cells (CTLs) are specifically cytotoxic for autologous tumor, when assayed in vitro. To examine their effectiveness in vivo, we tested the ability of these human T-cells to inhibit growth of human melanoma xenografts by using a Winn assay. Nude mice receiving specific CTLs (n = 10) demonstrated a dramatic inhibition of tumor growth. All treated mice were tumor-free at day 50 and nine remained tumor-free at day 65, vs. control mice (n = 10) with average tumor volumes of 321 mm3 and 808 mm3, respectively. To control for the possibility that a non-specific response to the human T-cells could inhibit tumor growth, an additional group received allospecific CTLs. There was no inhibition of tumor growth in this group (n = 8), with the average tumor volume of 2,768 mm3 at day 40 vs. 1,882 mm3 in the control group (n = 10). We conclude that these tumor-specific CTLs can inhibit tumor growth in vivo and may prove useful in the adoptive immunotherapy of melanoma.

Melanoma patient antibody responses to melanoma tumor-associated antigens defined by murine monoclonal antibodies.

Sera from melanoma patients undergoing immunization with three distinct immunogens were evaluated for development of IgG antibody specific for the melanoma tumor-associated antigens (TAAs) GD3 and 250-kd glycoprotein (high molecular weight; HMW). Of 99 patients receiving either irradiated allogeneic melanoma cells admixed with bacillus Calmette-Guérin, vaccinia viral oncolysate melanoma cell membranes, or cholesterol hemisuccinate-modified melanoma cells, 12 were determined to have responded against GD3 and 17 against the HMW TAA. This reactivity was measured using both direct binding to purified TAA and specific inhibition of binding of murine monoclonal antibodies R24 and Me-1-14 for GD3 and HMW TAA, respectively. Preimmune sera from these patients did not react with these TAAs, nor did preimmune sera or follow-up sera from melanoma patients electing not to receive immunotherapy. These results suggest that melanoma patients can be immunized against these TAAs as presented on the melanoma cell membrane in an immunotherapy setting and that immunization using purified TAAs might likewise be feasible.

In vivo tumor localization using tumor-specific monkey xenoantibody, alloantibody, and murine monoclonal xenoantibody.

Specific in vivo localization of antibodies reactive with human melanoma cell membrane tumor associated antigens (TAA) has been attempted using congenitally athymic nude mice bearing subcutaneous human melanoma tumor xenografts as the experimental model. IgG fractions were prepared from each of several immune and control sera. Antimelanoma antibody sources included human alloantibody obtained from melanoma patients immunized against allogeneic melanoma cells, a monkey antiserum raised by immunization against a single human melanoma cell line, and a murine monoclonal antimelanoma antibody-secreting hybridoma cell line. Localization of these radiolabeled antibodies and of control IgG preparations to tumor tissue was determined by whole body scintigraphy and by differential tissue counting. Compared with the different control IgG preparations, each of the antimelanoma IgG preparations exhibited significant specific accumulation within the melanoma tissue. However, variation existed in the ability of each antimelanoma IgG to tumor preparation to localize despite attempts to control model parameters such as tumor source, in vivo passage number and mass. This variation appears to reflect basic biologic differences between tumors in different animals and possibly differences in the antigen-binding capacities of each IgG preparation following radioiodination. This technique for tumor localization is very promising and has obvious potential for clinical application.