dinitrobenzenes and Melanoma

The author has devised a novel approach to the immunotherapy of cancer based on modification of autologous tumor cells with the hapten, dinitrophenyl (DNP). This technology is being developed by AVAX Technologies (MO, USA) as a treatment for melanoma under the brand name, M-Vax. The treatment program consists of multiple intradermal injections of DNP-modified autologous tumor cells mixed with bacille Calmette-Guerin as an immunological adjuvant. Administration of DNP vaccine to patients with metastatic melanoma induces a unique reaction--the development of inflammation in metastatic masses. Following DNP-vaccine treatment, almost all patients develop delayed-type hypersensitivity (DTH) to autologous, DNP-modified melanoma cells and about half also exhibit DTH to autologous, unmodified tumor cells. The toxicity of the vaccine is mild, consisting mainly of papules or pustules at the injection sites. Clinical trials have been conducted in two populations of melanoma patients: Stage IV with measurable metastases, and clinical Stage III patients rendered tumor-free by lymphadenectomy. There were 11 antitumor responses in 83 patients with measurable metastases: two complete, four partial and five mixed. In 214 Stage III patients the 5-year overall survival rate was 44%, which compares favorably with the reported surgical rate of 20-25%. In both populations, the induction of DTH to unmodified autologous tumor cells was associated with significantly longer survival. This is a platform technology that is adaptable to other human cancers and early trials indicate immunological activity in ovarian and renal cell carcinomas.

Topics: Adjuvants, Immunologic; Antigens, Neoplasm; Cancer Vaccines; Clinical Trials as Topic; Dinitrobenzenes; Haptens; Humans; Hypersensitivity, Delayed; Inflammation; Lung Neoplasms; Melanoma; Neoplasm Staging; T-Lymphocytes

We have reported that treatment of melanoma patients with a vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP), induced delayed-type hypersensitivity (DTH) to autologous, unmodified tumor cells. Moreover, this response was a significant and independent predictor of survival. We analyzed the vaccines prepared for 284 patients who were treated following resection of regional or distant metastases to determine whether the dose and composition correlated with immunological response. Regression analysis showed no significant association between the magnitude of this DTH response and the number of live (trypan blue-excluding) melanoma cells per dose. In fact, vaccines containing higher numbers or higher proportions of dead, but intact, tumor cells induced larger DTH responses to autologous unmodified tumor. The observation that dead tumor cells are immunogenic may be applicable to other cellular human cancer vaccines and underscores the need for applying pharmacological principles to cancer immunotherapy.

Topics: Cancer Vaccines; Cell Count; Cell Separation; Cell Survival; Dinitrobenzenes; Haptens; Humans; Hypersensitivity, Delayed; Injections, Intradermal; Melanoma; Neoplasm Metastasis; Survival Rate

We have devised a novel approach to active immunotherapy based on modification of autologous cancer cells with the hapten, dinitrophenyl (DNP). The treatment program consists of multiple intradermal injections of DNP-modified autologous tumor cells mixed with BCG. Administration of DNP-vaccine to patients with metastatic melanoma induces a unique reaction- the development of inflammation in metastatic masses. Histologically, this consists of infiltration of T lymphocytes, most of which are CD8+. These T cells usually produce interferon-gamma in situ. Moreover, they represent expansion of T-cell clones with novel T-cell receptor (TCR) structures. Occasionally, administration of DNP-vaccine results in regression of measurable metastases. The most common site of regression has been small lung metastases. Administration of DNP-vaccine to patients in the postsurgical adjuvant setting produces a more striking clinical effect. Of 62 patients with clinically evident stage III melanoma who had undergone lymphadenectomy, the 5-year relapse-free survival rate was 45% and the overall survival rate was 58%. These results appear to be better than those obtained with high-dose interferon, although a randomized phase III trial is required to prove that point. A recent phase I study suggests that this therapeutic approach is also applicable to stage III ovarian cancer. There appear to be no insurmountable impediments to applying this approach to much larger numbers of patients or to developing it as a standard cancer treatment.

Topics: Animals; Cancer Vaccines; Clinical Trials as Topic; Dinitrobenzenes; Female; Haptens; Humans; Hypersensitivity, Delayed; Immunotherapy, Active; Inflammation; Lung Neoplasms; Male; Melanoma; Mycobacterium bovis; Neoplasm Metastasis; Ovarian Neoplasms; T-Lymphocytes; Tumor Cells, Cultured

This paper is a report of response rate (RR) and survival of 34 metastatic melanoma patients who received a dinitrophenyl (DNP)-modified autologous melanoma cell vaccine. In all, 27 patients started the vaccine as a primary treatment for metastatic melanoma and seven started it as an adjuvant, with no evidence of disease at the time, but had developed new metastases. Interleukin-2 (IL-2) was administered in 24 out of the 34 patients: 19 who progressed on vaccine alone and five who had the combination from start. Interleukin-2 was administered in the intravenous, bolus high-dose regimen (seven patients) or as subcutaneous (s.c.) low-dose treatment (17). Overall response for the entire group was 35% (12 patients out of 34), 12% having a complete response (CR) and 23% a partial response (PR). However, only two patients had tumour responses while on the vaccine alone, whereas the other 10 demonstrated objective tumour regression following the combination with IL-2 (two CR, eight PR), lasting for a median duration of 6 months (range 3-50 months). Of the 12 responding patients, 11 attained strong skin reactivity to the s.c. injection of irradiated, unmodified autologous melanoma cells. None of the patients with a negative reactivity experienced any tumour response. Patients with positive skin reactions survived longer (median survival - 54 months). The results suggest enhanced RRs to the combination of IL-2 and autologous melanoma vaccine. Skin reactivity to unmodified autologous melanoma cells may be a predictor of response and improved survival, and therefore a criterion for further pursuing of immunotherapeutic strategies.

Topics: Adjuvants, Immunologic; Adolescent; Adult; Aged; Antineoplastic Agents; Cancer Vaccines; Child; Combined Modality Therapy; Dinitrobenzenes; Disease Progression; Female; Humans; Interleukin-2; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Skin Neoplasms; Survival Analysis; Treatment Outcome

Topics: Cyclophosphamide; Cytotoxicity, Immunologic; Dinitrobenzenes; Haptens; Humans; Immunotherapy, Active; Melanoma; Neoplasm Metastasis; Survival Analysis; T-Lymphocytes

Active immunotherapy of cancer with therapeutic vaccines has been the subject of experimental and clinical studies for at least 50 years. Our approach has employed 1) autologous, human cancer cells because of extensive evidence that tumor rejection antigens may differ between multiple tumors of the same histology; 2) the immunopotentiating drug, cyclophosphamide; and 3) haptens, particularly dinitrophenyl. Multiple clinical trials in 455 patients with melanoma and ovarian cancer have shown that administration of haptenized vaccines at the proper dosage-schedule regularly induces T cell-mediated immunity to autologous tumor cells as measured by delayed-type hypersensitivity. Moreover, the vaccine causes changes in the tumor site suggestive of an immune reaction, including inflammation and infiltration with CD8+ T lymphocytes that are activated and produce cytokines. The T cell response is oligoclonal, and dominant Vβ families differ between patients. Studies of measurable metastases show clinically important tumor regression. Commercial development of this technology is clearly feasible.

Topics: Antigens, Neoplasm; Cancer Vaccines; Dinitrobenzenes; Female; Humans; Inflammation; Melanoma

We have previously reported a clinical trial of a human cancer vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP), in patients with clinical stage III melanoma. Here we present a follow-up report expanded to 214 patients with 5-year follow-up.. Two hundred fourteen patients with clinical stage III melanoma (117 patients with stage IIIC and 97 patients with stage IIIB) who were melanoma-free after standard lymphadenectomy were treated with multiple intradermal injections of autologous, DNP-modified vaccine mixed with bacille Calmette-Guérin. Four vaccine dosage schedules were tested sequentially, all of which included low-dose cyclophosphamide. Patients were tested for delayed-type hypersensitivity (DTH) to autologous melanoma cells, both DNP-modified and unmodified, and to control materials.. The 5-year overall survival (OS) rate of the 214 patients was 44%. DTH responses to unmodified autologous melanoma were induced in 47% of patients. The OS of this DTH-positive group was double that of DTH-negative patients (59.3% v 29.3%; P <.001). In contrast, positive DTH responses to DNP-modified autologous melanoma cells and to purified protein derivative developed in almost all patients but did not affect OS. Surprisingly, the OS after relapse was also significantly longer in patients who developed positive DTH to unmodified tumor cells (25.2% v 12.3%; P <.001). Finally, the development of DTH was dependent on the schedule of administration of the vaccine, specifically, the timing of an induction dose administered at the beginning of the treatment program.. This study underscores the importance of the immunopharmacology of the autologous, DNP-modified vaccine and may be relevant to other cancer vaccine technologies.

Topics: Adjuvants, Immunologic; Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Alkylating; Cancer Vaccines; Combined Modality Therapy; Cyclophosphamide; Dinitrobenzenes; Female; Follow-Up Studies; Haptens; Humans; Hypersensitivity, Delayed; Male; Melanoma; Middle Aged; Mycobacterium bovis; Neoplasm Recurrence, Local; Skin Neoplasms; Survival Rate

Administration of a vaccine consisting of autologous melanoma cells modified with a hapten, dinitrophenyl (DNP), induces T cell infiltration of metastatic sites. We have reported an analysis of these infiltrating T cells, indicating that certain TCR-Vbeta gene segments are greatly overexpressed. In this study, we investigate the rearrangement of the TCR-Vbeta as well as the junctional diversity in T cells infiltrating melanoma metastases following treatment with DNP vaccine. In 19 of 26 control specimens, V-D-J length analysis showed the expected polyclonal patterns. In contrast, postvaccine tumors from 9 of 10 patients showed dominant peaks of V-D-J junction size in one or more Vbeta families. Dominant peaks were seen most frequently in six Vbeta families (Vbeta7, 12, 13, 14, 16, and 23) and were never seen in seven others. Further analysis of the oligoclonal Vbeta products showed dominant peaks in the J region as well. Of particular interest was the finding that Vbeta and Jbeta peaks were similar in inflamed metastases obtained at different times or from different sites from the same patient. Although 6 of 10 patients expressed HLA-A1, there was no common pattern of TCR rearrangements among them. Finally, the amplified PCR products from seven of these specimens were cloned and sequenced and the amino acid sequence of the complementarity-determining region 3 was deduced. In six of seven specimens, the same complementarity-determining region 3 sequence was repeated in at least two clones and in five of seven in at least three clones. Our study indicates that DNP vaccine induces the expansion of particular T cell clones that may be agents of its antitumor effects.

Topics: Cancer Vaccines; Clone Cells; Cloning, Molecular; Complementarity Determining Regions; Dinitrobenzenes; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Humans; Inflammation; Lymph Nodes; Lymphocytes, Tumor-Infiltrating; Melanoma; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocyte Subsets; Transcription, Genetic

A novel approach to active immunotherapy has been devised based on modification of autologous cancer cells with the hapten, dinitrophenyl (DNP). This technology is being developed by AVAX Technologies as a treatment for melanoma under the brand name, M-Vax(TM). The treatment program consists of multiple intradermal injections of DNP-modified autologous tumour cells mixed with Bacille Calmette-Guérin (BCG). DNP-vaccine administration to patients with metastatic melanoma induces a unique reaction - the development of inflammation in metastatic masses. The inflammation is mediated by IFN-gamma-producing T-lymphocytes, some of which represent expansion of novel clones. Following DNP-vaccine treatment, almost all patients develop delayed-type hypersensitivity (DTH) to autologous, DNP-modified melanoma cells; approximately half also exhibit DTH to autologous, unmodified tumour cells. The toxicity of the vaccine is mild, consisting mainly of papules or pustules at the injection sites. Clinical trials have been conducted in two populations of melanoma patients: stage IV with measurable metastases and clinical stage III patients, rendered tumour-free by lymphadenectomy. In 83 patients with measurable metastases, there were 11 antitumour responses: two complete responses (CRs), four partial responses (PRs) and five mixed. Both CRs and two of four PRs occurred in patients with lung metastases. In 214 stage III patients the 5-year overall survival rate was 46% (one nodal site = 48%, in-transit metastases = 50%, two nodal sites = 36%). In both populations, the induction of DTH to unmodified autologous tumour cells was associated with significantly longer survival. This technology is applicable to other human cancers and clinical trials have been initiated with ovarian adenocarcinoma. There appear to be no insurmountable impediments to applying this approach to much larger numbers of patients or to developing it as a standard cancer treatment.

Topics: Animals; BCG Vaccine; Cancer Vaccines; Combined Modality Therapy; Dinitrobenzenes; Haptens; Humans; Immunotherapy; Melanoma; Neoplasms

Active specific immunotherapy with dinitrophenyl (DNP)-modified autologous melanoma vaccine elicits inflammatory responses in metastatic tumor sites. Postsurgical adjuvant immunotherapy with this vaccine prolongs survival in stage III melanoma patients. We have reported that, after administration of DNP-modified melanoma vaccine, T cell responses to DNP-modified autologous tumor cells are demonstrable in vivo and in vitro. These responses are hapten specific and MHC restricted. To elucidate this phenomenon, we investigated the immune response to DNP-modified peptides eluted from autologous cells. Short peptides were extracted from DNP-modified and unmodified autologous melanoma cells by an acid elution technique and HPLC fractionation. Peptides were also extracted from DNP-modified and unmodified, EB virus-transformed, autologous B lymphoblasts. These various peptide fractions were loaded onto autologous B lymphoblasts and tested for ability to elicit a response by a DNP-specific T cell line as measured by IFN-gamma production. Unexpectedly, stimulatory activity of peptides from DNP-modified melanoma cells was confined to a single HPLC fraction. Spectrometric analysis of this fraction confirmed modification of peptides with DNP. A weaker T cell response was observed to a single HPLC fraction of DNP-modified peptides from the patient's B lymphoblasts. No T cell response was elicited by corresponding fractions of peptides eluted from unmodified melanoma cells or B lymphoblasts. These findings demonstrate the human T cell response to DNP-modified autologous melanoma cells is mediated by hapten-modified, MHC-associated peptides. Further investigation of these peptides could lead to a new strategy for peptide-based cancer immunotherapy.

Topics: Antigens, Neoplasm; Cancer Vaccines; Cell Line, Transformed; Dinitrobenzenes; Haptens; Humans; Immunotherapy; Lymphocyte Activation; Melanoma; Neoplasm Proteins; T-Lymphocytes; Tumor Cells, Cultured

Studies were conducted on oryzalin (3,5-dinitro-N,N-di(n-propyl)sulfanilamide), a widely used dinitroaniline sulfonamide herbicide, which was identified from plant extracts as an inhibitor of mitogen- and growth factor-mediated intracellular free Ca2+ ([Ca2+]i) signalling in mammalian cells.. Oryzalin inhibited vasopressin, bradykinin and platelet-derived growth factor [Ca2+]i signalling in Swiss 3T3 fibroblasts with IC50 values of 14, 16 and 18 microM, respectively. 45Ca2+ uptake into nonmitochondrial stores of saponin-permeabilized Swiss 3T3 cells was inhibited by oryzalin with an IC50 of 34 microM. Oryzalin inhibited colony formation of HT-29 colon carcinoma cells with an IC50 of 8 microM and inhibited the growth of a number of other cancer cell lines and primary human tumors in vitro with IC50 values in the range 3 to 22 microM. A number of oryzalin analogues were studied and an association was found between the ability to inhibit [Ca2+]i signalling and inhibition of the growth of HT-29 human colon cancer cells (P = 0.001) and of CCRF-CEM human leukemia cells (P = 0.016). Oryzalin at doses up to 600 mg/kg administered orally or subcutaneously daily to mice for 3 to 10 days beginning a day after tumor inoculation inhibited the growth of murine B16 melanoma by 63% but showed no appreciable activity when administered subcutaneously or intraperitoneally to mice beginning a number of days after tumor inoculation against a variety of human tumor xenografts. The peak plasma concentration of oryzalin following repeated subcutaneous administration of oryzalin at 600 mg/kg per day to mice was 37 microM and of its major metabolite N-depropyl oryzalin was 53 microM.. It is unlikely that the absence of significant antitumor activity of oryzalin is a result of the inability to achieve adequate plasma concentrations.

Topics: 3T3 Cells; Animals; Calcium; Calcium Channels; Carcinosarcoma; Cell Division; Cells, Cultured; Colonic Neoplasms; Dinitrobenzenes; Fibroblasts; Herbicides; Humans; In Vitro Techniques; Leukemia, Promyelocytic, Acute; Lung Neoplasms; Melanoma; Mice; Mice, Inbred Strains; Signal Transduction; Sulfanilamides

We have developed a novel approach to cancer immunotherapy-an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP). This approach elicits significant inflammatory responses in metastatic sites and some objective tumor responses. Post-surgical adjuvant immunotherapy with DNP-modified melanoma vaccine in a setting of micrometastatic disease produces significant survival prolongation in stage III melanoma patients. Histologically, the inflammatory responses of the tumor consist of infiltration by lymphocytes, the majority of which are CD8+, HLA-DR+ T cells. T cells from these lesions tend to have mRNA for interferon gamma. T cell receptor analysis suggests that the tumor-infiltrating T cells are clonally expanded. DNP-modified vaccine also induces T cells in the peripheral blood, which respond to DNP-modified autologous cells in a hapten-specific, MHC-restricted manner. Moreover, a T cell line generated from these lymphocytes responded to only a single HPLC fraction of MHC-associated, DNP-modified tumor peptides. Since inflammatory responses in metastases were not consistently associated with dramatic tumor regression, we considered the possibility of immunosuppression at the tumor site. We found that mRNA for the anti-inflammatory cytokine, interleukin-10 (IL-10) is expressed in most metastatic melanoma tissues and subsequently demonstrated that IL-10 protein is produced by melanoma cells. Thus the efficacy of DNP vaccine could be further enhanced by inhibition of IL-10 production or binding. Finally, we expect these results obtained with melanoma to be applicable to other human cancers.

Topics: Animals; Cancer Vaccines; Dinitrobenzenes; Disease-Free Survival; Haptens; Humans; Immunosuppression Therapy; Immunotherapy; Melanoma; Mice; Vaccines, Conjugate

Immunizing patients with metastatic melanoma by injection of autologous tumor cells modified by DNP induces inflammatory responses in metastatic masses, which is sometimes associated with tumor regression. To elucidate this phenomenon, we studied the immune response to DNP-modified cells in these patients. All developed DTH to DNP-modified autologous lymphocytes (mean +/- SE: 13.3 +/- 1.3 mm), but not to TNP-modified lymphocytes. Larger responses (21.9 +/- 3.6 mm) were elicited by DNP-modified autologous melanoma cells. In 8/11 patients tested, PBL proliferated in vitro when stimulated by autologous DNP-modified lymphocytes, and in 5 patients the stimulation resulted in production of interferon-gamma. DNP-modified autologous melanoma cells elicited lymphocyte responses as well. PBL from 1 patient were expanded by culture in IL2 and repeated restimulation with DNP-modified B lymphoblastoid cells. This T cell line proliferated and produced interferon-gamma but not IL4, when stimulated by autologous DNP-modified lymphocytes or melanoma cells. Both CD4+ and CD8+ subpopulations responded as determined by panning experiments and by testing of phenotypically homogeneous cultures obtained by limiting dilution. Studies of a stable CD8+ subline of the expanded T cells indicated that the response to DNP-modified cells was MHC-restricted, since it was blocked by antibody to class I determinants. Moreover, these T cells were able to respond to allogeneic DNP-modified stimulators that were matched at one or both HLA-A loci, but not to stimulators that were HLA-A mismatched. Finally, the CD8+ subline killed DNP-modified autologous melanoma cells, but not an HLA-A mismatched allogeneic melanoma, in a 6-hr 51Cr-release assay. These results may have significant implications for understanding the pathogenesis of drug-induced autoimmunity and for the development of new approaches to cancer immunotherapy.

Topics: Antibody Formation; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Cytotoxicity, Immunologic; Dinitrobenzenes; Haptens; HLA Antigens; Humans; Hypersensitivity, Delayed; Immunotherapy; Lymphocyte Activation; Lymphocytes; Major Histocompatibility Complex; Melanoma; T-Lymphocytes

Treatment of metastatic melanoma patients with an autologous vaccine modified by the hapten, dinitrophenyl (DNP), produces a striking immunological effect: the induction of clinically evident inflammatory responses in metastatic tumors. Histological examination shows these tumors to be infiltrated with T lymphocytes. We studied the expression of activation markers on those cells and compared them with matched peripheral blood lymphocytes (PBL) and with lymphocytes extracted from metastases before treatment with DNP-conjugated vaccine. The median fraction of cells that were T cells in post-vaccine tumors was 41%, as compared with 9% in pre-treatment tumors, and those T cells were predominantly CD8+ (mean CD8/CD4 ratio = 5.0). A high proportion of both pre- and post-treatment infiltrating T cells expressed HLA-DR (mean +/- SE = 48% +/- 4%), CD69 (56% +/- 7%), and ganglioside GD3 (68% +/- 5%). This distinguished them from matched PBL in which expression of those markers was significantly lower (HLA-DR = 10% +/- 2%; CD69 = 2% +/- 0.4%; GD3 = 49% +/- 4%). These changes were not accompanied by increased cell-surface expression of interleukin-2 (IL-2) receptors, either CD25 or p75, which were expressed by 1%-2% and 12% of tumor-infiltrating lymphocytes (TIL), respectively. The pattern of activation marker expression that we identified appears to be characteristic of tissue T cells with the memory phenotype. The low expression of IL-2 receptors could indicate functional impairment of TIL in situ, perhaps because of inhibitory molecules produced by melanoma cells.

Topics: Biomarkers; Dinitrobenzenes; Flow Cytometry; Gangliosides; Haptens; HLA-DR Antigens; Humans; Immunotherapy, Active; Inflammation; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Melanoma; Phenotype; T-Lymphocytes; Vaccines, Conjugate

Topics: Catechol Oxidase; Dinitrobenzenes; Humans; Melanoma; Monophenol Monooxygenase; Nitrobenzenes; Skin Neoplasms

The action of lonidamine, 1,(2,4 dichlorobenzyl)-1H-indazol-3-carboxylic acid, on protein synthesis of neoplastic cells growing both in vivo and in vitro has been investigated. Lonidamine decreases amino acid incorporation in all cells tested, although the inhibition is partially relieved by glucose. The inhibition of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake which, on the contrary, is enhanced by the drug. Tests on cell-free systems showed that lonidamine does not inhibit the tobacco mosaic virus (TMV)-mRNA-directed in vitro protein synthesis, thus indicating that protein synthetic machinery per se is not affected. The inhibition of the rate of protein synthesis achieved by lonidamine must be ascribed to an effect on energy-yielding processes with a mechanism similar to that observed in other metabolic inhibitors. Lonidamine, however, because of its capacity to inhibit both respiration and glycolysis in neoplastic cells, is effective at 10 to 20 times lower concentrations. DNP and oligomycin potentiate the inhibitory effect of lonidamine on the rate of protein synthesis. This finding substantiates the idea that neoplastic cells, including those growing in ascitic form, utilize mitochondrial oxidative phosphorylation as the main source of ATP for their biosynthetic processes.

Topics: Amino Acids; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Line; Cell-Free System; Dinitrobenzenes; Glycolysis; Humans; Indazoles; Kinetics; Lactates; Leukemia, Experimental; Male; Melanoma; Mice; Neoplasm Proteins; Oligomycins; Pyrazoles; Rabbits; Sarcoma, Experimental