Portrait of an autologous cancer vaccine: Then and now.

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 Vß families differ between patients. Studies of measurable metastases show clinically important tumor regression. Commercial development of this technology is clearly feasible.

Double-blinded, randomized phase II study using embolization with or without granulocyte-macrophage colony-stimulating factor in uveal melanoma with hepatic metastases.

PURPOSE: To investigate the effects of immunoembolization with granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with uveal melanoma (UM) with liver-only metastasis. MATERIALS AND METHODS: In this double-blind phase II clinical trial, patients were randomized to undergo immunoembolization or bland embolization (BE). Lobar treatment was performed with GM-CSF or normal saline solution mixed with ethiodized oil followed by embolization with gelatin sponge emulsified with iodinated contrast medium. Fifty-two patients (immunoembolization, n = 25; BE, n = 27) were enrolled. Response was assessed after every two treatments. The primary endpoint was overall response rate (ORR) of liver metastases. Progression-free survival (PFS), overall survival (OS), and immunologic responses were secondary endpoints. RESULTS: There were five partial responses in the immunoembolization group (ORR, 21.2%; 90% confidence interval [CI], 10.3%-30.5%) and three in the BE group (ORR, 16.7%; 90% CI, 6.3%-26.9%). Stable disease was seen in 12 patients in the immunoembolization group and 19 in the BE group. OS times were 21.5 months (95% CI, 18.5-24.8 mo) with immunoembolization and 17.2 months (95% CI, 11.9-22.4 mo) with BE. The degree of proinflammatory cytokine production was more robust after immunoembolization and correlated with time to "systemic" extrahepatic progression. In the immunoembolization group, interleukin (IL)-6 levels at 1 hour (P = .001) and IL-8 levels at 18 hours after the procedure (P < .001) were significant predictors of longer systemic PFS. Moreover, a dose-response pattern was evident between posttreatment serum cytokine concentrations and systemic PFS. CONCLUSIONS: Immunoembolization induced more robust inflammatory responses, which correlated with the delayed progression of extrahepatic systemic metastases.

A tale of two pities: autologous melanoma vaccines on the brink.

This paper reviews and compares two autologous vaccine technologies for human melanoma that failed to obtain marketing approval after 10-15 y of clinical development-the HSP vaccine invented by Srivastava and developed by the company, Antigenics, and the hapten-modified cellular vaccine invented by Berd and developed by AVAX Technologies. Both vaccines had a strong basic science background with a well-understood mechanism of action. The HSP vaccine failed in a phase III pivotal trial, while the haptenized cellular vaccine was never adequately tested in a phase III trial because of regulatory and financial problems. It is proposed that the phase I-II clinical trials of the HSP vaccine neglected to define optimal dose, schedule, and route of administration, which, together with safety, are the major reasons for doing such trials. Therefore, the phase III trial was bound to fail because it was based on insufficient immunopharmacological information. Developers of the haptenized cellular vaccine underestimated the manufacturing and regulatory hurdles inherent to that technology and were therefore unable to complete a pivotal trial. Valuable lessons can be learned by acknowledging the mistakes made in these attempts to bring forward new treatments that could have eased the burdens of melanoma patients.

Interleukin 6 mediates production of interleukin 10 in metastatic melanoma.

We previously reported that substantial amounts of IL-10, an immunomodulatory cytokine, are produced by cell suspensions of fresh human metastatic melanoma tissues. Production diminished with continuous culturing of cells, which suggests a pivotal interactive role between melanoma cells and the tumor microenvironment. In this study, we found that the culture media obtained from LPS-stimulated peripheral blood mononuclear cells induced IL-10 production by metastatic melanoma cells. Of the multiple cytokines present in the conditioned culture media, IL-6 was identified as the inducer of IL-10 production. A neutralizing antibody against IL-6 completely blocked the conditioned medium-induced IL-10 production. Metastatic melanoma cells that constitutively produce low amount of IL-10 increased IL-10 production in response to recombinant human IL-6 in a dose-dependent fashion. The response to exogenously added IL-6 was less significant in melanoma cells that produced high amounts of IL-6, probably due to pre-existing autocrine stimulation of IL-10 by endogenous IL-6. On the other hand, metastatic melanoma cells that do not constitutively produce IL-10 protein did not respond to exogenous IL-6. In IL-6-responsive melanoma cells, IL-6 increased STAT3 phosphorylation and inhibition of STAT3 signaling using siRNA or inhibitors for JAKs diminished IL-6-induced IL-10 production. In addition, inhibition of MEK and PI3K, but not mTOR, interfered with IL-10 production. Taken together, the data suggest that blocking of these signals leading to IL-10 production is a potential strategy to enhance an anti-melanoma immune response in metastatic melanoma.

Lymph node ratio is an important and independent prognostic factor for patients with stage III melanoma.

INTRODUCTION: The incidence of melanoma is dramatically increasing worldwide. We hypothesized that the ratio of metastatic to examined lymph node ratio (LNR) would be the most important prognostic factor for stage III patients. METHODS: We retrospectively reviewed our institutional database of melanoma patients and identified 168 patients who underwent lymph node dissection (LND) for stage III disease between 1993 and 2007. Patients were divided into three groups based on LNR (≤10%, n = 93; 10-≤25%, n = 45; and >25%, n = 30). Univariate and multivariate analysis was performed using Cox proportional hazards model. RESULTS: The median survival time of the entire group of patients was 34 months. The median number of positive nodes was 2 (range = 1, 55), and the median number of examined nodes was 22 (range = 5-123). Tumor characteristics of the primary melanoma (such as thickness, ulceration, and primary site) were not significant predictors of survival in this analysis. By univariate analysis, LNR was an important prognostic factor. Patients with LNR 10-25% and >25% had decreased survival compared to those patients with LNR ≤10% (HR = hazard ratio = 2.0 and 3.1, respectively; P ≤ 0.005). The number of positive lymph nodes also impacted on survival (P = 0.001). In multivariate analysis, LNR of 10-25% and >25% predicted survival (HR = 2.5 and 4.0, respectively). CONCLUSION: LNR is an important prognostic factor in patients undergoing LND for stage III melanoma. It can be used to stratify patients being considered for adjuvant therapy trials and should be evaluated using a larger prospective database.

Tumor-derived interleukin-10 as a prognostic factor in stage III patients undergoing adjuvant treatment with an autologous melanoma cell vaccine.

OBJECTIVES: Interleukin-10 (IL-10) downregulates T-cell-mediated immune responses. We studied the association between IL-10 production by freshly isolated melanoma cell suspensions in vitro and overall survival in patients undergoing adjuvant treatment with a vaccine prepared from the same autologous melanoma cells modified with a hapten, dinitrophenyl (DNP). METHODS: Forty-four patients with cutaneous melanoma (29 stage III and 15 stage IV) were prospectively evaluated. Tumor cells were extracted from metastatic deposits for production of DNP-modified autologous melanoma cell vaccine. Small aliquots of the melanoma cell suspensions were separated prior to vaccine processing and cultured overnight for IL-10 production. Based on a blind assessment of the distribution of IL-10 levels in the culture supernatants, a cutoff of 200 pg/ml was used to define high versus low IL-10 producers. Cox regression model was used for multivariate analysis. Overall survival was calculated using the Kaplan-Meier method, and survival curves were compared with the log-rank test. RESULTS: Out of 44 patients, 29 were low and 15 were high IL-10 producers. The median OS was significantly worse for high compared with low IL-10 producers (10.5 months vs. 42 months; P = 0.022). In stage III patients, the multivariate hazard ratio for high versus low IL-10 producers was 2.92 (95% CI, 1.04-8.20; P = 0.041). The corresponding hazard ratio in stage IV patients was 0.92 (95% CI, 1.04-8.20; P = 0.888). CONCLUSIONS: High IL-10 production in the tumor microenvironment could be a determinant of clinical outcomes in stage III melanoma patients receiving autologous melanoma cell vaccine.

High-dose immunoembolization: survival benefit in patients with hepatic metastases from uveal melanoma.

PURPOSE: To retrospectively evaluate prognostic factors for survival in patients with uveal melanoma who received chemoembolization (CE) with 1,3-bis (2-chloroethyl)-1-nitrosourea or immunoembolization (IE) with granulocyte-macrophage colony-stimulating factor (GM-CSF) for hepatic metastases. MATERIALS AND METHODS: Fifty-three consecutive patients with uveal melanoma were treated by using CE or IE in clinical trials approved by the Institutional Review Board. Prognostic factors associated with overall survival (OS) and progression-free survival (PFS) in the liver and extrahepatic (systemic) organs were retrospectively evaluated. Covariates of age, sex, preexisting extrahepatic metastases, liver enzyme levels, tumor volume, radiologic response in hepatic metastases, and treatment type were analyzed. RESULTS: Compared with CE, high-dose (>or=1500 microg of GM-CSF) IE resulted in significantly better OS (20.4 vs 9.8 months, P = .005) and systemic PFS (12.4 vs 4.8 months, P = .001) at univariate analysis. Overall, women outlived men (14.4 vs 9.8 months, P = .01). Patients who achieved regression of hepatic metastases after embolization lived much longer than did those who did not achieve regression (27.2 vs 9.9 months, P < .001). At multivariate analysis, prolonged OS was confirmed for women, patients who underwent high-dose IE, younger patients (age < 60 years), and patients with regression of hepatic metastases. Independent predictors of longer systemic PFS included high-dose IE, younger age, and regression of hepatic metastases. No covariate predicted liver PFS except for hepatic response. CONCLUSION: Treatment with high-dose IE prolonged survival of patients with uveal melanoma who received embolization of hepatic metastases and possibly delayed progression of extrahepatic metastases.

Human interleukin 10 receptor 1/IgG1-Fc fusion proteins: immunoadhesins for human IL-10 with therapeutic potential.

Interleukin 10 (IL-10) is produced by various types of human cancer, including malignant melanoma, and plays an important role in negative regulation of cell-mediated immune responses against tumors. We have developed chimeric molecules (immunoadhesins), combining the extracellular domain of human interleukin 10 receptor 1 (IL-10R1) with the Fc regions of human IgG1 heavy chain and investigated their capability of blocking the biological activities of human IL-10. Monomeric and dimeric immunoadhesins (IL-10R1/IgG1) constructs were tested for capturing human IL-10 and blocking its biological activities. Plasmid vectors that contained the IL-10 immunoadhesin constructs were directly transfected into human melanoma cell lines. Transfection of plasmid vectors into melanoma cell lines resulted in capturing of exogenously added as well as endogeneously produced IL-10. The supernatants obtained from an IL-10 non-producing melanoma cell line transfected with monomeric IL-10 immunoadhesin plasmids most efficiently captured exogenously added IL-10, compared to those obtained with the dimeric IL-10R1/IgG1 plasmid vector. Transfection of IL-10-producing melanoma cells with the monomeric IL-10 immunoadhesin plasmids totally captured endogenously produced IL-10 and enhanced T cell responses against allogeneic melanoma cells. Furthermore, purified monomeric IL-10 immunoadhesin protein showed IL-10 capturing efficacy compatible with that of IL-10-specific monoclonal antibodies. Collectively, these studies indicate that IL-10 immunoadhesins, especially in monomeric form, are potent inhibitors of biological activities of IL-10 and suggest that these molecules, alone or in conjunctions with other immunotherapeutic approaches, can be utilized for the immuno-targeting of IL-10 producing tumors.

Immunoembolization of malignant liver tumors, including uveal melanoma, using granulocyte-macrophage colony-stimulating factor.

PURPOSE: We conducted a phase I study to investigate the feasibility and safety of immunoembolization with granulocyte-macrophage colony-stimulating factor (GM-CSF; sargramostim) for malignant liver tumors, predominantly hepatic metastases from patients with primary uveal melanoma. PATIENTS AND METHODS: Thirty-nine patients with surgically unresectable malignant liver tumors, including 34 patients with primary uveal melanoma, were enrolled. Hepatic artery embolization accompanied an infusion of dose-escalated GM-CSF (25 to 2,000 microg) given every 4 weeks. Primary end points included dose-limiting toxicity and maximum tolerated dose (MTD). Patients who completed two cycles of treatments were monitored for hepatic antitumor response. Survival rates of patients were also monitored. RESULTS: MTD was not reached up to the dose level of 2,000 microg, and there were no treatment-related deaths. Thirty-one assessable patients with uveal melanoma demonstrated two complete responses, eight partial responses, and 10 occurrences of stable disease in their hepatic metastases. The median overall survival of intent-to-treat patients who had metastatic uveal melanoma was 14.4 months. Multivariate analyses indicated that female sex, high doses of GM-CSF (> or = 1,500 microg), and regression of hepatic metastases (complete and partial responses) were correlated to longer overall survival. Moreover, high doses of GM-CSF were associated with prolonged progression-free survival in extrahepatic sites. CONCLUSION: Immunoembolization with GM-CSF is safe and feasible in patients with hepatic metastasis from primary uveal melanoma. Encouraging preliminary efficacy and safety results warrant additional clinical study in metastatic uveal melanoma.

A pilot study of low-dose thalidomide and interferon alpha-2b in patients with metastatic melanoma who failed prior treatment.

Melanoma is a hypervascular tumor and angiogenesis plays a critical role in the development/progression of metastases. As various pathways are involved in tumor angiogenesis, a combination of agents with different antiangiogenesis activities is a reasonable approach. To determine the efficacy and toxicity of combination treatment with low-dose thalidomide and low-dose interferon (IFN) in patients with stage IV melanoma who failed prior treatment(s), fifteen patients with metastatic melanoma (nine cutaneous, six uveal) received oral thalidomide (200 mg daily) with subcutaneous interferon (IFN)-alpha2b (3 MIU, 3x/week). Stabilization or regression of metastases (as evidenced by computed tomographic measurement) was the primary endpoint of the study. Patients were evaluated monthly for toxicity and every 2 months for clinical response. At a median follow-up of 22.8 months (range, 12-32 months), one patient with metastatic cutaneous melanoma achieved partial response, three patients achieved stable disease (one uveal, two cutaneous), nine patients progressed, and two were not evaluable. The time to progression was 6 months for the patient with partial response, and 2, 5.5+ and 11 months for three patients with stable disease. The estimated median overall survival was 4.7 months (confidence interval, 2.2-9.9 months; range, 0.9-31.5 months), and median progression-free survival was 1.8 months (confidence interval, 1.5-3.0 months; range, 0.5-14 months). Grade 3 toxicities related to treatment included neutropenia (n=5), elevation of transaminases (n=2), and neuropathy (n=1). No treatment-related deaths were experienced. Thalidomide+IFN is a safe and tolerable palliative treatment for previously treated stage IV melanoma.

Whipsaw cancer treatments: the role of hormesis in endocrine and immune therapies.

In this report, we propose a philosophy of treatment that few physicians may be bold enough to actually embrace, but which we believe may eventually find a place in the oncologist's armamentarium. The proposal is based on two assumptions: (1) that many hormones and other biologicals have reverse effects in biologic systems depending on their dosage or concentration, a phenomenon called hormesis; and (2) that most malignant tumors have a large but slow-moving capacity to adapt to adverse conditions, probably by the selection of cellular variants. We suggest that the phenomenon of hormesis might be used to keep a tumor under hormonal and/or immunologic environments that are inimical to its growth and well-being.

Chemoembolization of the hepatic artery with BCNU for metastatic uveal melanoma: results of a phase II study.

Uveal melanoma is the most common primary intraocular malignancy in adults and the liver is the most common site for systemic metastases. We conducted a phase II clinical trial for patients with hepatic metastases from uveal melanoma using chemoembolization of the hepatic artery with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) dissolved in ethiodized oil. Gelatin sponge particles were used as a transiently occlusive agent. The responses in hepatic metastases, overall survival, time to progression and side-effects related to chemoembolization were evaluated. Thirty patients were enrolled. Twenty-four patients completed at least one treatment to all targeted liver metastases and were evaluable for hepatic response. Eighteen of these 24 patients experienced regression or stabilization of hepatic metastases for at least 6 weeks (one complete response in hepatic metastases; four partial responses; 13 stable disease). One of the 13 patients with stable disease was rendered free of disease by surgical removal of metastases after chemoembolization (surgical complete response). The overall response rates (complete and partial responses) for intention-to-treat patients and for patients who were evaluable for response were 16.7 and 20.4%, respectively. The median overall survival of the entire intention-to-treat group of patients was 5.2 months (range, 0.1-27.6 months), for patients with complete or partial response in hepatic metastases 21.9 months (range, 7.4-27.6 months), for patients with stable disease 8.7 months (range, 2.9-14.4 months) and for patients with progressive disease 3.3 months (range, 1.6-5.6 months). Importantly, 13 of the 18 patients who achieved complete response, partial response or stable disease subsequently developed progression of extrahepatic metastases with control of hepatic metastases. Chemoembolization with BCNU is a useful palliative treatment for the control of hepatic metastases in uveal melanoma patients. However, progression in extrahepatic sites after stabilization of hepatic metastases requires further improvement in the therapeutic approach to this disease.

M-Vax: an autologous, hapten-modified vaccine for human cancer.

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.

Combination of monocyte-derived dendritic cells and activated T cells which express CD40 ligand: a new approach to cancer immunotherapy.

Interactions between dendritic cells (DCs) and activated T cells are critically important for the establishment of an effective immune response. To develop the basis for a new DC-based cancer vaccine, we investigated cell-to-cell interactions between human monocyte-derived DCs and autologous T cells that are activated to express the CD40 ligand (CD40L). Peripheral blood monocytes were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) to induce differentiation of DCs. Activated T cells (ATs) consisted of autologous peripheral blood lymphocytes that had been activated with phytohemagglutinin (PHA) and then stimulated with calcium ionophore to up-regulate expression of CD40L. Coculture of these DCs and ATs induced significant production of interleukin 12 (IL-12) and also enhanced the production of interferon gamma (IFN-gamma). The production of IL-12 was blocked by an anti-CD40L antibody or by separation of the DC and AT fractions by a permeable membrane. Furthermore, coculture of DCs and ATs induced DCs to upregulate CD83 expression and stimulated migration of DCs toward the macrophage inflammatory protein 3-beta (MIP-3beta). ATs also migrated toward the MIP-3beta. These results suggest a combination of DCs and ATs as a potentially effective therapeutic strategy.

Immunopharmacologic analysis of an autologous, hapten-modified human melanoma vaccine.

PURPOSE: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: This study underscores the importance of the immunopharmacology of the autologous, DNP-modified vaccine and may be relevant to other cancer vaccine technologies.

Intracellular acidification abrogates the heat shock response and compromises survival of human melanoma cells.

This study tests the hypothesis that lowering intracellular pH (pHi) in melanoma cells grown at low extracellular pH (pHe) selectively abrogates 42 degrees C-induced heat shock protein (HSP) expression and reduces survival. Cells were acidified by a combination of a 0.2-pH-unit decrease in pHe coupled with the lactate/H+ transport inhibitor alpha-cyano-4-hydroxy-cinnamic acid (CNCn). A mild acute extracellular acidification was used to mimic the acute extracellular acidification observed in tumors that can be induced in vivo by oral glucose administration. CNCn blocks the activity of H(+)-linked monocarboxylate transporters (MCTs), particularly MCT isoform 1 (MCT-1). This transporter removes lactic acid from cells and has a high activity in DB-1 melanoma cells grown at low pHe. The effect of extracellular acidification combined with CNCn on pHi was measured in cells grown at pHe 6.7 and pHe 7.3. Cells grown at pHe 6.7 serve as an in vitro model for cells in an acidic tumor microenvironment. When cells were grown at pHe 6.7 and incubated with CNCn at pHe 6.5, the pHi decreased from 6.9 to below 6.5, and the 42 degrees C induction of HSP70 and HSP27 was blocked. The abrogation of HSP induction correlated positively with decreased clonogenic survival. In contrast, when cells growing at pHe 7.3 were acidified by a 0.2-pH unit to pHe 7.1, the inhibitor had less effect on pHi, which remained above 7.0. Under these conditions, the 42 degrees C-induction of HSPs was not inhibited, and cytotoxicity was not enhanced. These results indicate that a significant decrease in the pHi of melanoma cells can selectively sensitize the cells to 42 degrees C hyperthermia, possibly through the inhibition of HSP expression. This strategy could result in a therapeutic gain, because normal tissues, existing at a pHe above 7.0, would not be sensitized.

Contribution of dead cells to the immunogenicity of an autologous, hapten-modified melanoma vaccine.

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.

Regulation of intracellular pH in human melanoma: potential therapeutic implications.

Melanoma cells in vivo maintain intracellular pH (pHi) in a viable range despite an extracellular tumor pH (pHe) that is typically below 7.0. In general, three families of transporters are capable of removing metabolic protons, but the specific transporters responsible for the maintenance of pHi at low pHe in melanomas have not been identified. Although the transporters exist in most cells, an inhibitor would be predicted to have selectivity for cells located in an acidic tumor bed because cells in that environment would be expected to have transporters chronically activated. In this report, the levels and extent of expression of the Na+/H+ exchanger (NHE-1) and two of the H+-linked monocarboxylate transporters (MCTs) were evaluated in three melanoma cell lines. The effects of inhibitors of each transporter were tested at an extracellular pH (pHe) of 7.3, 6.7, or 6.5 in melanoma cells that were grown at pHe 7.3 or 6.7. The activity of MCT isoform 1 (MCT-1) was up-regulated in three melanoma cell lines at low pHe, but that of NHE-1 was not. Furthermore, NHE-1 activity was lower in the melanomas than in other normal and malignant cell lines that were tested. Reverse transcription-PCR using primers specific for MCT-1, MCT-4, and NHE-1 showed that expression of none of these transporters was reproducibly up-regulated at the level of transcription when cells were grown at pHe 6.7 instead of pHe 7.3. Ex vivo experiments using DB-1 human melanoma xenografts grown in severe combined immunodeficient mice found that MCT-1 and not NHE-1 was a major determinant of DB-1 tumor cell pHi. Taken together, the data indicate that MCTs are major determinants of pH regulation in melanoma. In contrast, keratinocytes and melanocytes under low pHe conditions relied on NHE-1. Inhibitors of MCTs thus have great potential to improve the effectiveness of chemotherapeutic drugs that work best at low pHi, such as alkylating agents and platinum-containing compounds, and they should be selective for cells in an acidic tumor bed. In most tissues, it is proposed that the NHE-1 could compensate for an inhibited MCT to prevent acidification, but in melanoma cells this did not occur. Therefore, MCT inhibitors may be particularly effective against malignant melanoma.

TCR rearrangement in lymphocytes infiltrating melanoma metastases after administration of autologous dinitrophenyl-modified vaccine.

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.