Clonal analysis of cytotoxic and regulatory T cell responses against human melanoma.

T cell-mediated immune response against autologous melanoma cells was analyzed, at population and clonal levels, in 31 patients with recurrent and/or metastatic disease. Fresh PBL and lymph node lymphocytes (LNL) from melanoma-involved nodes were not cytotoxic against the respective melanoma cells. When activated in in vitro coculture (IVC) against the autologous melanoma cells in the presence of IL-2, a majority of the activated PBL and LNL became cytotoxic against the autologous targets. The activated effector cells were cloned in limiting dilution microcultures, and growing clones were phenotypically defined and were functionally characterized for cytotoxicity and for potential regulatory function. Functional T cell clones were obtained from 15 of 31 cases. Of these, CTL responses exhibiting cytotoxicity restricted against the autologous melanoma were seen in four cases. All four CTL clones were CD3+, CD8+, and CD4-. Three of these four CTL clones were studied extensively. All three of these CTL clones expressed MHC class I-restricted cytotoxicity. mAb anti-CD3 blocked cytotoxicity in two and enhanced cytotoxicity in the other. Neither autologous sera nor autologous nonactivated fresh PBL modulated the cytotoxic functions of the CTL clones at the effector phase. T cell lines exhibiting regulatory function were obtained in 11 cases. The regulatory T cell lines were CD3+, CD4+, and CD8-. In three cases CD4+ clones amplified the cytotoxic response in the PBL in coculture, while in eight other cases the T cell lines downregulated the cytotoxic responses. Such T cell-mediated down-regulations were either restricted to the autologous system, induced by D/DR antigens expressed by the autologous or allogeneic melanoma cells, or induced by stimulus other than D/DR antigens. Taken together, these findings clearly demonstrate the existence of T cell-mediated cytotoxic and regulatory responses against human melanoma.

Tumor imaging of Dalton's lymphoma using antibody-desferal-Ga-67 complex.

Using desferal, a bifunctional chelating agent, Ga-67 was conjugated to the antibody, anti DLAA, specific for a 80 KD antigen associated with Dalton's lymphoma (DLAA) and was used for imaging of murine lymphomas induced by an 'air-pouch' technique. Scintiscan studies were carried out at 24 hr, 48 hr, 72 hr, after i.v. administration of Ga-67-anti DLAA-DF complex and compared with scintigrams obtained after administration of free Ga-67 citrate and suspension of Ga-67, DF, anti DLAA to different batches of tumor bearing mice. The study was also carried out using mouse IgG, a non-specific antibody, instead of anti DLAA. The results were compared with biodistribution studies. Scintigrams demonstrated that with anti DLAA there was maximum uptake of Ga-67 by the tumor providing the best images.

Tumor targeting potential of liposomes encapsulating Ga-67 and antibody to Dalton's lymphoma associated antigen (anti-DLAA).

Dalton's lymphoma (solid tumor) was induced in Swiss mice by an 'Air-pouch' technique using ascites cells. A liposomal technique for radioimmunodetection and localization using Ga-67 and antibody has been developed. Dalton's lymphoma associated antigen (DLAA) was isolated and purified by ammonium sulfate fractionation and chromatography. The antibody to this antigen, anti-DLAA, was prepared by immunization of rabbits and purified by CNBr activated chromatography. The specificity of this antibody to the solid tumor antigen was ascertained by agar gel diffusion. Scintiscan studies were carried out using controls and Dalton's lymphoma bearing mice at various time intervals after i.p. administration of 10-20 mu ci of Ga-67, Ga-67 Ga-67 as liposomes and Ga-67 incorporated with antibody to DLAA as liposomes. Extensive studies carried out using liposomes of different sizes and charges revealed that anti-DLAA significantly improved the tumor uptake of Ga-67, thereby facilitating better visualization of the tumor. Negative and neutral liposomes delivered maximum radioactivity at the target tissue, whereas positive liposomes did not cause significant tumor accumulation of 67Ga. Biodistribution studies showed maximum tumor-associated activity that is 15.43% of the injected dose per gram tumor tissue was achieved with Ga-67 anti-DLAA liposomes compared to 6.6% with Ga-67 administered as such. Injection of Ga-67 liposome resulted in minimum tumor-associated activity that is 5.33%, with maximum uptake by liver and spleen. This might be caused by accumulation of liposomes in these organs. Incorporation of anti-DLAA might significantly improve uptake of Ga-67 by target tissue, due to the specificity of the antigen antibody reaction.

Functionally different HTLV I-infected T cell lines with the same phenotype derived from a patient with melanoma.

Two autologous T cell lines infected with HTLV I are described. T cells from a patient with malignant melanoma were infected with HTLV I by co-culture with a HTLV I-producing T cell line, SLB I. Both T cell lines express identical phenotype (CD3+, CD4+, 4B4+, 2H4-) but they demonstrate marked differences in growth characteristics and function. One of these two lines, referred to as TFTx, established from the autologous tumor activated peripheral blood lymphocytes (PBL), grows in culture without any exogenous interleukin-2 (IL-2), secretes no detectable amount of IL-2 or gamma interferon (IFN) or tumor necrosis factor (TNF) alpha or beta. The other line (TFATx), established from a co-culture between the autologous PBL, lethally irradiated TFTx and the autologous melanoma cells TF-M, is IL-2-dependent for growth, secretes IFN gamma and TNF alpha and beta. TFTx exerts profound suppression of generation of cytotoxicity in the autologous PBL in co-culture with the autologous melanoma cells TF-M. In contrast, the TFATx enhances the cytotoxic response in similar co-culture. In addition to suppression of cytotoxic response, supernatant from TFTx suppresses the lectin-activated proliferation of PBL. In 4-h chromium release microcytotoxicity assays, neither line exhibits conventional characteristics of cytotoxic cells. Thus, phenotypically identical HTLV I-infected CD4+ T cell lines derived from the same individual exhibit opposite regulatory functions.

Clonal expansion of T cells following in vitro stimulation with autologous melanoma cells and interleukin-2 studied by molecular analysis of a T cell receptor.

Twelve autologous mixed peripheral blood (PBL) tumor cell interactions (MLTC) followed by in vitro expansion of the stimulated T cells in recombinant interleukin-2 (IL-2) were analyzed for the potential emergence of oligoclonal or monoclonal T cell populations in the PBL by Southern blot analysis of the T cell receptor (TCR) beta gene. The emergence of oligoclonal or monoclonal TCR beta gene rearranged populations was seen in 5 of the 12 cases. In 2 of these 5 cases only one dominantly rearranged band was observed. The emergence of oligoclonal or monoclonal T cell populations following stimulation with autologous melanoma cells was associated with predominant CD4 phenotype of the stimulated PBL exhibiting a varied degree of cytotoxicity toward the respective autologous melanoma cells. The evidence of emergence of monoclonal or oligoclonal T cell populations following stimulation with autologous tumor cells strongly supports the existence of T cell-mediated responses against autologous melanomas. Furthermore, cellular and molecular analyses of T cell responses in autologous mixed lymphocyte tumor cell interactions will provide valuable information on the nature of the T cell responses and on the pattern of gene segment usages by the T cells in response to the autologous tumor cells.

Surgical adjuvant active specific immunotherapy for patients with stage III melanoma: the final analysis of data from a phase III, randomized, double-blind, multicenter vaccinia melanoma oncolysate trial.

BACKGROUND: A phase III, randomized, double-blind, multicenter trial of active specific immunotherapy (ASI) using vaccinia melanoma oncolysate (VMO) was performed in patients with stage III (American Joint Commission on Cancer) melanoma to determine the efficacy of VMO to increase the disease-free interval (DFI) or overall survival (OS) in these patients. Two interim analyses of data from this trial were performed in May 1994 and June 1995. Although the results from these analyses showed no statistically significant improvement in DFI or OS in all patients using VMO, two subsets-men aged 44-57 years with one to five positive nodes and all patients with clinical stage I and pathologic stage II disease-showed an overall survival advantage with VMO therapy. A final analysis of data from this trial was performed in May 1996 and is reported here. The design of future melanoma vaccine trials is discussed based on information learned from this first randomized, multicenter trial of ASI therapy. STUDY DESIGN: A polyvalent VMO was prepared using melanoma cells derived from four melanoma cell lines and vaccinia vaccine virus (V). Patients were accrued from 11 United States institutions and were randomized by the Statistical Center at the University of Alabama, Birmingham. Two hundred fifty patients were randomized to treatment with either VMO (1 U containing 2 mg of total protein derived from 5 x 10(6) melanoma cells and 10(5.6) 50% tissue culture infectious dose of vaccinia virus) or control V (1 U containing 10(5.4) 50% tissue culture infectious dose of vaccinia virus) once a week for 13 weeks and then once every 2 weeks for a total of 12 months, or until recurrence. Patient data were collected by the Statistical Center and analyzed as of May 1996 for DFI and OS using Wilcoxon test and log-rank analysis. RESULTS: Two hundred seventeen patients were found to be eligible according to the inclusion criteria. Data from these patients were analyzed for DFI and OS after a median followup of 46.3 months (50.2 months for VMO and 41.3 months for V). This final analysis showed no statistically significant increase in either DFI (p = 0.61) or OS (p = 0.79) of patients treated with VMO (n = 104) compared with V (n = 113). At 2-, 3-, and 5-year intervals, 47.8%, 43.8%, and 41.7% of patients treated with VMO were disease-free, respectively, compared with 51.2%, 44.8%, and 40.4% of patients treated with V. At the same intervals, 70.0%, 60.0%, and 48.6% of patients treated with VMO survived, compared with 65.4%, 55.6%, and 48.2% of patients treated with V. In a retrospective subset analysis, male patients aged 44-57 years (n = 20) with one to five positive nodes showed 18.9%, 26.82%, and 21.3% improvement in survival at 2-, 3-, and 5-year intervals, respectively, after treatment with VMO when compared with V (n = 18) (p = 0.046). CONCLUSIONS: This study was a randomized, multicenter, placebo-controlled evaluation of an active specific immunotherapeutic agent to increase the DFI or OS of patients with stage III melanoma in a surgical adjuvant setting. In this trial, ASI with VMO when compared with V showed no difference in either DFI or OS. In a retrospective subset analysis, however, a subset of men with one to five positive nodes, between the ages of 44 and 57 years, showed a survival advantage with VMO. This result suggests that one must include a detailed subset analysis in the design of future trials of ASI for patients with American Joint Commission on Cancer stage III melanoma. An appropriate control arm also must be included in ASI trials.

Overview analysis of adjuvant therapies for melanoma--a special reference to results from vaccinia melanoma oncolysate adjuvant therapy trials.

A phase III, randomized, double-blind, multi-institutional vaccinia melanoma oncolysate (VMO) trial was performed for patients with stage III (AJCC) melanoma. When compared with the control vaccinia virus (V) therapy, VMO therapy did not show clinical efficacy in the final analysis of data from this trial. However, the data did allude to significant therapeutic efficacy with VMO therapy if it had been compared with an observation arm. Therefore, a comparative overview statistical analysis was performed to identify the therapeutic efficacy of VMO. This review compares VMO results with data from the treatment and observation arms of other prominent randomized anti-melanoma biologic trials (i.e., ECOG EST 1684; SWOG, IFN-gamma (J. Natl. Cancer Inst. 87 (1995) 1710); WHO IFN-alfa-2a (ASCO 14 (1995) 410); Mayo IFN-alfa-2a (J. Clin. Oncol. 13 (1995) 2776); French IFN-alfa-2a (ASCO 15 (1996) 437). The analysis was carried out comparing the disease-free interval (DFI) and overall survival (OS). The analysis shows that the VMO results are fairly comparable to the results of the treatment arms from the ECOG and Mayo trials at the 5-year mark; percent DFI 0.37, 0.37, and 0.4, percent OS 0.48, 0.46, 0.47, respectively. In some cases, VMO DFI is superior to the observation arms from other studies; ECOG, Mayo, and WHO; 0.37 versus 0.26, 0.3, 0.27 (4 years), respectively. These comparative results suggest that the vaccinia arm is not a true observation arm in the VMO trial, and the VMO could have shown an enhanced efficacy had the trial included a no-treatment observation control arm.

Active specific immunotherapy with vaccinia melanoma oncolysate.

Based on antitumor effects observed with vaccinia virus infected tumor cell lysate in animal models, adjuvant immunotherapeutic clinical trials were undertaken in patients with melanoma using vaccinia virus infected melanoma oncolysate (VMO). Preliminary clinical trials showed that the VMO is safe except minimal side effects such as mild fever, pain, and tenderness at the site of VMO injection, and mild lymphadenopathy. The effective dose of VMO was investigated in a following trial using 0.05 to 2.0 mg doses of VMO. Clinical responses and laboratory monitoring of melanoma-specific antibody responses decided the 2 mg dose of VMO is optimal for future trials. In all the clinical trials, patients showed moderate responses and their postimmune sera contained melanoma-specific antibodies. In a Phase II clinical trial completed August 1985 19 of 39 stage II patients had a disease-free mean survival time of 24.6 months, statistically significant compared with historical controls. Because of compelling evidence of significant clinical responses in patients treated with VMO adjuvant immunotherapy in the Phase II trial, a prospective randomized multi-institutional double-blind Phase III adjuvant VMO immunotherapeutic trial using adjuvant therapy of vaccinia virus alone as control, was recently initiated. Results of this trial are anxiously anticipated.

Combinatorial antibodies against human malignant melanoma.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma antigens in patients. We have attempted to identify melanoma-associated antigens recognized by patients' B cells using an antibody phage display approach. Antibody display on filamentous phages allows direct screening of cDNA libraries for expression of cell-surface-reactive antibodies, without the need for antibody production and purification using bacteria or eukaryotic cell systems. This approach was used to identify melanoma-associated cell-surface antigens recognized by patients' B cells. Antibodies produced by the B cells of a melanoma patient (in remission for > 7 years following periodic vaccination with allogeneic melanoma cell vaccine) were displayed as Fabs on the surfaces of filamentous phages. A library of 10(8) phages was absorbed to normal melanocytes, followed by phage binding to and elution from melanoma cells (human lymphocyte antigen nonmatched and vaccine melanoma cells). Phages were further selected for reactivities with tunicamycin-treated melanoma cells. These procedures resulted in a > 10(6)-fold enrichment of tumor-specific phages from the original phage library. One phage-Fab bound to melanoma cells, other tumor cells, and a few normal cells in cultured cell lines and in tissue sections.

T-cell clones that react against autologous human tumors.

T cells (derived from peripheral blood lymphocytes [PBL], lymph nodes or tumor tissues and restimulated with autologous tumor cells and expanded in interleukin-2 [IL-2]), when cloned, produce three functional classes of clone. Class I T-cell clones exhibit the phenotype of alpha/beta cytotoxic T lymphocytes (CD3+, CD8+, CD4-, WT31+), use their CD3-alpha/beta complexes for cognate function, and lyse the autologous tumor cells specifically in a major histocompatibility complex (MHC) Class I-restricted manner. The second class of T cell clone expresses identical phenotype but exhibits a rather broad cytotoxic profile against the autologous and allogeneic tumor cells derived from tumors with similar and/or dissimilar histologies. Although these CTL clones can, at times, show MHC Class I-restricted killing and use their T-cell receptors (TCR) complexes for function, activation via certain accessory molecules, particularly lymphocyte-function associated (LFA-1) antigens, might induce their broad cytotoxic behavior. The nature of the tumor antigen recognized by the Class I antigen-specific CTL clones remains unknown. It is evident, however, that more than one antigen can be associated with a given tumor and they are recognized by different CTL clones from individual patients. The third class of T-cell clone is usually of CD4+ alpha/beta T cells (CD3+, CD4+, CD8-, WT31) and these T-cell clones exhibit no cytotoxicity toward the autologous or allogeneic target cells. When tested for potential regulatory property, one type of CD4+ T-cell clone exhibits the characteristics of helper T cells. This type induces or amplifies cytotoxic response in fresh PBL by elaborating interleukin-2 (IL-2) and interferon-gamma). These helper T-cell clones can proliferate against the autologous tumor cells and demonstrate functional specificity for the autologous tumor cells. The other type of CD4+ T-cell clone exhibits the phenotype of the helper T-cell clone (CD3+, CD4+, CD8-, WT31+) but suppresses the cytotoxic response of the autologous PBL in co-culture in the presence of the autologous tumor cells and exogenous IL-2. In some situations, these CD4+ suppressor T-cell clones exhibit considerable specificity for the autologous tumor cells. They do not suppress the cytotoxic response against allogeneic targets or against EBV-infected autologous lymphoblastoid cells. Furthermore, they specifically up-regulate their IL-2 receptors (IL-2R) when stimulated by the autologous tumor cells or with autologous tumor cell-pulsed antigen-presenting cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunotherapy of a vaccinia colon oncolysate prepared with interleukin-2 gene-encoded vaccinia virus and interferon-alpha increases the survival of mice bearing syngeneic colon adenocarcinoma.

Therapeutic effect of a vaccinia colon oncolysate prepared with interleukin-2 (IL-2) gene-encoded vaccinia virus (IL-2VCO) in combination with recombinant interferon-alpha (IFN-alpha) was studied in a syngeneic murine CC-36 colon hepatic metastasis model. Treatment with this IL-2VCO+IFN-alpha produced a higher survival rate (90% on day 60 after tumor transplantation) in mice having CC-36 hepatic metastases when compared to treatment with IFN-alpha (0%), VCO+IFN-alpha (0%), or IL-2VCO (11%). The only treatment that produced a survival rate similar to the survival rate of IL-2VCO+IFN-alpha was VCO+IL-2 + IFN-alpha (survival rate was 67%). The cause of the prolonged survival with the IL-2VCO+IFN-alpha treatment was identified as the reduction of CC-36 hepatic metastases (mean liver weight 1.31 g and mean tumor nodules 2). This reduction was significant when compared to IL-2VCO (1.58 g and 11), VCO+IFN-alpha (1.96 g and 53), and IFN-alpha (2.24 g and 91) treatments. The mechanism of the induction of antitumor response by the VCO+IL-2+IFN-alpha treatment was analyzed by measuring the direct cytotoxic activity of IFN-alpha on CC-36 tumor cells and by measuring the induction of cytolytic T-cell activity against CC-36 tumor cells. Results suggest that IFN-alpha produced minimal direct cytotoxic activity against CC-36 cells; however, the IL-2VCO+IFN-alpha combination therapy induced an enhanced cytolytic T-cell activity against CC-36 tumor cells (85.1% at E:T 100:1) when compared to other treatments (IL-2VCO 26.3%, VCO+IFN-alpha 13.4%, and IFN-alpha alone 6.3%). In addition, the role of T-cell subsets for the induction of antitumor immune response was analyzed in a survival study that used CD8-positive T cell-depleted mice. It was found that the survival rate was affected in mice depleted with CD8-positive T cells and treated with IL-2VCO+IFN-alpha when compared to control mice which had no T-cell depletion and were treated with IL-2VCO+IFN-alpha. This study suggests that the addition of IFN-alpha along with IL-2VCO increased the survival rate of mice having CC-36 hepatic metastases through the induction of CD8-positive T cells. Furthermore, this study confirms that IL-2VV can be used as a substitute for recombinant IL-2 in cytokine-augmented active specific immunotherapy.

Therapeutic effect of a vaccinia colon oncolysate prepared with interleukin-2-gene encoded vaccinia virus studied in a syngeneic CC-36 murine colon hepatic metastasis model.

Vaccinia CC-36 murine colon oncolysate (VCO) prepared with interleukin-2-gene encoded recombinant vaccinia virus (IL-2VCO) was used in the treatment of a syngeneic murine colon adenocarcinoma (CC-36) hepatic metastasis to test the beneficial effect of the interleukin-2-gene encoded vaccinia virus over a control recombinant vaccinia virus in producing a vaccinia oncolysate tumor cell vaccine. Results suggest that the IL-2VCO treatment significantly reduced the hepatic tumor burden in comparison with the controls that received either IL-2-gene-encoded recombinant vaccinia virus or a plain recombinant vaccinia virus or vaccinia oncolysate prepared with the plain recombinant virus. The survival of mice treated with IL-2VCO was also improved in comparison with mice treated with other preparations. The induction of a cytolytic T lymphocyte response was examined to elucidate the mechanism of the induction of antitumor responses in IL-2VCO-treated mice. Fresh peripheral blood lymphocytes (PBL) isolated from IL-2VCO-treated mice showed a higher cytolytic activity against CC-36 tumor cell target when compared to PBL from the mice of other treatment groups, suggesting that the IL-2VCO induced an antitumor cytolytic T lymphocyte response. These results suggest that a vaccinia oncolysate, prepared with recombinant vaccinia virus encoding an immunomodulating cytokine gene will enhance antitumor responses in the host.

Adjuvant immunotherapy for patients with melanoma: are patients with melanoma of the head and neck candidates for this therapy?

BACKGROUND: Although a wealth of information is available on adjuvant immunotherapy for melanoma, little is known about adjuvant immunotherapy for head and neck melanoma. Interestingly, a few immunotherapy clinical trials report the observation of clinical responses in a subset of patients with head and neck melanoma. METHOD: An up-to-date literature search was performed to identify the current information on adjuvant immunotherapy for patients with melanoma, including head and neck melanoma. Moreover, a retrospective analysis of a subset of primary head and neck melanoma was performed using data from a phase III, randomized, double-blind, multi-institutinal, vaccinia melanoma oncolysate adjuvant immunotherapy trial that was performed in our laboratory for patients with stage III (AJCC) melanoma. RESULTS: In a passive immunotherapy trial with an antibody to melanoma ganglioside antigen GM2, a complete regression was observed in one patient with lesions of the right cheek. In three active specific immunotherapy trials, including our phase III trial, a subset of patients with head and neck primary melanoma showed a longer disease-free and overall survival with immunotherapy. Moreover, these clinical responses were correlated to the induction of immune response, delayed-type hypersensitivity response and melanoma-specific antibody response. CONCLUSIONS: The above results therefore suggest that patients with head and neck melanoma clinically respond to immunotherapy. However, these results need to be confirmed in a prospectively randomized trial for patients with head and neck melanoma.

Active specific adjuvant immunotherapy with vaccinia melanoma oncolysate.

Vaccinia melanoma oncolysate is an investigative agent for active specific adjuvant immunotherapy. Viral oncolysate work in animals has led to application of this treatment in humans. Success of initial trials in treatment of melanoma has led to a phase III randomized, prospective, double-blind, multi-institutional trial, which is currently under way.

Prospects for gene therapy and lymphokine therapy for metastatic melanoma.

Conventional treatment of cancer, especially for patients with metastatic melanoma tumor, is often ineffective. Immunotherapy and recently introduced gene therapy have revolutionized the treatments of patients with metastatic melanoma tumor. Use of biological response modifiers, such as interleukins and interferons, have been found to enhance therapeutic benefits to patients with malignant melanoma. Initial studies with a high-dose interleukin-2 (IL-2) therapy have proved effective in patients with melanoma tumor, although a variety of systemic toxicities were observed. A low-dose IL-2 continuous infusion has shown a similar response in patients with melanoma tumor, but produced lesser toxicity. The low-dose IL-2 therapy has been studied with an adoptive transfer combined with either autologous lymphokine activated killer cells or autologous tumor infiltrating lymphocytes (TIL). IL-2 in combination with chemotherapeutic agents such as flavone acetic acid, dacarbazine, and cyclophosphamide have also been studied in patients with metastatic melanoma. Results have shown a moderate response in patients with metastatic melanoma. TIL therapy, however, has been shown to result in higher objective regression due to potent tumor-specific killing and tumor-specific targeting characters of the TIL. The tumor targeting nature of the TIL creates the possibility of using TIL as a vehicle to deliver gene product specifically to tumor tissue. Safety and toxicity of gene-transduced TIL were addressed by the use of neomycin-resistant, gene-transduced TIL in patients with metastatic melanoma. We also investigated the use of vaccinia oncolysate therapy by using the viral oncolysate prepared with IL-2 gene encoded vaccinia virus. Preliminary studies with murine hepatic metastases colon model have shown encouraging results.

Colon cancer cell vaccine prepared with replication-deficient vaccinia viruses encoding B7.1 and interleukin-2 induce antitumor response in syngeneic mice.

A replication-deficient recombinant vaccinia virus, NYVAC, was developed by deleting 18 open reading frames in the vaccinia virus genome. Recombinant NYVAC, encoding the murine T cell co-stimulatory gene B7.1 (CD 80) (NYVAC-B7.1) and the murine interleukin-2 gene (NYVAC-IL-2), were prepared and the expression of B7.1 and the secretion of IL-2 were respectively confirmed in vitro. The use of these viruses to prepare a potent tumor cell vaccine was studied in a syngeneic murine CC-36 colon adenocarcinoma model. Mice were immunized on days 1 and 8 with 10(6) irradiated CC-36 cells that were infected with 10(7) plaque-forming units of either NYVAC-B7.1, NYVAC-IL-2 or a control virus, NYVAC-HR, which encodes a vaccinia virus host-range gene. These mice were then challenged with 10(8) viable CC-36 tumor cells on day 15. All mice (10/10) in a group that had received no vaccination and all mice (20/20) in a group that had received a control vaccine of CC-36/NYVAC-HR developed tumor 4-weeks after tumor cell challenge. Interestingly, only 16/20 mice in a group that had received CC-36/ NYVAC-B7.1 showed the development of tumor after the same interval. The protection against tumor development and the reduction in tumor burden (as mean tumor diameter, 4 weeks after tumor challenge) were significant in this group when compared to groups that were either unvaccinated or vaccinated with CC-36/NYVAC-HR (mean tumor diameter = 6.51+/-3.2 mm compared to 26.5+/-0.9 mm or 26.2+/-1.8 mm respectively) (P = < 0.05). The protection against tumor in a group of mice that received CC-36/ NYVAC-IL-2 vaccination was similar to that in the unvaccinated group or the group receiving a CC-36/NYVAC-HR control vaccination. However, in a survival experiment, mice that received either CC36/NYVAC-B7.1 or CC-36/ NYVAC-IL-2 vaccination on the day of tumor transplantation survived significantly longer than mice that had not been vaccinated (median survival 60+ days, 60+ days or 23.5 days respectively) (P = < 0.05). Interestingly, when a therapeutic tumor vaccination was performed on day 4 after tumor transplantation, mice that had been vaccinated with either CC36/NYVAC-B7.1 or CC-36/NYVAC-IL-2 did not show an improved survival when compared to mice in the control that had not been vaccinated (median survival 28 days compared to 26 days or 25 days respectively). However, mice that had received a therapeutic vaccination with CC-36 cells infected with both NYVAC-B7.1 and NYVAC-IL-2, 4 days after tumor transplantation, survived significantly longer than control mice that had not received any vaccination (median survival 29.5 days compared to 25 days respectively) (P<0.05). These results suggest that a replication-deficient recombinant NYVAC encoding the B7.1 gene and NYVAC encoding the IL-2 gene can be used to produce an effective vaccinia-virus-augmented tumor cell vaccine.

Favorable clinical responses in subsets of patients from a randomized, multi-institutional melanoma vaccine trial.

BACKGROUND: A phase III, randomized, double-blind, multi-institutional trial was performed evaluating active specific immunotherapy using vaccinia melanoma oncolysate (VMO) in the surgical adjuvant setting in patients with stage II melanoma (UICC staging). The first interim analysis showed no significant difference in disease-free and overall survival. The data were further analyzed to identify subsets of patients with improved outcome when treated with VMO. METHODS: Patients received either VMO or placebo of live vaccinia vaccine virus (V), once a week for 13 weeks and then once every 2 weeks for an additional 39 weeks or until recurrence. Having stratified patients according to sex, age, number of positive nodes, tumor thickness, and clinical stage, data were analyzed for disease-free survival and overall survival. RESULTS: Male patients showed a 17% difference in overall survival at 4 years when treated with VMO (p = 0.19). A subset of male patients < 57 years of age with one to five positive nodes showed a 30% difference at 4 years with VMO (p = 0.06). Patients with clinical stage I but pathological stage II disease (both male and female), who had undergone prophylactic node dissection, showed a 23% difference in survival at 3 years with VMO (p = 0.11). CONCLUSIONS: This subset analysis shows encouraging survival benefit in certain subsets of patients and an increasing trend in overall survival. Further follow-up of this phase III trial from a second interim analysis will be forthcoming.

Autologous melanoma-induced activation of regulatory T cells that suppress cytotoxic response.

The host immune response toward autologous human cancer is subject to regulation by the immunoregulatory network. We show that certain CD4+ T cell clones, derived from melanoma involved lymph node lymphocytes and from PBL stimulated by autologous melanoma cells, selectively down-regulated the induction of cytotoxic immune response of PBL against the respective autologous melanoma cells in two autologous systems. In both systems, only the generation of cytotoxic response against the autologous melanoma cells were suppressed. Cytotoxic response against EBV-infected autologous lymphoblastoid cell line in one case and cytotoxic responses against allogeneic targets in the other were not affected. In addition to suppressor activity selectively expressed against the autologous melanoma cells, the T cell clones up-regulated their Tac receptors when cocultured with the autologous melanoma cells and APC. These results support the existence of a putative tumor Ag-driven activation of regulatory T cells that affect cytotoxic immune response, in vitro, against autologous human melanoma.

A phase III randomized, double-blind multiinstitutional trial of vaccinia melanoma oncolysate-active specific immunotherapy for patients with stage II melanoma.

BACKGROUND: In a Phase II trial, surgical adjuvant active specific immunotherapy using a live vaccinia virus-augmented allogeneic polyvalent melanoma cell lysate, vaccinia melanoma oncolysate (VMO), produced a significant disease free interval (DFI) in patients with International Union Against Cancer Stage II melanoma with positive lymph nodes. Therefore, a Phase III randomized prospective, double-blind, multiinstitutional, surgical adjuvant VMO trial was performed to determine the efficacy of VMO to increase the DFI and the overall survival in this group of patients with Stage II disease. METHODS: Two hundred and fifty patients with Stage II melanoma were divided into two postsurgical groups. One group received VMO (total protein equals 2 mg/ml) and the other received the placebo of live vaccinia vaccine virus (V) (10(5.4) TCID50/ml), an adjuvant component of the VMO. Patients initially received these biologics once a week for 13 weeks and then once every 2 weeks for an additional 39 weeks or until recurrence. All surviving patients have been followed for at least 30 months. RESULTS: Statistical analysis of survival data (n = 217) for this first interim analysis shows that there is no statistically significant (P = 0.99) increase in DFI of patients treated with VMO (n = 104) when compared with V (n = 113). The median DFI is 38.0 months for patients treated with VMO and 37.0 months for patients treated with V. At 2- and 4-year intervals, 70 and 38%, respectively, of patients treated with VMO vs. 66 and 36%, respectively, of patients treated with V were free of melanoma. The median overall survival is not available because the patients treated with VMO have not yet reached the 50% mark and the median overall survival is 45.0 months for patients treated with V. At 2- and 4-year intervals, 70 and 38%, respectively, of VMO-treated patients survived when compared with 66 and 36%, respectively, of patients treated with V. Although the overall survival of patients treated with VMO is not statistically significant (P = 0.88) at this point, there is an increasing trend in the overall survival of patients treated with VMO; a 10% increase at the 4-year time point. Moreover, in the subset analysis, VMO-treated male patients (n = 63) showed a 17% improvement in survival at 4-year time point when compared with male patients treated with V (n = 67) (P = 0.19) at the same time point and male patients (n = 20) between the ages of 44 and 57 having 1-5 positive lymph nodes showed a 37% difference in overall survival at the 4-year time point when compared with those patients treated with V (n = 18) (P = 0.13) at the same time point. CONCLUSION: In this first interim analysis, active specific immunotherapy with VMO vs. V showed no difference in the disease free interval or overall survival. Subset analyses likewise showed no significant differences in outcome but the data suggest a potential difference in immunoreactivity between male and female patients with melanoma that awaits further follow up and may merit further investigation.