High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens.

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or <10(-5) of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported. To begin assessing their contribution to vaccine-induced rejection, we evaluated their blood frequency in five vaccinated patients. The antitumor cytotoxic T lymphocyte (CTL) precursors ranged from 10(-4) to 3 x 10(-3), which is 10-10,000 times higher than the anti-vaccine CTL in the same patient. High frequencies were also observed before vaccination. In a patient showing nearly complete regression after vaccination with a MAGE-3 antigen, we observed a remarkably focused antitumoral response. A majority of CTL precursors (CTLp's) recognized antigens encoded by MAGE-C2, another cancer-germline gene. Others recognized gp100 antigens. CTLp's recognizing MAGE-C2 and gp100 antigens were already present before vaccination, but new clonotypes appeared afterwards. These results suggest that a spontaneous antitumor T cell response, which has become ineffective, can be reawakened by vaccination and contribute to tumor rejection. This notion is reinforced by the frequencies of anti-vaccine and antitumor CTLs observed inside metastases, as presented by Lurquin et al. (Lurquin, C., B. Lethe, V. Corbiere, I. Theate, N. van Baren, P.G. Coulie, and T. Boon. 2004. J. Exp. Med. 201:249-257).

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery.

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an "all in vivo" strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful "all in vivo" vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.

A phase II study of the cancer vaccine TG4010 alone and in combination with cytokines in patients with metastatic renal clear-cell carcinoma: clinical and immunological findings.

MUC1 over-expression in renal clear-cell carcinoma (RCC) is associated with poor prognosis. This phase II study determined the efficacy and tolerability of TG4010, a cancer vaccine based on a modified vaccinia virus expressing MUC1 and interleukin-2, in combination with cytokines, as first-line therapy in metastatic RCC. Thirty-seven patients with progressive, MUC1-positive RCC received TG4010 10(8) pfu/inj weekly for 6 weeks, then every 3 weeks until progression, when TG4010 was continued in combination with interferon-α2a and interleukin-2. Assessments included clinical response (primary endpoint), safety, time to treatment failure (TTF), overall survival (OS), and immune response. No objective clinical responses occurred. Five of the 27 evaluable patients (18%) had stable disease for >6 months with TG4010 alone and six of 20 patients (30%) had stable disease for >6 months with TG4010 plus cytokines. Median TTF was 4.1, 3.6, and 9.3 months for monotherapy, combination therapy, and overall, respectively. Median OS was 19.3 months for all patients and 22.4 months combination therapy recipients. The most frequent TG4010-related adverse events were minor-to-moderate injection-site reactions, fatigue, and flu-like symptoms. Six of 28 patients showed a MUC1 CD4+ T cell proliferative response during therapy. Anti-MUC1 CD8+ T cells were detected before and after therapy in 3 and 4 patients, respectively. MUC1-specific CD8+ T cell responses were associated with longer survival. Therapy with TG4010 plus cytokines appears to be feasible and well tolerated in patients with metastatic RCC. However, these data should be interpreted with caution, as additional prospective studies are necessary to clarify the clinical efficacy of this therapy.

Strong vaccine responses during chemotherapy are associated with prolonged cancer survival.

Therapeutic cancer vaccines have effectively induced durable regressions of premalignant oncogenic human papilloma virus type 16 (HPV16)-induced anogenital lesions. However, the treatment of HPV16-induced cancers requires appropriate countermeasures to overcome cancer-induced immune suppression. We previously showed that standard-of-care carboplatin/paclitaxel chemotherapy can reduce abnormally high numbers of immunosuppressive myeloid cells in patients, allowing the development of much stronger therapeutic HPV16 vaccine (ISA101)-induced tumor immunity. We now show the clinical effects of ISA101 vaccination during chemotherapy in 77 patients with advanced, recurrent, or metastatic cervical cancer in a dose assessment study of ISA101. Tumor regressions were observed in 43% of 72 evaluable patients. The depletion of myeloid suppressive cells by carboplatin/paclitaxel was associated with detection of low frequency of spontaneous HPV16-specific immunity in 21 of 62 tested patients. Patients mounted type 1 T cell responses to the vaccine across all doses. The group of patients with higher than median vaccine-induced immune responses lived longer, with a flat tail on the survival curve. This demonstrates that chemoimmunotherapy can be exploited to the benefit of patients with advanced cancer based on a defined mode of action.

Apoptosis-related mitochondrial dysfunction defines human monocyte-derived dendritic cells with impaired immuno-stimulatory capacities.

The death of dendritic cells (DCs) can potentially influence immune responses by affecting the duration of DC stimulation of lymphocytes. Here, we report that cultured mature monocyte-derived DCs manifest early mitochondrial damage (i.e. within 24 hrs), characterized by mitochondrial membrane potential (psi Delta m) disruption and mitochondrial release of pro-apoptotic factors, followed by reactive oxygen species (ROS) production and activation of caspases. Afterwards, DCs with mitochondrial alterations are condemned to undergo apoptosis and necrosis. Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated. Importantly, pre-apoptotic DCs (characterized by a low Delta psi m) showed a modified phenotype, with down-regulation of HLA-DR and of the co-stimulatory molecules CD80 and CD86. Moreover, sorted viable low psi Delta m DCs were unable to activate allogeneic T cells, indicating that pre-apoptotic DCs have already lost some of their immuno-stimulatory capabilities long before any detectable signs of death occur. Perturbations to mitochondrial respiration with rotenone identified the same modifications to DC immune functions. These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC. Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.

Intralesional adenovirus-mediated interleukin-2 gene transfer for advanced solid cancers and melanoma.

Numerous preclinical and clinical studies have shown that interleukin-2 (IL-2) induces regression of metastatic tumors. We have conducted a phase I/II, multicenter, open-label, dose-escalating study to evaluate the safety, efficacy, and biological effects of repeated intratumoral injections of adenovirus-IL-2 (TG1024) in patients with advanced solid tumors and melanoma. Thirty five patients (twenty-five with metastatic melanoma and ten with other solid tumors) were treated in eight successive cohorts at dose levels ranging from 3 x 10(8) to 3 x 10(11) viral particles (vp). Intratumoral TG1024 injections in combination with dacarbazine (DTIC) were tested in metastatic melanoma in one cohort. No clinical responses were observed at doses below 3 x 10(11) vp. Six local objective responses were recorded in patients receiving 3 x 10(11) vp per treatment [five in metastatic melanoma and one in metastatic squamous cell carcinoma (SCC) of the skin], of which two were complete responses (CRs). Most of the common side effects were injection site reactions and flu-like syndrome. TG1024 dose intensification across cohorts resulted in increased serum IL-2 levels after the injection. Intratumoral TG1024 injection induced pronounced inflammation of the treated lesion, with predominant CD8(+), TIA+ lymphocytic infiltrate. Our results show that intratumoral injections of TG1024 are safe and well tolerated. The clinical activity of TG1024 observed in this study warrants further investigations.

Influence of heat stress on human monocyte-derived dendritic cell functions with immunotherapeutic potential for antitumor vaccines.

Mild heat stress can modulate the activities of immune cells, including dendritic cells (DC) and theoretically, would constitute an innovative approach capable of enhancing the antitumor functions of DC. Therefore, we tested the effects of mild heat stress on the physiology and viability of human monocyte-derived DC, the major type of DC used in tumor immunotherapy trials. We first designed a heat-stress protocol consisting of repetitive, sublethal heat shocks throughout the generation of DC. Using this protocol, we observed that heat stress did not perturb the morphology and the phenotype of immature or mature DC or the capacities of immature DC to uptake antigens efficiently. It is noteworthy that in response to heat stress, mature DC produced higher levels of IL-12p70 and TNF-alpha, which are two cytokines involved in the stimulation of inflammatory reaction, whereas IL-10 production remained low. After heat-stress exposure, mature DC have the full ability to stimulate naive T cells with Th1 response polarization (high IFN-gamma and low IL-4 production) in an allogeneic MLR. It is interesting that heat stress enhanced the migratory capacities of DC in response to MIP-3beta/CCL19. Finally, heat stress partly protected DC from apoptosis induced by cytokine withdrawal. Overall, these findings validate the feasibility of improving immune response by heating human monocyte-derived DC and provide a strong rationale for using mild heat stress in combination with DC vaccination to increase antitumor response.

Dacarbazine, cisplatin, and interferon-alfa-2b with or without interleukin-2 in metastatic melanoma: a randomized phase III trial (18951) of the European Organisation for Research and Treatment of Cancer Melanoma Group.

BACKGROUND: Based on phase II trial results, chemoimmunotherapy combinations have become the preferred treatment for patients with metastatic melanoma in many institutions. This study was performed to determine whether interleukin-2 (IL-2) as a component of chemoimmunotherapy influences survival of patients with metastatic melanoma. PATIENTS AND METHODS: Patients with advanced metastatic melanoma were randomly assigned to receive dacarbazine 250 mg/m2 and cisplatin 30 mg/m2 on days 1 to 3 combined with interferon-alfa-2b 10 x 10(6) U/m2 subcutaneously on days 1 through 5 without (arm A) or with (arm B) a high-dose intravenous decrescendo regimen of IL-2 on days 5 through 10 (18 x 10(6) U/m2/6 hours, 18 x 10(6) U/m2/12 hours, 18 x 10(6) U/m2/24 hours, and 4.5 x 10(6) U/m2 for 3 x 24 hours). Treatment cycles were repeated in the absence of disease progression every 28 days to a maximum of four cycles. RESULTS: Three hundred sixty-three patients with advanced metastatic melanoma were accrued. The median survival was 9 months in both arms, with a 2-year survival rate of 12.9% and 17.6% in arms A and B, respectively (P = .32; hazard ratio, 0.90; 95% CI, 0.72 to 1.11). There was also no statistically significant difference regarding progression-free survival (median, 3.0 v 3.9 months) and response rate (22.8% v 20.8%). CONCLUSION: Despite its activity in melanoma as a single agent or in combination with interferon-alfa-2b, the chosen schedule of IL-2 added to the chemoimmunotherapy combination had no clinically relevant activity.

Highly successful therapeutic vaccinations combining dendritic cells and tumor cells secreting granulocyte macrophage colony-stimulating factor.

In an attempt to induce potent immune antitumor activities, we investigated, within the rat 9L gliosarcoma model, distal therapeutic vaccinations associating three therapies: dendritic cell vaccination, intratumoral granulocyte macrophage colony-stimulating factor (GM-CSF) gene transfer, and tumor apoptosis induction. Vaccines of dendritic cells coinjected with processed GM-CSF secreting 9L cells induced systemic responses, resulting in the complete regression of distant preimplanted 9L tumor masses in, with the best strategy, 94% of male rats. All of the cured rats developed a long-term resistance to a rechallenge with parental cells. The curative responses were correlated with the detection of elevated specific cytotoxic activities and a CD4+, CD8+ T cell-, and natural killer (NK) cell-mediated IFN-gamma production. The survival rate of the rat seemed more directly linked to the amount of GM-CSF secreted by the transduced tumor cells, which in turn depended on the toxicity of the apoptosis-inducing treatment, than to the level of apoptosis induced. Unexpectedly, alive GM-CSF secreting 9L cells became apoptotic when injected in vivo. Thus we documented the positive role of apoptosis in the induction of therapeutic antitumor responses by comparing, at equal GM-CSF exogenous supply, the effects of dendritic cells coinjected with apoptotic or necrotic 9L cells. The data showed the superior therapeutic efficiency of combined vaccines containing apoptotic tumor cells. In conclusion, vaccinations with dendritic cells associated with apoptotic tumor cells secreting GM-CSF show a very high therapeutic potency that should show promise for the treatment of human cancer.

Immune characterization of clinical grade-dendritic cells generated from cancer patients and genetically modified by an ALVAC vector carrying MAGE minigenes.

Gene delivery into dendritic cells (DC) is most efficiently achieved by viral vectors. Recombinant canarypox viruses (ALVAC) were validated safe and efficient in humans. We aimed firstly to evaluate DC transduction by ALVAC vectors, then to investigate if such infection induced or not the maturation of the DC, and finally to assess the efficiency of ALVAC-MAGE-transduced DC to activate specific CTL clones. Clinical grade DC from melanoma patients were generated from blood monocytes and infected with a recombinant ALVAC virus encoding either a marker gene (EGFP) or the MAGE-1-MAGE-3 minigenes. According to the patient-donor, 22+/-16% of immature DC were successfully transduced. Flow cytometry analysis of surface markers expressed on DC after ALVAC infection did not reveal a mature phenotype. Moreover, ALVAC transduction did not interfere with the capacity of the DC to further mature under poly:IC stimulation. But most importantly, our results demonstrated that DC from HLA-A1 patient-donors infected with the recombinant ALVAC MAGE-1-MAGE-3 minigenes virus were capable of activating a MAGE 3/A1 CTL clone more efficiently than same DC loaded with MAGE 3/A1 peptide, as shown by increased IFN-gamma secretion. These results could be the basis for the development of a new clinical strategy in melanoma patient's immunotherapy.

Construction and production of oncotropic vectors, derived from MVM(p), that share reduced sequence homology with helper plasmids.

The production of currently available vectors derived from autonomous parvoviruses requires the expression of capsid proteins in trans, from helper sequences. Cotransfection of a helper plasmid always generates significant amounts of replication-competent virus (RCV) that can be reduced by the integration of helper sequences into a packaging cell line. Although stocks of minute virus of mice (MVM)-based vectors with no detectable RCV could be produced by transfection into packaging cells; the latter appear after one or two rounds of replication, precluding further amplification of the vector stock. Indeed, once RCVs become detectable, they are efficiently amplified and rapidly take over the culture. Theoretically RCV-free vector stocks could be produced if all homology between vector and helper DNA is eliminated, thus preventing homologous recombination. We constructed new vectors based on the structure of spontaneously occurring defective particles of MVM. Based on published observations related to the size of vectors and the sequence of the viral origin of replication, these vectors were modified by the insertion of foreign DNA sequences downstream of the transgene and by the introduction of a consensus NS-1 nick site near the origin of replication to optimize their production. In one of the vectors the inserted fragment of mouse genomic DNA had a synergistic effect with the modified origin of replication in increasing vector production.

Granulocyte macrophage-colony stimulating factor gene transfer to induce a protective anti-tumoral immune response against the 9L rat gliosarcoma model.

The injection of irradiated tumor cells genetically engineered to express the granolocyte macrophage-colony stimulating factor (GM-CSF) is reported as stimulating antitumoral immunity in several animal models. We used the 9L gliosarcoma rat model to investigate the potency of this strategy in relation to the central nervous system. After in vitro transduction experiments to generate 9L murine cells producing GM-CSF (9LmGM-CSF cells), we found that with the exception of one rat subcutaneously (s.c.) grafted with 108 9LmGM-CSF cells, syngenic rats s.c. injected (dorsal route) with 106-108 viable 9LmGM-CSF cells did not develop s.c. 9L gliosarcomas, while rats s.c. grafted with the same number of naive 9L cells died between 25 and 45 days postgraft. Intracerebral stereotactic injections of 4.104 naive 9L (without s.c. 9LmGM-CSF treatment) killed all the rats within 18-24 days, while s.c. grafting with 108 9LmGM-CSF cells wholly prevented the development of 9L brain gliosarcomas. These results outline the feasibility of the GM-CSF gene transfer approach in glioma (or at least gliosarcoma) gene therapy and could therefore serve as a basis for the development of an adjuvant treatment of glioblastomas using GM-CSF-producing tumor cell vaccines.

Assessment of in vivo chemotherapy-induced DNA damage in a p53-mutated rat tumor by micronuclei assay.

Production of DNA damage is the basis of cancer treatments such as chemo- and radiotherapy. Such treatments induce mitotic catastrophe, a form of cell death resulting from abnormal mitosis and leading to the formation of interphase cells with multiple micronuclei. In this study, we compared apoptosis induction and micronuclei formation to assess the DNA damage provoked in vivo by cytotoxic agents in established 9L rat gliosarcoma tumors expressing a mutated p53 gene. Results from TUNEL assays revealed the efficiency of local gamma-irradiation at the tumor site to induce apoptosis within 9L tumor mass. However, little or no apoptosis was detected after systemic (ip) injection of cisplatin (1 mg/kg). Interestingly, the micronuclei assays showed that not only gamma-irradiation but also cisplatin treatment led to an increase in the emergence of binucleated cells with micronuclei. Apoptosis induction and micronuclei emergence are thus not absolutely correlated. However, micronuclei assays, rarely performed on solid tumors, appear more sensitive than apoptosis assays in evaluating DNA damage linked to chemotherapy.

A novel method for the titration of recombinant virus stocks by ELISPOT assay.

The development of vectors for gene therapy requires the definition of quality control parameters such as titration, contamination, transduction efficiency and biological effects in defined model systems. For most viral vectors, the classical titration by plaque formation is not applicable, because vectors are defective for replication and packaging cell lines are not always available. In particular, for vectors derived from the autonomous parvovirus MVM(p), the titration method used currently is based on the amplification of the viral genome inside an infected cell, which can then be revealed with a specific radioactive probe (J. Virol. 63 (1989) 1023). In situ hybridization allows to titrate wild-type virus as well as vectors, using probes that are specific for the substituted viral genes or for the transgene, respectively. This method is, however, time consuming, making the simultaneous titration of large numbers of samples difficult. The use of a radioactive probe requires an adequate facility. An ELISPOT method that allows for rapid titration of up to 23 vector stocks in one 96 well dish was devised. This method is based on the actual expression of the transgene. Compared to in situ hybridization, titers obtained by the ELISPOT method were in general equivalent or higher. However, for some vector stocks the ELISPOT titers were repeatedly lower, indicating that in situ hybridization does not give an accurate measure of transducing units. Our model system is recombinant parvovirus MVM expressing human IL2, but the method should be adaptable to other vectors expressing transgenes that are secreted and for which antibodies are available.

Minocycline-induced activation of tetracycline-responsive promoter.

Minocycline has been suggested to be an anti-apoptotic compound and an anti-inflammatory agent in various models of neurodegeneration. In the present study, using a stable cell line expressing green fluorescent protein under the control of a tetracycline-responsive promoter, we demonstrate that minocycline is able to promote tetracycline-controlled gene expression although it needs longer time and higher concentration to reach the effect obtained with the classical inducer doxycycline. Furthermore, the extinction of the system after antibiotics removal is faster when using minocycline. Interestingly, minocycline displays lower cytotoxicity than doxycycline. It is thus tempting to speculate that combining the intrinsic neuroprotective activity of minocycline with its ability to induce tetracycline-regulatable promoters would be greatly beneficial for neuroprotective/neurorestaurative gene therapy.

IL-10 in vivo gene expression in a cell-induced animal model of proliferative vitreoretinopathy.

Due to inhibitory activities on cell-mediated immune responses, interleukin-10 (IL-10) has been proposed as a good candidate to treat inflammatory eye disease and proliferative vitreoretinopathy (PVR). In this study we evaluate the effect of human IL-10 (hIL-10) expression in a cell-induced animal model of PVR. Rabbit dermal fibroblasts were genetically modified by infection with retroviral particles carrying the neomycin resistance gene (neoR) alone or in combination with the hIL-10 gene. PVR was induced in rabbits by intravitreal injections of RDF hIL-10 or RDF neo. Some rabbits received instead injections of soluble recombinant hIL-10 (rhIL-10). PVR was graded by fundoscopy. Eyes were enucleated for histology at day 28. ELISA was performed to measure hIL-10 production in RDF supernatants and in vitreous samples, 24 h after injection. Results showed that in vitro hIL-10 production by RDF was 24,500 pg/10(6) cells/ml/24 h. In vivo IL-10 secretion was detected in all rabbits injected with RDF hIL-10 but was undetectable in control rabbits. Similar clinical grades of PVR were found in rabbits injected with RDF hIL-10 or RDF neo. Histology showed that all eyes injected with RDF hIL-10 had significant inflammatory infiltration whereas only one control eye was clearly inflamed. Rabbits injected with soluble rhIL-10 had normal fundoscopy and normal histology. In conclusion, our results show that in vivo, in a cell-induced model of PVR, hIL-10 has no effect in the clinical progression of PVR. Histology, however, shows that pro-inflammatory effects seem to overcome its suppressive properties.

Survival benefit of adding Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) at the different time-points of treatment of ovarian cancer: review of evidence.

The standard treatment for advanced ovarian cancer consists in complete cytoreductive surgery (CRS) and intravenous combination chemotherapy with a platinum compound and a taxane. Although response rates to initial therapy are high, many patients will recur and die of peritoneal carcinomatosis. The addition of Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) to the standard therapy aims at increasing survival by reducing peritoneal recurrence. This review describes the survival results of HIPEC at the different time-points of the treatment of ovarian cancer: at upfront CRS, at interval CRS, at consolidation CRS after complete response to initial therapy, at secondary CRS after incomplete response, at salvage CRS for recurrence and as palliative treatment without CRS for unresectable ovarian cancer with chemotherapy resistant ascites. The available evidence suggests that a potential survival benefit of adding HIPEC may be largest in the settings of secondary CRS for stage III ovarian cancer and salvage CRS for recurrent ovarian cancer, two time-points representing failure of initial standard therapy. There is much less evidence for a potential benefit of HIPEC for less advanced stages (I-II) and for earlier time-points in the treatment of ovarian cancer (upfront, interval and consolidation). Postoperative mortality is not higher after CRS and HIPEC (0.7%) than after CRS only (1.4%). Four randomised trials are ongoing and their results are eagerly awaited. Palliative HIPEC without CRS might be used more in patients with incapacitating ascites due to recurrent ovarian cancer which has become resistant to systemic chemotherapy.

Synergy between dendritic cells and GM-CSF-secreting tumor cells for the treatment of a murine renal cell carcinoma.

Dendritic cell (DC) immunotherapy for cancer certainly holds promises but definitely needs improvements, especially for enhancing tumor-specific responses able to eradicate preexisting tumors. To this end, we investigated here, for the treatment of a preimplanted murine renal cell carcinoma Renca, a new vaccination approach combining injection of DC and granulocyte macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cells. When treatment by either DC or Renca-mGM-CSF cells alone had no therapeutic effect at all, combined vaccines induced therapeutic response in 50% of the tumor-bearing mice, in a GM-CSF dose-dependent manner. Importantly, all these cured mice were protected against a rechallenge with parental Renca cells, indicating the generation of memory immune response. The combined vaccines induced elevated cytotoxic responses in all the cured mice and half of the uncured ones and a stronger systemic CD4+ T-cell-mediated interferon-gamma production in the cured vaccinated mice as compared with uncured ones. In conclusion, vaccines associating DC and GM-CSF-secreting tumor cells induce high therapeutic effect in mice with preexisting renal cell carcinoma that are correlated to the induction of specific CD8 and CD4+ T-cell responses. This original vaccination approach should be further evaluated in a clinical trial for the treatment of metastatic human renal cell carcinoma.

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses, high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration.

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy-combining dendritic cells and irradiated GM-CSF secreting tumor cells-to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-gamma secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.

A phase II study of Tg4010 (Mva-Muc1-Il2) in association with chemotherapy in patients with stage III/IV Non-small cell lung cancer.

BACKGROUND: TG4010 is a recombinant viral vector expressing both the tumor-associated antigen MUC1 and Interleukine-2. This vector is based on the modified virus of Ankara, a significantly attenuated strain of vaccinia virus. TG4010 has been designed to induce or amplify a cellular immune response directed against tumor cells expressing MUC1. METHODS: A multicenter, randomized phase II study has explored two schedules of the combination of TG4010 with first line chemotherapy in patients with stage IIIB/IV non-small cell lung cancer. In Arm 1, TG4010 was combined upfront with cisplatin (100 mg/m day 1) and vinorelbine (25 mg/m day 1 and day 8). In Arm 2, patients were treated with TG4010 monotherapy until disease progression, followed by TG4010 plus the same chemotherapy as in Arm1. Response rate was evaluated according to RECIST. Median time to progression and median overall survival were calculated according to the Kaplan-Meier method. RESULTS: Sixty-five patients were enrolled, 44 in Arm 1 and 21 in Arm 2, in accordance with the two stage Simon design of the statistical plan. In Arm 1, partial response was observed in 13 patients out of 37 evaluable patients (29.5% of the intent to treat population, 35.1% of the evaluable patients). In Arm 2, two patients experienced stable disease for more than 6 months with TG4010 alone (up to 211 days), in the subsequent combination with chemotherapy, one complete and one partial response were observed out of 14 evaluable patients. Arm 2 did not meet the criteria for moving forward to second stage. The median time to progression was 4.8 months for Arm 1. The median overall survival was 12.7 months for Arm 1 and 14.9 for Arm 2. One year survival rate was 53% for Arm 1 and 60% for Arm 2. TG4010 was well tolerated, mild to moderate injection site reactions, flu-like symptoms, and fatigue being the most frequent adverse reactions. A MUC1-specific cellular immune response was observed in lymphocyte samples from all responding patients evaluable for immunology. CONCLUSIONS: The combination of TG4010 with standard chemotherapy in advanced non-small cell lung cancer is feasible and shows encouraging results. A randomized study evaluating the addition of TG4010 to first line chemotherapy in this population is in progress.