Analysis of a rare melanoma patient with a spontaneous CTL response to a MAGE-A3 peptide presented by HLA-A1

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5×10⁻⁷ of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.     

Intravenous and intradermal TriMix-dendritic cell therapy results in a broad T-cell response and durable tumor response in a chemorefractory stage IV-M1c melanoma patient

Dendritic cells (DCs) electroporated with mRNA encoding CD70, CD40L and a constitutively active toll-like receptor 4 (TriMix-DC) have an increased T-cell stimulatory capacity. In a prospective phase IB clinical trial, we treated melanoma patients with intradermal and intravenous injections of autologous TriMix-DC co-electroporated with mRNA encoding full-length MAGE-A3, MAGE-C2, tyrosinase and gp100. We report here the immunological and clinical results obtained in one patient with a particularly favorable outcome. This patient had stage IV-M1c melanoma with documented progression during dacarbazine chemotherapy and received 5 TriMix-DC injections. Following DC therapy, a broad CD8(+) T-cell response against multiple epitopes derived from all four treatment antigens was found in the blood and among T cells derived from DTH biopsy. In addition, CD4(+) T cells recognizing different MAGE-A3-derived epitopes were detected in DTH-derived cells. A spontaneous anti-MAGE-C2 CD8(+) T-cell response was present prior to TriMix-DC therapy and increased during treatment. The tumor response was assessed with 18-fluorodeoxyglucose-positron emission/computed tomography. We documented a partial tumor response according to RECIST criteria with a marked reduction in (18)F-FDG-uptake by lung, lymph node and bone metastases. The patient remains free from progression after 12 months of follow-up. This case report indicates that administration of autologous TriMix-DC by the combined intradermal and intravenous route can mediate a durable objective tumor response accompanied by a broad T-cell response in a chemorefractory stage IV-M1c melanoma patient.     

A case report: Immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy

 The first use of granulocyte/macrophage-colony-stimulating-factor-transduced, lethally irradiated, autologous melanoma cells as a therapeutic vaccine in a patient with rapidly progressive, widely disseminated malignant melanoma resulted in the generation of a novel antitumour immune response associated with partial, albeit temporary, clinical benefit. An initially negative reaction to non-transduced, autologous melanoma cells was converted to a delayed-type hypersensitivity (DTH) reaction of increasing magnitude following successive vaccinations. While intradermal vaccine sites showed prominent dendritic cell accrual, DTH sites revealed a striking influx of eosinophils in addition to activated/memory T lymphocytes and macrophages, recalling the histology of challenge tumour cell rejection in immune mice. Cytotoxic T lymphocytes (CTL) reactive with autologous melanoma cells were detectable at high frequency after vaccination, not only in limiting-dilution analysis, but also in bulk culture without added cytokines. Clonal analysis of CTL showed a conversion from a purely CD8⁺ response to a high proportion of CD4⁺ clones following vaccination. A prominent acute-phase response manifested by a five- to tenfold increase in C-reactive protein was observed, as was a systemic eosinophilia. Vaccination resulted in the regression of axillary lymphatic metastases, stabilisation of pulmonary metastases, and a dramatic, reversible increase in cerebral oedema associated with multiple central nervous system metastases; however, lesions in the adrenal glands, pancreas and spleen proved refractory. The antitumour effects and immune response were not detectable 2 months following the last vaccination. Irradiation of the extensive cerebral metastases resulted in rapid deterioration and death of the patient. Received: 20 September 1996 / Accepted: 5 December 1996     

Polyclonal CTL responses observed in melanoma patients vaccinated with dendritic cells pulsed with a MAGE-3.A1 peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3(168-176) peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 x 10(-6) to 9 x 10(-4) of CD8(+) blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.     

Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.     

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1.

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.     

Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200

Background and Objective Immune escape by tumors can occur by multiple mechanisms, each a significant barrier to immunotherapy. We previously demonstrated that upregulation of the immunosuppressive molecule CD200 on chronic lymphocytic leukemia cells inhibits Th1 cytokine production required for an effective cytotoxic T cell response. CD200 expression on human tumor cells in animal models prevents human lymphocytes from rejecting the tumor; treatment with an antagonistic anti-CD200 antibody restored lymphocyte-mediated tumor growth inhibition. The current study evaluated CD200 expression on solid cancers, and its effect on immune response in vitro. Methods and Results CD200 protein was expressed on the surface of 5/8 ovarian cancer, 2/4 melanoma, 2/2 neuroblastoma and 2/3 renal carcinoma cell lines tested, but CD200 was absent on prostate, lung, breast, astrocytoma, or glioblastoma cell lines. Evaluation of patient samples by immunohistochemistry showed strong, membrane-associated CD200 staining on malignant cells of melanoma (4/4), ovarian cancer (3/3) and clear cell renal cell carcinoma (ccRCC) (2/3), but also on normal ovary and kidney. CD200 expression on melanoma metastases was determined by RT-QPCR, and was found to be significantly higher in jejunum metastases (2/2) and lung metastases (2/6) than in normal samples. Addition of CD200-expressing, but not CD200-negative solid tumor cell lines to mixed lymphocyte reactions downregulated the production of Th1 cytokines. Inclusion of antagonistic anti-CD200 antibody restored Th1 cytokine responses. Conclusion These data suggest that melanoma, ccRCC and ovarian tumor cells can express CD200, thereby potentially suppressing anti-tumor immune responses. CD200 blockade with an antagonistic antibody may permit an effective anti-tumor immune response in these solid tumor types.     

Phase 1 clinical trial of irradiated autologous melanoma cells adenovirally transduced with human GM-CSF gene

The objective of this study was to determine the safety and antitumor activity of an autologous GM-CSF-secreting melanoma cell vaccine that was engineered ex vivo with recombinant replication-incompetent adenovirus harboring a human GM-CSF gene (Adv/hGM-CSF). Melanoma samples were surgically obtained from 30 patients (15 female and 15 male, ages ranging from 23 to 87) and were processed for vaccine preparation. Due to stringent eligibility criteria, 9 out of 30 patients were enrolled in the phase 1 clinical trial (FDA IND7677). Melanoma cell lines established from surgical specimens of 9 patients were transduced with Adv/hGM-CSF (MOI of 100) and subsequently irradiated at 35 Gy. These cell lines secreted human GM-CSF in vitro at an average rate of 80-424 ng/10(6) cells/24 h. All patients were intradermally and subcutaneously injected at several sites with irradiated autologous melanoma cells (2x10(6)-1x10(7) in 300 microl saline), 2-10 times, at intervals of 4-8 weeks. None of the patients vaccinated showed any serious adverse systemic response. Three patients (nos.1, 6 and 7) demonstrated local reaction (erythema) to the vaccination. Tumor-specific CTL assays performed in the absence of K562 cells showed that the levels of CTLs in peripheral blood of 5 patients increased following vaccination, whereas those in one patient declined. Levels of CTLs assayed in the presence of K562 cells were considerably lower than those assayed in the absence of K562 cells, but were also found to increase following vaccination in the peripheral blood of 6 patients. A patient who had been vaccinated 10 times (patient 1) responded to the vaccination by apparent reduction in size of metastatic tumor in the lung. Immunohistochemical examination of the vaccination sites of patient 1, biopsied after the 3rd and 4th vaccination. showed that the vaccination sites responded with infiltration of inflammatory cells, such as T cells (CD3+, CD8+), macrophages and dendritic cells (CD83+), for a period up to about 8 days. These data suggest that repeated vaccinations with irradiated autologous GM-CSF-producing tumor cells were well tolerated by patients and led to the activation of an antitumor immune response in some patients.     

Cytolytic T lymphocytes recognize an antigen encoded by MAGE-A10 on a human melanoma.

From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254-262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.     

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.     

MAGE-A1-, MAGE-A10-, and gp100-derived peptides are immunogenic when combined with granulocyte-macrophage colony-stimulating factor and montanide ISA-51 adjuvant and administered as part of a multipeptide vaccine for melanoma

Twelve peptides derived from melanocyte differentiation proteins and cancer-testis Ags were combined and administered in a single mixture to patients with resected stage IIB, III, or IV melanoma. Five of the 12 peptides included in this mixture had not previously been evaluated for their immunogenicity in vivo following vaccination. We report in this study that at least three of these five peptides (MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622)) are immunogenic when administered with GM-CSF in Montanide ISA-51 adjuvant. T cells secreting IFN-gamma in response to peptide-pulsed target cells were detected in peripheral blood and in the sentinel immunized node, the node draining a vaccine site, after three weekly injections. The magnitude of response typically reached a maximum after two vaccines, and though sometimes diminished thereafter, those responses typically were still detectable 6 wks after the last vaccines. Most importantly, tumor cell lines expressing the appropriate HLA-A restriction element and MAGE-A1, MAGE-A10, or gp100 proteins were lysed by corresponding CTL. This report supports the continued use of the MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622) epitopes in peptide-based melanoma vaccines and thus expands the list of immunogenic peptide Ags available for human use. Cancer-testis Ags are expressed in multiple types of cancer; thus the MAGE-A1(96-104) and MAGE-A10(254-262) peptides may be considered for inclusion in vaccines against cancers of other histologic types, in addition to melanoma.     

Monoclonal anti-MAGE-3 CTL responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus

We have analyzed the T cell responses of HLA-A1 metastatic melanoma patients with detectable disease, following vaccination with a recombinant ALVAC virus, which bears short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA-A1. To evaluate the anti-MAGE CTL responses, we resorted to antigenic stimulation of blood lymphocytes under limiting dilution conditions, followed by tetramer analysis and cloning of the tetramer-positive cells. The clones were tested for their specific lytic ability and their TCR sequences were obtained. Four patients who showed tumor regression were analyzed, and an anti-MAGE-3.A1 CTL response was observed in three of these patients. Postvaccination frequencies of anti-MAGE-3.A1 CTL were 3 x 10(-6), 3 x 10(-3), and 3 x 10(-7) of the blood CD8 T cells, respectively. These three responses were monoclonal. No anti-MAGE-1.A1 CTL response was observed. These results indicate that, like peptide immunization, ALVAC immunization produces monoclonal responses. They also suggest that low-level antivaccine CTL responses can initiate a tumor regression process. Taken together, our analysis of anti-MAGE-3.A1 T cell responses following peptide or ALVAC vaccination shows a degree of correlation between CTL response and tumor regression, but firm conclusions will require larger numbers.     

Induction of immune response against NY-ESO-1 by CHP-NY-ESO-1 vaccination and immune regulation in a melanoma patient

BACKGROUND: NY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro. AIM: In this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites. RESULTS: Ten days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-gamma secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4(+)CD25(+)Foxp3(+) Tregs was observed in PBMC but significantly increased numbers of CD4(+)CD25(+)Foxp3(+) Tregs and CD68(+) immunoregulatory macrophages were detected at the local tumor sites. CD4(+)CD25(+)Foxp3(+) Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site. CONCLUSIONS: Our observations indicate induction of specific humoral and cellular immune responses against NY-ESO-1 after CHP-NY-ESO-1 vaccination in a melanoma patient. The concomitant appearance of regulatory T cells and of immune regulatory macrophages and cytokines at the local tumor sites in this patient may explain immune escape.     

Monitoring of anti-vaccine CD4 T cell frequencies in melanoma patients vaccinated with a MAGE-3 protein

Quantitative evaluation of T cell responses of patients receiving antitumoral vaccination with a protein is difficult because of the large number of possible HLA-peptide combinations that could be targeted by the response. To evaluate the responses of patients vaccinated with protein MAGE-3, we have developed an approach that involves overnight stimulation of blood T cells with autologous dendritic cells loaded with the protein, sorting by flow cytometry of the T cells that produce IFN-gamma, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN-gamma upon stimulation with the Ag. An important criterion is that T cell clones must recognize not only stimulator cells loaded with the protein, but also stimulator cells transduced with the MAGE-3 gene, so as to exclude the T cells that recognize contaminants generated by the protein production system. Using this approach it is possible to measure T cell frequencies as low as 10(-6). We analyzed the frequencies of anti-vaccine CD4 T cells in five metastatic melanoma patients who had been injected with a MAGE-3 protein without adjuvant and showed evidence of tumor regression. Anti-MAGE-3 CD4 T cells were detected in one of the five patients. The frequency of the anti-MAGE-3 CD4 T cells was estimated at 1/60,000 of the CD4 T cells in postvaccination blood samples, representing at least an 80-fold increase in the frequency found before immunization. The frequencies of one anti-MAGE-3 CD4 T cell clonotype were confirmed by PCR analysis on blood lymphocytes. The 13 anti-MAGE-3 clones, which corresponded to five different TCR clonotypes, recognized the same peptide presented by HLA-DR1.     

Divergent roles for CD4+ T cells in the priming and effector/memory phases of adoptive immunotherapy

The requirement for CD4(+) Th cells in the cross-priming of antitumor CTL is well accepted in tumor immunology. Here we report that the requirement for T cell help can be replaced by local production of GM-CSF at the vaccine site. Experiments using mice in which CD4(+) T cells were eliminated, either by Ab depletion or by gene knockout of the MHC class II beta-chain (MHC II KO), revealed that priming of therapeutic CD8(+) effector T cells following vaccination with a GM-CSF-transduced B16BL6-D5 tumor cell line occurred independently of CD4(+) T cell help. The adoptive transfer of CD8(+) effector T cells, but not CD4(+) effector T cells, led to complete regression of pulmonary metastases. Regression of pulmonary metastases did not require either host T cells or NK cells. Transfer of CD8(+) effector T cells alone could cure wild-type animals of systemic tumor; the majority of tumor-bearing mice survived long term after treatment (>100 days). In contrast, adoptive transfer of CD8(+) T cells to tumor-bearing MHC II KO mice improved survival, but eventually all MHC II KO mice succumbed to metastatic disease. WT mice cured by adoptive transfer of CD8(+) T cells were resistant to tumor challenge. Resistance was mediated by CD8(+) T cells in mice at 50 days, while both CD4(+) and CD8(+) T cells were important for protection in mice challenged 150 days following adoptive transfer. Thus, in this tumor model CD4(+) Th cells are not required for the priming phase of CD8(+) effector T cells; however, they are critical for both the complete elimination of tumor and the maintenance of a long term protective antitumor memory response in vivo.     

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.     

Lack of tumor recognition by cytolytic T lymphocyte clones recognizing peptide 195–203 encoded by gene <Emphasis Type="Italic">MAGE-A3</Emphasis> and presented by HLA-A24 molecules

Gene MAGE-A3 encodes tumor-specific antigenic peptides recognized by T cells on many tumors. MAGE-A3 peptides presented by HLA class I molecules have been identified using CD8 lymphocytes stimulated with cells that either expressed gene MAGE-A3 or were pulsed with candidate peptides. One antigen identified with the latter method is peptide MAGE-A3(195-203) IMPKAGLLI, presented by HLA-A24 molecules. It has been used to vaccinate advanced cancer patients. Here, we have used HLA/peptide tetramers to detect T cells recognizing this peptide. Their frequency was estimated to be 2 x 10(-8) of the blood CD8 cells in non-cancerous HLA-A24(+) individuals, which is tenfold lower than the reported frequencies of T cells against other MAGE peptides. In the blood of a patient vaccinated with MAGE-A3, the estimated frequency was 5 x 10(-7). Anti-MAGE-3.A24 cytolytic T cell clones were derived, that lysed peptide-pulsed cells with half-maximal effect at the low concentration of 500 pM. However, these CTL did not recognize a panel of HLA-A24(+) tumor cells that expressed MAGE-A3 at levels similar to those found in HLA-A1(+) tumor cells recognized by anti-MAGE-3.A1 CTLs. Furthermore, 293-EBNA cells transfected with MAGE-A3 and HLA-A24 constructs were hardly recognized by the anti-MAGE-3.A24 CTL clones. These results suggest that peptide MAGE-A3(195-203) is poorly processed and is not an appropriate target for cancer immunotherapy.     

Induction of anti-tumor immunity by vaccination with dendritic cells pulsed with anti-CD44 IgG opsonized tumor cells

Due to the pivotal role that dendritic cells (DC) play in eliciting and maintaining functional anti-tumor T cell responses, these APC have been exploited against tumors. DC express several receptors for the Fc portion of IgG (Fcγ receptors) that mediate the internalization of antigen-IgG complexes and promote efficient MHC class I and II restricted antigen presentation. In this study, the efficacy of vaccination with DC pulsed with apoptotic B16 melanoma cells opsonized with an anti-CD44 IgG (B16-CD44) was explored. Immature bone marrow derived DC grown in vitro with IL-4 and GM-CSF were pulsed with B16-CD44. After 48 h of pulsing, maturation of DC was demonstrated by production of IL-12 and upregulation of CD80 and CD40 expression. To test the efficacy of vaccination with DC+B16-CD44, mice were vaccinated subcutaneously Lymphocytes from mice vaccinated with DC+B16-CD44 produced IFN-γ in response to B16 melanoma lysates as well as an MHC class I restricted B16 melanoma-associated peptide, indicating B16 specific CD8 T cell activation. Upon challenge with viable B16 cells, all mice vaccinated with DC alone developed tumor compared to 40% of mice vaccinated with DC+B16-CD44; 60% of the latter mice remained tumor free for at least 8 months. In addition, established lung tumors and distant metastases were significantly reduced in mice treated with DC+B16-CD44. Lastly, delayed growth of established subcutaneous tumors was induced by combination therapy with anti-CD44 antibodies followed by DC injection. This study demonstrates the efficacy of targeting tumor antigens to DC via Fcγ receptors.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.