Analysis of T-cell responses in metastatic melanoma patients vaccinated with dendritic cells pulsed with tumor lysates

In melanoma patients, CD8⁺ cytotoxic T cells have been found recognizing self-proteins of which the expression is restricted to the melanocytic lineage. These melanocyte differentiation antigens are expressed in normal melanocytes as well as in 80–100% of primary and metastatic melanoma. In this report, six HLA-A*0201–subtyped metastatic melanoma patients vaccinated with dendritic cells (DCs) pulsed with autologous tumor lysates and keyhole limpet hemocyanin (KLH) were screened for the presence of CD8⁺ T cells specific for three HLA-A*0201–binding peptides derived from the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase. For this purpose, nonstimulated as well as in vitro peptide-stimulated peripheral blood mononuclear cells (PBMCs) were tested for peptide-specific IFN- release by enzyme-linked immunosorbent spot (ELISpot) assays. Furthermore, expression of the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase in tumor lesions was analyzed by immunohistochemistry before and after vaccination. We also used the ELISpot technique to investigate whether KLH-specific T cells were induced and whether these cells released type 1 (IFN-) and/or type 2 (IL-13) cytokines. Our data show induction of CD8⁺ T cells specific for the melanosomal peptides MART-1/Melan-A_27–35 or tyrosinase_1–9, as well as IFN-–releasing KLH-specific T cells, in two of six vaccinated melanoma patients, but do not support an association between the induction of these T cells and clinical responses.     

Phase I/II study of treatment with matured dendritic cells with or without low dose IL-2 in patients with disseminated melanoma

Background In the present study, we have examined whether treatment of patients with metastatic melanoma with matured dendritic cell (DC) vaccines with or without low dose IL-2 may improve treatment outcomes. Methods Sixteen patients received DC vaccines (DCs) sensitized with autologous melanoma lysates and 18 patients received DCs sensitized with peptides from gp100, MART-1, tyrosinase, MAGE-3.A2, MAGE-A10 and NA17. IL-2 was given subcutaneously (sc) at 1 MU/m² on the second day after each injection for 5–14 days in half of each group. DCs were given by intranodal injection. Results There were 2 partial responses (PR) and 3 with stable disease (SD) in the nine patients receiving DCs + peptides + IL-2, and 1 PR and 1 SD in nine patients treated with DCs + peptides without IL-2. There were only two patients with SD in the group receiving DCs + autologous lysates and no IL-2. Median overall survival for all patients was very good at 18.5 months but this was most probably due to selection of a favourable group of patients for the study. There was no significant difference in survival between the groups by log rank analysis. Treatment was not associated with significant side effects. The quality and yield of the DCs in the preparations were generally good. Conclusions We conclude that mature DC preparations may be superior to immature DC preparations for presentation of melanoma peptides and that IL-2 may increase clinical responses to the DCs plus peptides. However, in our view the low response rates do not justify the cost and complexity of this treatment approach.     

Phase I/II study of immunotherapy with T-cell peptide epitopes in patients with stage IV melanoma

Previous studies in small groups of patients suggested that immunization of melanoma patients with peptide epitopes recognized by T cells could induce regression of melanoma. This approach was tested in 36 patients with stage IV melanoma. The (MHC class I–restricted) peptides were from gp100, MART-1, tyrosinase, and MAGE-3. The gp100 and MART-1 peptides had been modified to increase their immunogenicity. In half the patients (groups 3 and 4) the peptides were given in the adjuvant Montanide-ISA-720, and half the patients in both groups were given GM-CSF s.c. for 4 days following each injection. Treatment was well tolerated except for two severe erythematous responses to Montanide-ISA-720 and marked inflammatory responses at sites of GM-CSF administration in three patients. There were no objective clinical responses but stabilization of disease for periods from 3 to 12 months were seen in seven patients. Five of these were patients given the peptides in Montanide-ISA-720. Delayed-type hypersensitivity (DTH) skin test responses were also seen mainly in the patients given the peptides in Montanide-ISA-720. GM-CSF did not increase DTH responses in patients in the latter group but may have increased DTH responses in those not given peptides in Montanide-ISA-720. Inflammatory responses around s.c. metastases or regional lymph nodes were observed in two patients. These results suggest that the peptides are more effective when given in the adjuvant Montanide-ISA-720. Nevertheless, results from this study, together with those from a number of comparable studies, indicate that peptide vaccines are currently of minimal benefit to patients and support the need for ongoing development of new strategies in treatment of this disease.     

Long-term outcomes in patients with metastatic melanoma vaccinated with melanoma peptide-pulsed CD34+ progenitor-derived dendritic cells

Between March 1999 and May 2000, 18 HLA-A*0201⁺ patients with metastatic melanoma were enrolled in a phase I trial using a dendritic cell (DC) vaccine generated by culturing CD34⁺ hematopoietic progenitors. This vaccine includes Langerhans cells. The DC vaccine was loaded with four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100), Influenza matrix peptide (Flu-MP), and keyhole limpet hemocyanin (KLH). Ten patients received eight vaccinations, one patient received six vaccinations, one patient received five vaccinations, and six patients received four vaccinations. Peptide-specific immunity was measured by IFN-γ production and tetramer staining in blood mononuclear cells. The estimated median overall survival was 20 months (range: 2–83), and the median event-free survival was 7 months (range: 2–83). As of August 2005, four patients are alive (three patients had M1a disease and one patient had M1c disease). Three of them have had no additional therapy since trial completion; two of them had solitary lymph node metastasis, and one patient had liver metastasis. Patients who survived longer were those who mounted melanoma peptide-specific immunity to at least two melanoma peptides. The present results therefore justify the design of larger follow-up studies to assess the immunological and clinical outcomes in patients with metastatic melanoma vaccinated with peptide-pulsed CD34-derived DCs.Joseph W. Fay and A. Karolina Palucka have equally contributed to this work     

Dendritic cells derived from metastatic cancer patients vaccinated with allogeneic dendritic cell–autologous tumor cell hybrids express more CD86 and induce higher levels of interferon-gamma in mixed lymphocyte reactions

Dendritic cells (DCs) are highly effective antigen-presenting cells that, when derived from cancer patients, seem to be functionally deficient. Herein, we show that vaccination with allogeneic DC–autologous tumor cell hybrids affects the phenotype and improves the function of monocyte-derived DCs (Mo-DCs) from cancer patients. Mononuclear cells were isolated from patients peripheral blood by density gradient centrifugation, and adherent cells were cultured in medium containing GM-CSF plus IL-4 and, after 5 days, TNF-. After 2 more days, Mo-DCs were harvested and their CD80, CD86, and CD83 expression was assessed by flow cytometry. They were also used as stimulators in mixed lymphocyte reactions (MLR), where IFN- production was measured by ELISA. Mo-DCs from unvaccinated patients expressed significantly lower levels of CD86, and tended to express lower levels of CD83 than Mo-DCs from healthy donors. However, Mo-DCs generated after hybrid cell vaccination presented increased expression of the same markers and induced significantly higher levels of IFN- in MLR. These results indicate that the use of allogeneic DC–based cancer vaccines induces recovery of DC function in metastatic cancer patients and, therefore, could precede the use of autologous DCs for vaccine preparation. Such an approach could be relevant and should be investigated in clinical trials.     

Optimizing dendritic cell-based anticancer immunotherapy: maturation state does have clinical impact

Tumour immunotherapy using dendritic cells (DCs) is a new therapeutic approach, which has been applied to a variety of different cancers over the last 5 years. Here we discuss the clinical results of these trials in relation to the different protocols used to generate DCs, and in particular the effect that DC maturation state has had on clinical responses. In ten different melanoma trials a total of 167 patients have been treated, resulting in 9 complete tumour regressions, 24 partial regressions, 26 patients with stable disease, and 108 with progressive disease. Favourable response, defined as any outcome other than progressive disease, was not associated with previous chemotherapy, but was significantly correlated (p<0.001) with the addition of TNF- for the maturation of DCs in vitro. Hence DC maturation state has had an impact on clinical responses to therapy. However, TNF- is not the only molecule capable of inducing DC maturation, and strategies for improving clinical responses by optimizing DC maturation are discussed.     

Allogeneic dendritic cell vaccination against metastatic renal cell carcinoma with or without cyclophosphamide

In this phase I/II study, we evaluated the feasibility, safety and efficacy of allogeneic dendritic cells (DCs) with or without cyclophosphamide in the treatment of patients with metastatic renal cell carcinoma (RCC). Immunomagnetic beads were used to isolate CD14⁺ monocytes from healthy donor leukapheresis products, and CD83⁺ antigen-pulsed monocyte-derived DCs (moDCs) loaded with tumor lysate and keyhole limpet hemocyanin (KLH) were generated. Twelve patients were treated with allogeneic moDCs alone, while ten patients also received cyclophosphamide on days 4 and 3 prior to vaccination. Of the 22 patients enrolled, 20 received full treatment consisting of at least three vaccinations at monthly intervals. Two mixed responses with substantial tumor regression were observed. In 3 patients, disease stabilization occurred, in 13 patients disease progressed and 4 patients were lost to follow-up. Overall, immune responses against KLH and tumor lysate were weak or absent; however, the strongest increases in antigen-independent and KLH-specific responses were observed in the 2 patients with mixed responses. In addition, 1 of them showed a substantial increase in oncofetal antigen (OFA)-specific IFN- production. Importantly, the 2 mixed responders and 1 patient with stable disease belonged to the cyclophosphamide group. Median overall survival in the cyclophosphamide group was 23.2 and 20.3 months in the group that received allogeneic moDCs alone. Allogeneic immunotherapy with moDCs is feasible and well tolerated. However, the immunogenicity of allogeneic moDCs is clearly less pronounced than that of autologous moDC immunotherapy. Cyclophosphamide may have the capacity to augment DC-induced antitumor immunity.     

Pilot study of treatment of biochemotherapy-refractory stage IV melanoma patients with autologous dendritic cells pulsed with a heterologous melanoma cell line lysate

Eleven AJCC stage IV melanoma patients with progressive disease after treatment with biochemotherapy were treated with autologous dendritic cells pulsed with heterologous tumor cell lysates. The vaccine used mature DCs (CD1a⁺⁺⁺, CD40⁺⁺, CD80⁺⁺, CD83⁺, and CD86⁺⁺⁺) generated from peripheral blood monocytes in the presence of GM-CSF and IL-4. After 7 days, DCs were matured with a defined cocktail of cytokines (IL-1+IL-6+TNF-+PGE₂) and simultaneously pulsed with lysates of heterologous melanoma cell lines, for 2 days. A total of 4×10⁶ DCs was injected monthly under ultrasound control in an inguinal lymph node of normal appearance. The study was closed when all patients died as a consequence of tumor progression. No sign of toxicity was observed during the study. One patient experienced a partial response lasting 5 months, and two patients showed a mixed response which lasted 3 months. The median survival of the whole group was 7.3 months (range 3–14 months). This vaccination program had specific antitumoral activity in highly pretreated and large tumor burden stage IV melanoma patients and was well tolerated. The clinical responses and the median survival of the group of patients, together with the low toxicity of our DC vaccine, suggest that this approach could be applied to earlier AJCC stage IV melanoma patients.     

Induction of tumor-specific T-cell responses by vaccination with tumor lysate-loaded dendritic cells in colorectal cancer patients with carcinoembryonic-antigen positive tumors

Background Dendritic cells (DCs) are the most effective antigen-presenting cells. In the last decade, the use of DCs for immunotherapy of cancer patients has been vastly increased. High endocytic capacity together with a unique capability of initiating primary T-cell responses have made DCs the most potent candidates for this purpose. Although DC vaccination occasionally leads to tumor regression, clinical efficacy, and immunogenicity of DCs in clinical trials has not been yet clarified. The present study evaluated the safety and effectiveness of tumor-lysate loaded DC vaccines in advanced colorectal cancer (CRC) patients with carcinoembryonic antigen (CEA) positive tumors. Results Six patients HLA-A*0201-positive were vaccinated with autologous DCs loaded with tumor lysates (TL) together with tetanus toxoid antigen, hepatitis B, and influenza matrix peptides. Two additional patients were injected with DCs that were generated from their sibling or parent with one haplotype mismatch. All patients received the vaccines every 2 weeks, with a total of three intra-nodal injections per patient. The results indicated that DC vaccination was safe and well tolerated by the patients. Specific immune responses were detected and in some patients, transient stabilization or even reduction of CEA levels were observed. The injection of haplotype mismatched HLA-A*0201-positive DCs resulted in some enhancement of the anti-tumor response in vitro and led to stabilization/reduction of CEA levels in the serum, compared to the use of autologous DCs. Conclusion Altogether, these results suggest that TL-pulsed DCs may be an effective vaccine method in CRC patients. Elimination of regulatory mechanisms as well as adjustment of the vaccination protocol may improve the efficacy of DC vaccination. An erratum to this article can be found at     

Autologous dendritic cell vaccine for estrogen receptor (ER)/progestin receptor (PR) double-negative breast cancer

Purpose

A wealth of preclinical information, as well as a modest amount of clinical information, indicates that dendritic cell vaccines have therapeutic potential. The aim of this work was to assess the immune response, disease progression, and post-treatment survival of ER/PR double-negative stage II/IIIA breast cancer patients vaccinated with autologous dendritic cells pulsed with autologous tumor lysates.

Methods

Dendritic cell (DC) vaccines were generated from CD14+ precursors pulsed with autologous tumor lysates. DCs were matured with defined factors that induced surface marker and cytokine production. Individuals were immunized intradermally four times. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and lymphocyte subsets were determined for the evaluation of the therapeutic efficiency. Overall survival and disease progression rates were analyzed using Kaplan–Meier curves and compared with those of contemporaneous patients who were not administered DC vaccines.

Results

There were no unanticipated or serious adverse effects. DC vaccines elicited Th1 cytokine secretion and increased NK cells, CD8+ IFN-γ+ cells but decreased the percentage of CD3+ T cells and CD3+ HLA-DR+ T cells in the peripheral blood. Approximately 58% (18/31) of patients had a DTH-positive reaction. There was no difference in overall survival between the patients with and without DC vaccine. The 3-year progression-free survival was significantly prolonged: 76.9% versus 31.0% (with vs. without DC vaccine, p?Conclusion Our findings strongly suggest that tumor lysate-pulsed DCs provide a standardized and widely applicable source of breast cancer antigens that are very effective in evoking anti-breast cancer immune responses.     

Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell–based cancer vaccination

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated apoptotic tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I– and class II–restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-, and TNF- by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 K^b /SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN- and TNF- production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.Abbreviations DC Dendritic cell - FSC Forward scatter - SSC Side scatter - TC Tumor cells This work was supported by Grant 9853 from the Leukaemia Research Fund, UK; a JRC studentship from GKT; and the Lewis Family Research Trust     

Wild-type and modified gp100 peptide-pulsed dendritic cell vaccination of advanced melanoma patients can lead to long-term clinical responses independent of the peptide used

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients. Several strategies have been employed to load DC with antigen, including peptide loading. To increase immunogenicity of peptides, major histocompatibility complex (MHC) class I binding affinity and stability of peptide-MHC complexes at the cell surface may be improved by modification of the amino acid sequence. In this study, we compared the capacity of DC loaded with wild-type versus modified gp100 peptides with higher binding affinities to induce an immune and clinical response in advanced melanoma patients. Metastatic HLA-A2.1(+) melanoma patients were vaccinated intravenously (on average 25?×?10(6) DC) and intradermally (on average 11?×?10(6) DC) with mature DC loaded with keyhole limpet hemocyanin (KLH) together with tyrosinase peptide and either wild-type (15 patients) or modified (12 patients) gp100 peptides. All vaccinated patients showed a pronounced proliferative T cell or humoral response against KLH. Gp100-specific T cell responses were monitored in post-treatment delayed type hypersensitivity (DTH) skin biopsies by tetramer and functional analysis. Antigen-specific T cells were found in 2 of 15 patients vaccinated with wild-type gp100-loaded DC, versus 1 of 12 patients vaccinated with modified peptide-loaded DC. These three patients also had the best clinical response, with long-term (>8?years) complete responses in two patients, one in each group. We conclude that vaccination with peptide-loaded DC can result in long-term clinical responses in a minority of metastatic melanoma patients, and that the use of modified as compared to wild-type gp100 peptides for DC loading does not result in a relevant enhanced immune responses.     

Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution

Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.     

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses

Introduction

Immunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH⁺ with respect to DTH^? unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood.

Materials and methods

Healthy donors and melanoma patient’s lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release.

Results/discussion

Peripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4⁺ TGF-β⁺) regulatory T lymphocytes compared with healthy donors. Notably, DTH⁺ patients showed a threefold reduction of Th3 cells compared with DTH^? patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH⁺ with respect to DTH^? patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response.

Conclusions

Our results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.     

Immuno-gene therapy of cancer with tumour-mRNA transfected dendritic cells

We have developed immuno-gene therapy for malignant melanoma and prostate cancer. The therapy is based on monocyte-derived dendritic cells (DCs) that are transfected with autologous melanoma-mRNA or mRNA from three prostate cancer cell lines (DU-145, LN-CaP and PC-3). A broad spectrum of tumour-associated antigens will be included in both DC-vaccines. The use of autologous melanoma-mRNA moreover allows targeting of individual tumour antigens that are specific to each patient. Effective protocols have been established for mRNA-transfection by square wave electroporation and for the generation of clinical grade DCs. A full scale preclinical evaluation demonstrated in vitro T cell responses in 6/6 advanced melanoma patients. The responses were specific to antigens encoded by the transfected tumour-mRNA. Recently, we have conducted two phase I/II trials, in advanced malignant melanoma and androgen-resistant prostate cancer. Successful vaccine preparations were obtained for all 41 patients elected. No serious adverse effects were observed. Specific T cell responses (T cell proliferation and/or IFNγ ELISPOT) were demonstrated in 9/19 evaluable melanoma patients and in 12/19 prostate cancer patients. The response rates were higher for patients receiving intradermal vaccination, compared to intranodal injection. Thirteen prostate cancer patients developed a decrease in log-slope PSA. The PSA-response was significantly related to the T cell response (P=0.002). We conclude that the DC-vaccine is feasible and safe, and that T cell responses are elicited in about 50% of patients.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer”, held in Shenzhen, China, on 9–11 December 2005.     

Ionizing radiation affects human MART-1 melanoma antigen processing and presentation by dendritic cells

Radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes. In this study, we demonstrate the noncytotoxic effects of ionizing radiation on MHC class I Ag presentation by bone marrow-derived dendritic cells (DCs) that have divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-1(27-35) that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-1(27-35) also protected mice against growth of B16-A2/K(b) tumors in HLA-A2.1/K(b) transgenic mice. Taken together, these results suggest that radiation modulates MHC class I-mediated antitumor immunity by functionally affecting DC Ag presentation pathways.     

Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters

Purpose

Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.

Experimental design

Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.

Results

KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20?μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9?%, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.

Conclusions

We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.     

T cell responses in melanoma patients after vaccination with tumor-mRNA transfected dendritic cells

We have developed an individualized melanoma vaccine based on autologous dendritic cells (DCs) transfected with autologous tumor-mRNA. The vaccine targets the unique spectrum of tumor antigens in each patient and may recruit multiple T cell clones. In a recent phase I/II trial, we demonstrated T cell responses against vaccine antigens in 9/19 patients evaluable by T cell assays. Here, we report a follow-up study that was conducted to characterize interesting T cell responses and to investigate the effects of long-term booster vaccination. Two patients were selected for continued vaccine therapy. The clinical follow-up suggested a favorable clinical development in both patients. The immunological data (T cell proliferation/IFNgamma ELISPOT/Bioplex cytokine assays) indicated sustained T cell responses and suggested an enhancing effect of booster vaccinations. Both CD4(+) and CD8(+) T cell responses were demonstrated. From post-vaccination samples, we generated 39 T cell clones that responded specifically to stimulation by mRNA-transfected DCs and 12 clones that responded to mock-transfected DCs. These data clearly indicate a two-component vaccine response, against transfected and non-transfected antigens. T cell receptor (TCR) clonotype mapping, performed on 11 tDC-specific clones, demonstrated that 10/11 clones had different TCRs. The results thus indicate a broad spectrum T cell response against antigens encoded by the transfected tumor-mRNA. We generally observed mixed Th1/Th2 cytokine profiles, even in T cell clones that were confirmed to be derived from a single cell. This finding suggests that cytokine patterns after cancer vaccination may be more complex than indicated by the classic Th1/Th2 dichotomy.     

Immunogenicity of dendritic cells pulsed with MAGE3, Survivin and B-cell maturation antigen mRNA for vaccination of multiple myeloma patients

The introduction of autologous stem cell transplantation (SCT) and novel drugs has improved overall survival in multiple myeloma (MM) patients. However, minimal residual disease (MRD) remains and most patients eventually relapse. Myeloma plasma cells express tumor-associated antigens (TAA), which are interesting targets for immunotherapy. In this phase 1 study, we investigated the safety and immunological effects of TAA-mRNA-loaded dendritic cell (DC) vaccination for treatment for MRD in MM after SCT. Mature monocyte-derived DCs were pulsed with keyhole limpet hemocyanin (KLH) and electroporated with MAGE3, Survivin or B-cell maturation antigen (BCMA) mRNA. Twelve patients were vaccinated three times with intravenous (5–22 × 10⁶ DCs) and intradermal vaccines (4–11 × 10⁶ DCs), at biweekly intervals. Immunological responses were monitored in blood and delayed-type hypersensitivity (DTH) biopsies. All patients developed strong anti-KLH T-cell responses, but not KLH antibodies. In 2 patients, vaccine-specific T cells were detected in DTH biopsies. In one patient, we found MAGE3-specific CD4⁺ and CD8⁺ T cells, and CD3⁺ T cells reactive against BCMA and Survivin. In the other patient, we detected low numbers of MAGE3 and BCMA-reactive CD8⁺ T cells. Vaccination was well tolerated with limited toxicity. These findings illustrate that TAA-mRNA-electroporated mature DCs are capable of inducing TAA-T-cell responses in MM patients after SCT.