A melanoma helper peptide vaccine increases Th1 cytokine production by leukocytes in peripheral blood and immunized lymph nodes.

BACKGROUND: Cancers produce soluble and cell-associated molecules that can suppress or alter antitumor immunity. Preclinical studies suggest the disease burden may alter the cytokine profile of helper T cell responses to cancer antigens. We studied cytokine production by helper T cells responding to vaccination with 6 melanoma helper peptides (6MHP) in blood and lymph nodes. METHODS: Twenty-three patients with stage IIIB-IV melanoma received a 6MHP vaccine. Antigen-reactive T cells from blood and draining lymph nodes were cultured, exposed to antigen, and then supernatants (days 2 and 5) were assayed for Th1 and Th2 cytokines. Results from 4 time points were compared to pre-vaccine levels. RESULTS: Cytokine responses to vaccinating peptides were observed in 83% of patients. Th1 favoring responses were most common (17 of 19 responders). The most abundant cytokines produced were IFN-γ and IL-5 in the PBMC's. IL-2 responses predominated in cells obtained from draining lymph nodes in 2-day culture but not in 5-day cultures. Patients with clinically measurable disease produced similar levels of total cytokine and similar degree of Th1 polarization as patients with no evidence of disease (NED). CONCLUSIONS: The MHC class II-associated peptides used in this study induced helper T cells with a Th1-biased cytokine response in both PBMC and sentinel immunized nodes. Most patients can mount a Th1 dominant response to these peptides. Future studies are needed to test newer vaccine adjuvants in combination with these peptides. TRIAL REGISTRATION: CDR0000378171, Clinicaltrials: NCT00089219.

Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine.

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function.

BACKGROUND: Clinical trials of immunologic therapies provide opportunities to study the cellular and molecular effects of those therapies and may permit identification of biomarkers of response. When the trials are performed at multiple centers, transport and storage of clinical specimens become important variables that may affect lymphocyte viability and function in blood and tissue specimens. The effect of temperature during storage and shipment of peripheral blood on subsequent processing, recovery, and function of lymphocytes is understudied and represents the focus of this study. METHODS: Peripheral blood samples (n = 285) from patients enrolled in 2 clinical trials of a melanoma vaccine were shipped from clinical centers 250 or 1100 miles to a central laboratory at the sponsoring institution. The yield of peripheral blood mononuclear cells (PBMC) collected before and after cryostorage was correlated with temperatures encountered during shipment. Also, to simulate shipping of whole blood, heparinized blood from healthy donors was collected and stored at 15 °C, 22 °C, 30 °C, or 40 °C, for varied intervals before isolation of PBMC. Specimen integrity was assessed by measures of yield, recovery, viability, and function of isolated lymphocytes. Several packaging systems were also evaluated during simulated shipping for the ability to maintain the internal temperature in adverse temperatures over time. RESULTS: Blood specimen containers experienced temperatures during shipment ranging from -1 to 35 °C. Exposure to temperatures above room temperature (22 °C) resulted in greater yields of PBMC. Reduced cell recovery following cryo-preservation as well as decreased viability and immune function were observed in specimens exposed to 15 °C or 40 °C for greater than 8 hours when compared to storage at 22 °C. There was a trend toward improved preservation of blood specimen integrity stored at 30 °C prior to processing for all time points tested. Internal temperatures of blood shipping containers were maintained longer in an acceptable range when warm packs were included. CONCLUSIONS: Blood packages shipped overnight by commercial carrier may encounter extreme seasonal temperatures. Therefore, considerations in the design of shipping containers should include protecting against extreme ambient temperature deviations and maintaining specimen temperature above 22 °C or preferably near 30 °C.

The use of gamma-irradiation and ultraviolet-irradiation in the preparation of human melanoma cells for use in autologous whole-cell vaccines.

BACKGROUND: Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37) were gamma-irradiated (200 Gy), approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV)-irradiation as a possible adjunct to, or replacement for gamma-irradiation. METHODS: Melanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression. RESULTS: UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100. CONCLUSION: These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.

Evaluation of the sentinel immunized node for immune monitoring of cancer vaccines.

BACKGROUND: We hypothesized that lymph nodes draining sites of cutaneous vaccination could be identified by sentinel node biopsy techniques, and that measuring T-cell response with lymphocytes obtained from these lymph nodes would provide a more sensitive measure of immunogenicity than would the same measurement made with peripheral blood lymphocytes (PBL). METHODS: ELISpot analysis was used to determine the magnitude of vaccine-specific T-cell response in the sentinel immunized nodes (SIN), random lymph nodes, and peripheral blood lymphocytes (PBL) obtained from patients enrolled in clinical trials of experimental melanoma vaccines. RESULTS: The SIN biopsy was successful in 97% of cases and morbidity was very low. The T-cell response to vaccination was detected with greater sensitivity in the SIN (57%) than in PBL (39%), and evaluation of T-cell responses in the SIN and the PBL together yielded T-cell responses in 63% of patients. When the T-cell responses from a SIN and a random lymph node were compared in four patients, immune responses were detected to one of the vaccine peptides in three of these four patients. In all of those cases, responses were present in the SIN but absent from the random lymph node. CONCLUSION: Measurements of T-cell responsiveness to cutaneous immunization are more frequently positive in the SIN than they are in the PBL, however evaluation of both the SIN and PBL permit a more sensitive measure of T-cell immunogenicity than use of either single source.

Helper T-cell responses and clinical activity of a melanoma vaccine with multiple peptides from MAGE and melanocytic differentiation antigens.

PURPOSE: A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase. PATIENTS AND METHODS: Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors. RESULTS: Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years. CONCLUSION: Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.