Twenty-five patients with high-risk resected stages IIB, III, and IV melanoma were immunized with a vaccine consisting of the minimal epitope, immunodominant 9-amino acid peptide derived from the MART-1 tumor antigen (AAGIGILTV) complexed with incomplete Freund’s adjuvant. The last three patients received the MART-127–35 peptide with incomplete Freund’s adjuvant mixed with CRL 1005, a block copolymer adjuvant. Patients were immunized with increasing doses of the MART-127–35 peptide in a Phase I trial to evaluate the toxicity, tolerability, and immune responses to the vaccine. Immunizations were administered every 3 weeks for a total of four injections, preceded by leukapheresis to obtain peripheral blood mononuclear cells for immune analyses, followed by a post-vaccine leukapheresis 3 weeks after the fourth vaccination. Skin testing with peptide and standard delayed-type hypersensitivity skin test reagents was also performed before and after vaccinations. Local pain and granuloma formation were observed in the majority of patients, as were fevers or lethargy of grade 1 or 2. No vaccine-related grade III/IV toxicity was observed. The vaccine was felt to be well tolerated. Twelve of 25 patients were anergic to skin testing at the initiation of the trial, and 13 of 25 developed a positive skin test response to the MART-127–35 peptide. Immune responses were measured by release of IFN-γ in an ELISA assay by effector cells after multiple restimulations of peripheral blood mononuclear cells in the presence of MART-127–35 peptide-pulsed antigen-presenting cells. An ELISPOT assay was also developed to measure more quantitatively the change in numbers of peptide-specific effector cells after vaccination. Ten of 22 patients demonstrated an immune response to peptide-pulsed targets or tumor cells by ELISA assay after vaccination, as did 12 of 20 patients by ELISPOT. Nine of 25 patients have relapsed with a median of 16 months of follow-up, and 3 patients in this high-risk group have died. Immune response by ELISA correlated with prolonged relapse-free survival. These data suggest a significant proportion of patients with resected melanoma mount an antigen-specific immune response against a peptide vaccine and support further development of peptide vaccines for melanoma.

The earliest hint that tumor cells were immunogenic, or expressed antigens that were recognized by the immune system, came from the work of Priehn and Main (1), who demonstrated that mice immunized with tumor cells from UV- or carcinogen-induced tumors would reject a subsequent challenge of live tumor cells from the parental tumor but not an unrelated tumor. Rejection of antigen-expressing tumor cells was mediated by specific host cytolytic T cells in UV-induced tumors (2). Tumor cells, therefore, expressed antigens that were recognized by the immune system of the tumor-bearing host. There is an accumulating body of evidence to suggest that many tumors in experimental model systems and from cancer patients express molecules that are recognized by T cells. The molecular cloning of a tumor-specific antigen expressed by a murine cell line that has been mutagenized has been described, as well as the cloning of a naturally occurring tumor-specific antigen expressed by the murine mastocytoma P815 (3, 4).

In patients bearing metastatic melanomas, a number of groups have demonstrated the existence of antitumor CTL responses. PBMCs,3 as well as TILs, contain populations of cells and individual clones that demonstrate tumor specificity; they lyse autologous tumor cells but not natural killer targets, allogeneic tumor cells, or autologous fibroblasts (5, 6, 7). Tumor-specific TILs that mediate partial and complete regressions of metastatic melanoma after adoptive transfer with IL-2 as well as melanoma-specific CTL clones raised from the peripheral blood of melanoma patients have been used in cloning strategies to identify antigens including MAGE-1 and MAGE-3, GAGE-1, MART-1, gp100, gp75 (TRP-2), tyrosinase, mutated p16, and E-cadherin (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), which expands the repertoire of molecules to use in a vaccine strategy for melanoma. Eight or nine amino acid peptide epitopes have been shown to be displayed in association with class I MHC molecules for recognition by T cells (21, 22), and tumor cells have been shown to express these naturally processed epitopes. We immunized patients with resected melanoma at high risk of harboring microscopic disease to augment T-cell immunity against a known tumor antigen. In this report, we describe the results of a Phase I clinical trial in which the minimal epitope immunodominant 9-amino acid peptide derived from a melanoma differentiation antigen, MART-1, was combined with an oleic oil-based adjuvant, Montanide ISA 51, or IFA to immunize patients with resected melanoma at high risk of harboring microscopic disease and thus at high risk of relapse. Twenty-two patients received the MART-127–35 peptide with IFA, and 3 had a block copolymer adjuvant added to the peptide-IFA combination. The toxicity, tolerability, and specific immune responses to the vaccine were measured, as well as baseline nonspecific immunological parameters.

Trial Eligibility.

All patients had resected stages IIB, III, and IV melanoma by the 1988 modified American Joint Commission on Cancer staging system and were rendered free of disease surgically. They were required to have a magnetic resonance image or computed tomography scan of the head, and computed tomography scans of the chest, abdomen, and pelvis showing no indication of disease within 4 weeks of initiating therapy to verify that they were clinically free of melanoma. Eligibility criteria included age 18 or greater, creatinine <1.4 mg/dl, bilirubin <1.5 mg/dl, platelets of 100,000/mm3 or more, hemoglobin ≥9 g/dl, and total WBC of ≥3,000/mm3. HIV, hepatitis C antibody, and hepatitis B surface antigen were required to be negative, and all patients were HLA-A2 positive by a microcytotoxicity assay. All patients were required to comprehend and sign an informed consent form approved by the National Cancer Institute and the Los Angeles County and University of Southern California Institutional Review Board.

Peptide.

The MART-127–35 peptide (AAGIGILTV) vaccine was administered as outpatient therapy. The bulk peptide was supplied by Chiron Mimetope, Inc., and the finished injectable dosage form was manufactured by the Monoclonal Antibody/Recombinant Protein Production Facility, NCI (Frederick, MD). Peptide was provided by Cancer Therapy Evaluation Program/NCI (Bethesda, MD) under an Investigational New Drug application held by the NCI as the trifluroacetate salt in DMSO. The vials of peptide contained no preservative.

Adjuvants.

Montanide ISA-51 (IFA) was manufactured by Seppic, Inc. and supplied as glass ampules containing 3 ml of sterile adjuvant solution without preservative.

CRL 1005 is a nonionic block copolymer consisting of two chemical components: hydrophobic polyoxypropylene and hydrophilic polyoxyethylene. The copolymer forms small (500 nm–2 μm) particles that combine with protein and peptide antigens. It was manufactured and supplied by Vaxcell, Inc. (Norcross, GA) as 75 mg/ml CRL 1005 in a 2.5-mg vial without preservative.

Vaccine Preparation and Administration.

An appropriate amount of MART-127–35 was diluted with sterile DMSO (RIMSO, Gaithersburg, MD) and added in a 1:1 volume to Montanide ISA-51 and then mixed in a Vortex mixer (Fisher, Inc., Alameda, CA) for 10 min at room temperature. The resulting emulsion was injected deeply s.c. in the lateral thigh in a volume of 1 or 2 ml using a glass syringe. s.c. as opposed to intradermal administration was chosen because of the large volume of injectate (up to 2 ml). Alternating thighs were used for a total of four injections, which were done 3 weeks apart. Twenty-three patients had a leukopheresis with an exchange of ∼5 liters of blood volume performed within 2 weeks before beginning vaccinations and 3 weeks after the final vaccination to collect PBMCs, which were frozen for future analysis. Two patients could not have leukopheresis performed because of poor venous access. Skin tests were performed using 50 μg of the MART-127–35 peptide in DMSO injected intradermally in a volume of 100 μl using a tuberculin syringe and a 27-gauge needle, with 100 μl of 100% DMSO injected at a separate site as a control. Candida extract, mumps, and trichophyton provided a positive control, and saline was a negative control for assessment of DTH. At least 5 mm of induration or erythema above and beyond that shown by DMSO alone read 48 h after intradermal injection was required to score a MART-1 skin test as positive.

Dose Escalation.

Patients received escalating doses of peptide with IFA, starting with the initial cohort at 300 μg/dose, then 1000 μg/dose, and 2000 μg/dose. Four patients received 300 μg, 4 received 1000 μg, and 17 patients were treated at the 2000-μg dose. The last three patients at the 2000-μg dose received 25 mg of block copolymer adjuvant CRL 1005 in addition to the IFA with the MART-127–35 peptide at 2000 μg.

Screening for Vitiligo and Eye Changes.

All patients had a complete skin exam prior to therapy and at each visit for vaccination to screen for vitiligo. Slit lamp exams and iris photos were done by an ophthalmologist prior to starting therapy in all patients, and hand held ophthalmoscopic retinal and iris exams were performed at each vaccination visit to assess ocular toxicity. No patient had evidence of vitiligo or ocular toxicity.

Preparation of PBMC Specimens.

Pheresis samples were processed to purify PBMCs by sedimentation on a Ficoll-Hypaque cushion (Pharmacia, Alameda, CA) with extensive washing in HBSS. Cells were frozen in 40% human AB serum (Gemini Bioproducts, Calabasas, CA), 50% RPMI (Life Technologies, Inc., Grand Island, NY), and 10% DMSO (Sigma) and stored in a liquid nitrogen freezer at −168°C until use.

Proliferation Assays.

Assays were performed by incubating 105 thawed PBMCs in wells of a round-bottomed, 96-well plate (Corning, Inc., Oneonta, NY) in sextuplicate in a total volume of 200 μl of RPMI 1640 with 10% human AB serum. Various reagents were then added, and the plates were incubated in a 5% CO2 incubator at 37°C for 5 days. One μCi of tritiated thymidine was then added to each well in a volume of 20 μl and again incubated at 37°C for 16 h. The contents of each well were harvested using a Skatron harvester and counted in a liquid scintillation β counter. Results are presented as the mean of five to six determinations/point.

CASTA is a preparation of proteins derived from Candida albicans obtained from Greer Labs (Lenoir, NC). PHA was obtained from Sigma. Peptides used for in vitro studies were synthesized at the USC/Norris Cancer Center Core Peptide Facility.

Cytokine Assays.

Assays were performed using peptide-stimulated T cells as effector cells. Peptide-stimulated T cells were produced by incubating 2 × 105 thawed PBMCs with MART-127–35 or FLU-MI peptide-pulsed dendritic cells that were irradiated with 6000 rads at a 1:3 ratio in wells of a 24-well plate (Corning). Cells were plated in IMEM media with 10% human AB serum. Two days later, IL-2 (kindly provided by Chiron, Emeryville, CA) was added at 50 IU/ml. Fresh IL-2 was added every 3–4 days. After 10 days, the T cells were restimulated with thawed autologous PBMCs pulsed with 10 μg/ml of MART-127–35 peptide at 37°C for 2 h and irradiated with 3000 rads. IL-2 was again added 48 h later at 50 IU/ml, Tcells were restimulated with peptide-pulsed PBMCs every 7 days, and after four restimulations were harvested for immune assays. The performance of cytokine release assays after two or three restimulations invariably resulted in high nonspecific backgrounds. For the cytokine release assay, 105 peptide-stimulated T cells were harvested at least 5 days after the last restimulation and incubated with 105 T2 cells pulsed with 10 μg/ml MART-1 peptide or 624-mel cells as targets in a total volume of 1 ml of RPMI medium without serum for 18 h in a 5% CO2 incubator at 37°C. Neither the effectors nor the targets were irradiated. Supernatants were collected, spun briefly at 14,000 × g to pellet cells and debris, and frozen at −80°C until assays were done. IFN-γ was detected in supernatants using an antihuman IFN-γ Quantikine ELISA kit (R and D Systems, Minneapolis, MN).

ELISPOT Assays.

Assays were performed with 300,000, 100,000, 30,000, and 10,000 effectors/well, with a constant 100,000 targets in triplicates. The effectors were bulk CTLs after one or two restimulations in vitro with peptide-pulsed antigen-presenting cells. Nitrocellulose 96-well plates were coated with anti-IFN-γ antibodies and incubated overnight at room temperature. Plates were washed and incubated at 37°C with blocking buffer. T2 target cells (105) pulsed with peptides were added to the wells, and then serial dilutions of effectors were added for a total volume of 200 μl/well. The plate was incubated overnight at 37°C and then washed extensively. Biotinylated secondary anti-IFN-γ antibody was added, and the plate was incubated overnight at 4°C. Plates were again washed extensively, and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium reagent followed by Streptavidin alkaline-phosphatase was added. The reaction was halted by washing under running distilled water, and the plates were dried overnight at room temperature. Spots were enumerated by counting under a microscope using a computer-controlled mechanical stage and a digital camera (Olympus Optical, Kagoshima, Japan) input to a Micron 2000 Pentium II computer using Image Pro Plus software (Media Cybernetics, Silver Spring, MD). Counts were the means of triplicates.

Statistics.

The association between post-vaccine ELISA cytokine release and time to relapse was calculated using the post-vaccine level of IFN-γ or the difference of post-vaccine minus pre-vaccine levels of IFN-γ released as continuous variables. Kaplan-Meier plots were constructed, and the log-rank test was used to calculate Ps.

Demographics.

A total of 25 patients with stages IIB, III, and IV resected melanoma were treated in this Phase I trial. The demographic details of this group of high-risk patients is shown in Table 1. The median age of the 12 men and 13 women was 52. Fifteen patients had resected stage III disease, mostly lymph nodal recurrences after adjuvant IFN therapy, and 5 each had resected stage IIB or IV disease. Twenty-one had cutaneous melanoma, and 4 had ocular melanoma. The median time since diagnosis of the primary lesion for the whole group was 2.5 years. Eight of the patients had failed previous IFN-α, and two had a cellular vaccine. Three patients failed to be leukopheresed after finishing the series of four vaccinations, one because of progressive disease, and two because of inadequate venous access, leaving 22 patients with blood samples collected for evaluation both before and after vaccination.

Toxicities.

All 25 patients were evaluable for toxicity. The overall toxicities of the MART-127–35 vaccine are shown in Table 2. The MART-127–35 vaccine was generally well tolerated, but almost all patients (23 of 25) had grade I or II local tenderness and pain at the injection sites. Fifteen patients developed granulomata at the injection sites, although none needed to be resected because of symptoms. One patient had a granuloma resected because of suspicion that it might represent a recurrence, and effector cells were grown from the resected granuloma tissue for a cytokine assay to determine antigen specificity as described below. Fatigue was observed in 14 patients but was not dose related. The only grade III toxicities were neutropenia that was transient and not felt to be vaccine related in one patient at the 1000-μg dose and grade III nausea in one patient at the 2000-μg dose level. Nausea was observed in five patients, and fevers, invariably low grade, were observed in three patients. Six patients experienced headaches, and six had arthralgia. Toxicity was not dose related, with no appreciable differences noted between the 300-, 1000-, and 2000-μg doses. Patients were screened for vitiligo and ocular toxicity as indicated in “Materials and Methods,” and none were observed. In conclusion, the toxicity of the MART-127–35/IFA vaccine was modest and not dose related, and the vaccine was felt to be well tolerated, with only one episode of grade III vaccine-related toxicity. Greater and more prolonged granuloma formation appeared to occur in the three patients that received the block copolymer CRL 1005 added to the MART-127–35/IFA vaccine.

DTH Skin Test Results.

Skin test reactivity to a panel of recall antigens was assessed prior to and after MART-127–35 vaccine therapy. The data at the bottom of Table 1 show that, surprisingly, 12 of 25 patients tested were anergic to recall antigens prior to MART-127–35 vaccination. DTH to the MART-127–35 peptide was also assessed by skin testing, and 2 of 25 patients reacted to the vaccine peptide prior to vaccination. Twenty-five patients were tested after MART-127–35 vaccination for DTH to the peptide after baseline reactivity to DMSO was subtracted as background, and 15 of 25 were positive, i.e., 13 of 25 developed antigen-specific DTH reactivity after vaccination, defined as at least 5 mm of induration and/or erythema after subtraction of the DMSO alone control, including seven patients who were anergic to the panel of recall antigens (Candida, mumps, and trichophyton) prior to and after vaccination. Skin test reactivity to the MART-127–35 peptide did not appear to be dose related, nor did it correlate in any way with relapse of disease or survival (data not shown). As shown in Table 1, 6 of 11 patients with an immune response by ELISA also had a positive skin test to MART-127–35.

Proliferation Assays.

Proliferation of patient PBMCs in response to MART-127–35 was tested prior to and after the series of four vaccinations to assess whether a proliferative response had been successfully induced to that class I-restricted peptide. Immune parameters were measured in the 25 patients that received the MART-127–35/IFA vaccine. The proliferation of PBMCs in response to PHA, a mitogenic stimulus, as well as in response to CASTA, a C. albicans protein extract, was also assessed as an overall measure of immune status prior to and after vaccination. The results are shown in Table 3, in which 24 patients were tested, showing for the whole group no overall evidence of a proliferative response to the MART-127–35 peptide when pulsed onto PBMCs at 1 μg/ml (7047 ± 2367 to 7892 ± 2491 cpm) or 10 μg/ml (6567 ± 2011 to 8188 ± 2536 cpm) without further restimulation. No changes were seen in proliferative responses to PHA (84,163 ± 20,610 to 89,634 ± 15,901 cpm) or C. albicans proteins (37,835 ± 13,265 to 45,441 ± 10,883 cpm). In several cases, the proliferation of PBMCs to MART-127–35 peptide decreased appreciably after vaccination, without a clear reason.

Cytokine Release Immune Assays in MART-1-vaccinated Patients.

MART-127–35-specific immunity was measured in 22 patients who had pre- and post-vaccination PBMC samples available by measuring antigen-specific release of IFN-γ by ELISA from effector cells restimulated weekly four times with peptide-pulsed irradiated PBMC stimulators. Effectors were incubated for 18 h with control HLA-A2+ T2 cells, MART-127–35 peptide-pulsed T2 cells, or 624-mel, a HLA-A2-positive, MART-1-positive melanoma cell line, as described in detail in “Materials and Methods.” The results of pre- and post-vaccine cytokine release assays for those 22 patients are shown in Table 4. A total of 11 of 22 patients, at all dose levels, showed evidence of increased reactivity to MART-127–35 peptide-pulsed T2 targets or MART-1-positive target 624-mel after vaccination, with release of IFN-γ secreted per 105 cells/ml that ranged from 100 to 3000 pg/ml. The cytokine release had to be at least 100 pg/ml above the T2 unpulsed control to be scored as positive, which represented two SDs from the mean of the T2 unpulsed controls. Two patients at the 300-μg dose level, 1 at the 1000-μg dose level, and 6 at the 2000-μg dose level, including one of three who had the block copolymer added to their vaccine, had increased ELISA reactivity to MART-127–35-pulsed T2 cells. Two additional patients, one each at the 300- and 1000-μg cohorts, had reactivity to 624-mel. Seven of nine ELISA responders had increased cytokine release to both T2 cells pulsed with the MART-127–35 peptide and 624-mel cells. Four patients, including two responders by ELISA, had detectable reactivity prior to vaccination. All of the cytokine release assays were repeated at least once, with similar results. For cytokine release assays, background release of unpulsed targets incubated with effectors ranged from 0 to 60% of MART-1 peptide-pulsed targets incubated with effectors and are shown in the leftward column of Table 4; the middle column represents actual MART-1-specific release without subtraction of any background. The observation that reactivity was seen with MART-127–35 peptide-pulsed T2 targets as well as HLA-A2-positive, MART-1-positive cell line 624-mel suggested that the increased immune effectors detected in the peripheral blood could recognize naturally processed MART-127–35 peptide on the surface of a tumor cell line. To verify that increased MART-127–35-specific reactivity measured by specific cytokine release after vaccination was antigen specific, and that patients showing no or minimal (<100 pg/ml) cytokine release could react to an influenza stimulus, PBMCs from selected patients who had evidence either of a clearly positive response or no change in MART-127–35-specific cytokine release were subjected to a cross-specificity assay in which pre- and post-vaccine PBMC samples were split into two and stimulated as above four times weekly in the presence of a HLA-A2-restricted influenza virus matrix protein or MART-127–35-peptide pulsed onto irradiated PBMC stimulators and then used as effectors in a cytokine release assay as in Table 4, with targets consisting of T2 cells pulsed with FLU peptide or T2 cells pulsed with the MART-127–35 peptide. A positive signal in FLU-specific release and no MART-1 specific signal was expected for FLU-stimulated effector cells both pre- and post-vaccination for all patients. No FLU-specific reactivity was expected for MART-127–35-stimulated effector cells for any patients pre- or post-vaccination, but increased MART-127–35-specific reactivity by MART-127–35-stimulated effector cells after vaccination was expected for patients who had an immune response in Table 4. In the left panel of Fig. 1, for the pre- and post-vaccine sample pair from patient 6, FLU-specific cytokine release was observed both pre- and post-vaccine, but significant MART-127–35-specific reactivity was seen only after vaccination, as shown also in Table 4. The data in the right panel of Fig. 1 suggest that for the pre- and post-vaccine sample pair from patient 11, there was no MART-127–35-specific response post vaccine, reproducing the result in Table 4. but that good FLU-specific release was observed pre- and post-vaccine as a positive control. These data confirm the MART-1 specificity of the cytokine release data shown in Table 4 for patients 6 and 11 in a repeated experiment and demonstrate that patients without a MART-127–35 response still have the ability to mount a FLU-specific immune response.

ELISPOT Immune Assays in MART-1-vaccinated Patients.

As a further measure of immune response to the MART-127–35 peptide vaccine, we devised an ELISPOT assay that detected the presence of single E:T cell interactions by immobilization of E:T pairs and detection of IFN-γ after two restimulations with peptide-pulsed stimulators ex vivo. This assay did not directly measure antigen-specific IFN-γ-releasing effector cells in fresh blood but yielded a semiquantitative assessment of the presence of antigen-specific effector cells after minimal restimulation. The data in Table 5 show that 11 of 20 patients tested demonstrated an immune response to MART-127–35 after vaccination, although background reactivities to FLU peptide-pulsed or unpulsed T2 targets were higher than in the cytokine release assay, and not all patients that had evidence of boosted immunity by ELISA also had a positive response by ELISPOT. These data do support the notion that an augmented MART-127–35-specific CTL response can be detected in peripheral blood cells after vaccination with the MART-127–35 peptide plus adjuvant with a quantitative single-cell ELISPOT assay. However, only 3 of 11 ELISPOT responders had a response by ELISA, and 6 of the 12 ELISPOT responders have relapsed, with a median of 16 months of follow-up. There was no relationship between ELISPOT response and time to relapse.

Correlation between Immune Response and Relapse-free Survival.

Although it was not a prospectively determined end point of this Phase I study, a correlation was made between relapse-free survival and immune response by cytokine release ELISA at the median of 16 months of follow-up, at which time 3 of 25 patients had died and 16 of 25 remained free of disease. The association between the two continuous variables was calculated using the log-rank test, indicating that relapse-free survival time correlated with post-vaccine ELISA assay (P < 0.003) and also was associated with the difference between pre- and post-vaccine values (P < 0.04). When the patients were arbitrarily grouped into those with a “strong” cytokine response (>100 pg/ml post-vaccine compared with pre-vaccine), “weak” cytokine response (<100 pg/ml post-vaccine compared with pre-vaccine), or no response (0 pg/ml post-vaccine compared with pre-vaccine), it is intriguing to note that all eight patients in the “strong” group were alive and free of progression, whereas the nine relapsed patients (including the three who had died from disease) were distributed between the no response or “weak response” groups.

Endogenously synthesized antigens of virtually all mammalian cells are processed and converted to small epitope peptides that are displayed on the cell surface in association with class I MHC molecules (21, 22, 23, 24). Peptides bind with different consensus motifs based on preferred NH2 (positions 1 and 2) and COOH (positions 8, 9, and 10) amino acids that localize the peptide to the MHC “cleft” or peptide-binding groove (25, 26, 27). The binding ability of epitope peptides define class I MHC restriction and T-cell receptor specificity for any protein. Tumor-associated or tumor-specific antigenic proteins present MHC-restricted peptide epitopes at the cell surface for T-cell recognition (28). To provide a strong antigen-specific stimulus in this vaccine trial, we used an epitope peptide derived from MART-1, an melanoma antigen recognized by T cells (10, 11). The principal goal of this immunization strategy was to augment antigen-specific T-cell responses in patients to eliminate tumor and prevent relapse in individuals with microscopic tumor burdens, as has been shown in experimental murine models.

Antigens present on melanomas can be broadly divided into three categories; one is the cancer/testis group expressed by a large variety of tumors, of which the MAGE, BAGE, and GAGE gene families are examples (29). The second is a group of mutated normal genes uniquely present on individual tumors; β-catenin, HLA-A variants, and p16 are examples (19, 20). The third category is differentiation antigens that are expressed by melanomas as well as normal melanocytes; MART-1/Melan A, tyrosinase, gp75, TRP-2, and gp100/pMel 17 are examples (10, 11, 12, 13, 14, 15, 16, 17). The tumor-restricted distribution of the first two groups make them attractive targets for immunotherapy, but there is little evidence of immune reactivity to those antigens in most melanoma patients. In contrast, the differentiation antigens, although expressed in normal tissue, clearly provoke an immune response in melanoma patients. Cytolytic T cells from peripheral blood, or which infiltrate tumors from HLA-A2-positive patients, recognize an antigen or group of antigens on HLA-A2 melanoma cells and fresh tumors (6, 30, 31, 32). MART-1 was defined as a gene product recognized by CTL clones from peripheral blood of a melanoma patient and by CTLs derived from a melanoma patient’s TILs, in whom cellular therapy had induced a partial regression of metastatic disease (10, 11), suggesting that it might be a target recognized by T cells with antitumor potential. The TILs that recognized MART-1 as well as TILs from a number of other melanoma patients reacted with virtually all melanoma cell lines that expressed HLA-A2, and transfection of the A2 gene into other non-A2-expressing melanoma lines increased their sensitivity to TIL lysis (33). This suggested that MART-1 was a common A2-restricted melanoma antigen recognized by CTLs. MART-1 was expressed by virtually all metastatic melanoma lesions, a majority of cells lines derived from metastatic melanomas, and also by melanocytes, but not by any other normal tissue. The MART-1 gene encoded a putative protein of Mr 26,000 with sequences that matched the known HLA-A2 binding motifs. The nonamer sequence AAGIGILTV, representing residues 27–35 of the MART-1 protein, bound most strongly to HLA-A2 (34). This peptide stimulated the growth of specific CTLs from the PBMCs of melanoma patients and of normal persons (35). Multiple restimulations of PBMCs with MART-127–35 peptide resulted in cultures of MART-1-specific CTLs derived from 11 of 12 melanoma patients (36). These CTLs lysed fresh uncultured melanoma cells and were 100-fold more lytically active against melanoma cells than TILs grown in high-dose IL-2. The majority of TILs grown from patients with melanoma are capable of recognizing the MART-127–35 peptide, and some of those TIL cultures induced regression of metastatic melanoma after adoptive transfer with IL-2. The repertoire of Vβ T-cell receptor molecules from TILs and peripheral blood-derived CTL lines that are MART-1 specific are quite skewed (37, 38, 39).

Peptides derived from MART-1 were eluted from melanoma cells, suggesting that MART-127–35 is a naturally occurring antigen on fresh tumors (40, 41, 42). A protein database analysis demonstrated that sequences conforming to the MART-1 A2 binding motif and possessing features important for CTL recognition occurred frequently in proteins (43), and that a peptide derived from glycoprotein C of herpes simplex virus could sensitize target cells to lysis by MART-127–35-specific CTLs (43). These data suggest that epitope mimicry by normal or other commonly occurring proteins may account for the frequency of CTLs detected against melanoma antigens like MART-1.

Greater MART-127–35-reactive CTL activity has been demonstrated in the peripheral blood of melanoma patients compared with normal persons, suggesting that a tumor-related “priming” effect has occurred (44), and in a clinical trial of MART-127–35 peptide with adjuvant in patients with metastatic melanoma, a boost in MART-127–35-specific immunity was observed in a significant proportion of patients, but without clinical responses (45). Clinical benefit for a MART-1 peptide vaccine has been observed in a trial that included multiple peptides with granulocyte/macrophage-colony stimulating factor for metastatic melanoma, with 5 of 26 patients showing a clinical response that correlated with augmented MART-1-specific CTLs in at least three cases (46). The MART-1 peptide was used with several other peptides to pulse autologous dendritic cells, which were adoptively transferred by intralymph nodal and s.c. injections, resulting in a 25% response rate in patients with metastatic melanoma (47).

The overlapping MART-126–35 peptide has been shown to be more immunogenic than the 27–35 epitope, and a single amino acid modification to the 26–35 peptide rendered it a stronger binder to A2.1 and even more immunogenic (48, 49). This peptide is a prime candidate for future clinical vaccine trials. The A2.1-restricted peptide in this study has been shown to bind to multiple other HLA-A2 subtypes, as well as allele A45, but no other MART-1-specific peptides have been shown to elicit specific immune responses in vitro in patients bearing other HLA class I alleles (50, 51, 52, 53).

Western blotting as well as immunohistochemical staining using MART-1 antibodies have established that MART-1 is a transmembrane protein component of the melanosome complex (54, 55). Reverse transcription-PCR analysis has shown that MART-1 mRNA is present in virtually 100% of metastatic melanoma lesions, yet immunohistochemical staining has shown that there is considerable heterogeneity in MART-1 expression on primary and metastatic lesions, with 60–90% of all lesions staining positively (56, 57, 58, 59, 60). In one study, deletion of MART-1 expression, as well as transporter associated with antigen processing (TAP) transporter expression, rendered cells transparent to CTL recognition, suggesting that loss of MART-1 may be a mechanism for immune evasion (61).

The data presented in this report suggest that ∼50% of patients have demonstrated augmented, antigen-specific T-cell reactivity after receiving a MART-127–35/IFA vaccine. The use of cytokine release assays with IFN-γ and the use of an automated ELISPOT assay yielded semiquantitative information about increases in antigen-specific effector cells in circulating PBMCs after vaccination. The cross-specificity ELISA data from selected patients suggest that the CTLs generated from post-vaccine PBMCs are truly antigen specific, but the ELISPOT data are consistent with a fairly low frequency of precursor CTLs after vaccination. A statistical analysis of the IFN-γ ELISA data, albeit with small numbers, provided a provocative hint that there was a correlation between ELISA response and relapse-free survival. The correlation of antigen-specific ELISA assay with a desired clinical end point is encouraging. However, there was no clear relationship between DTH reactivity or ELISPOT response and relapse-free survival, which suggests a cautious interpretation for the data. No increases in MART-127–35-specific proliferation were seen after one restimulation of PBMCs, also consistent with a low frequency of antigen-specific T cells.

The MART-1 antigen is also expressed by normal melanocytes (31, 62), and the use of a MART-127–35 epitope peptide vaccine had the potential to induce autoimmune reactions. It is not known whether normal melanocytes effectively present the MART-127–35 epitope peptide to T cells in vivo, and previous clinical experience with the adoptive transfer of CTLs that were highly MART-1 reactive and mediated regression of tumor did not indicate the onset of any autoimmune damage to skin, brain, inner ear, or retina, where melanocyte lineage cells are located. As in the present study, patients with metastatic melanoma that received a MART-127–35 peptide vaccine did not demonstrate any evidence of ocular or other toxicity.4 None of the 25 patients with resected stages IIB/III/IV melanoma that received MART-127–35 peptide vaccine with Montanide ISA-51 in the present study exhibited vitiligo, which has been observed in melanoma patients receiving immunotherapy with IL-2 or chemotherapy combined with IL-2 (63). No ocular problems nor any evidence of autoimmune pathology have occurred in any patients on this trial with a median follow-up of 16 months. Toxicity was confined to mostly local pain, edema, and formation of granuloma, none of which became infected or required surgical intervention.

None of the three patients who died and none of nine relapsed patients showed evidence of increased immunity to the MART-127–35 vaccine. Eleven of 16 patients who are free of disease showed an immune response, as evidenced by increased release of IFN-γ after exposure of PBMCs to MART-127–35 peptide-pulsed antigen presenting cells or MART-1-expressing tumor cell line 624-mel. To determine whether augmented MART-127–35-specific release of IFN-γ post-vaccination was associated with prolonged time to relapse, we used Kaplan-Meier plots and the log-rank test. The log-rank test based on this model was used with cytokine values grouped into thirds prior to the analysis to calculate P for the significance of the association. P between the level of immune response post-vaccine and relapse-free survival was 0.01, and for the difference between pre- and post-vaccine cytokine values, P was 0.009. The number of vaccinated patients is too small to draw a statistically meaningful general conclusion from the lack of immune responses in relapsed patients. The data in this trial, however, support the idea of a follow-up trial using multiple peptides derived from MART-1, gp100, and tyrosinase, which will be used to vaccinate high-risk melanoma patients rendered free of disease with a more prolonged schedule of immunizations in association with novel adjuvants (64, 65). An important clinical end point of a larger follow-up trial will be the correlation between immune response and time to relapse to determine whether augmented peptide-induced immunity has the potential to result in clinical benefit.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

This work was supported by Grant FD-001-11001 from the Food and Drug Administration’s Orphan Drug Program and in part by Cancer Center Study Grant 5P30-CA14089 from the National Cancer Institute.

                
3

The abbreviations used are: PBMC, peripheral blood mononuclear cell; TIL, tumor-infiltrating lymphocyte; IL, interleukin; HLA, human leukocyte antigen; NCI, National Cancer Institute; FLU, influenza; DTH, delayed-type hypersensitivity; PHA, phytohemagglutinin; IFA, incomplete Freund’s adjuvant.

        
4

F. Marincola, personal communication.

Fig. 1.

The experiment in Fig. 1 describes a cross specificity analysis of PBMC samples from one patient with a positive immune response (Patient 6) and one patient with a negative response (Patient 11) from Table 4. Effector cells were prepared as described in “Materials and Methods” by restimulation of PBMC samples that were split into two aliquots. One aliquot was restimulated in the presence of HLA-A2+ FLU peptide-pulsed stimulator cells, and the other was restimulated in the presence of MART-127–35 peptide-pulsed stimulator cells. One hundred thousand resulting effector cells were plated after the fourth restimulation in vitro with 100,000 targets consisting of either HLA-A2+ T2 cells pulsed with the FLU peptide or T2 cells pulsed with MART-127–35 peptide (both peptides at a concentration of 10 μg/ml overnight at 37°C) in a 24-well plate in complete medium for 18 h in a volume of 1 ml. The supernatant was harvested and then spun in a microcentrifuge at 14,000 × g for 30 s to pellet cells and debris. Supernatants were removed and used to measure IFN-γ release using a commercial ELISA kit as described in “Materials and Methods.” Figures shown are the means of duplicate values of IFN-γ. Similar results were obtained in repeated experiments for both patients. Left, Patient 6 (a responder to the MART vaccine): the first pair of columns from the left shows the cytokine release response to FLU restimulated PBMCs pre-vaccination (Pre-vaccine Flu) by either FLU-pulsed T2 targets (□) or MART-127–35-pulsed targets (▪). *, zero level of cytokine release. The cytokine release response to MART-127–35-restimulated PBMCs for the two targets is shown in the second group (Pre-vaccine MART-1), and for FLU or MART-127–35-restimulated PBMCs post-vaccine (Post-vaccine Flu or Post-vaccine MART-1) in which a FLU-specific response was detected pre- and post-vaccine, as shown, and where a MART-1-specific response was detected post- but not pre-vaccine Similar data are shown for MART-1 nonresponding patient 11 (right), in which a FLU-specific response was detected pre- and post-vaccine, as shown, but no response to MART-127–35.

Fig. 1.

The experiment in Fig. 1 describes a cross specificity analysis of PBMC samples from one patient with a positive immune response (Patient 6) and one patient with a negative response (Patient 11) from Table 4. Effector cells were prepared as described in “Materials and Methods” by restimulation of PBMC samples that were split into two aliquots. One aliquot was restimulated in the presence of HLA-A2+ FLU peptide-pulsed stimulator cells, and the other was restimulated in the presence of MART-127–35 peptide-pulsed stimulator cells. One hundred thousand resulting effector cells were plated after the fourth restimulation in vitro with 100,000 targets consisting of either HLA-A2+ T2 cells pulsed with the FLU peptide or T2 cells pulsed with MART-127–35 peptide (both peptides at a concentration of 10 μg/ml overnight at 37°C) in a 24-well plate in complete medium for 18 h in a volume of 1 ml. The supernatant was harvested and then spun in a microcentrifuge at 14,000 × g for 30 s to pellet cells and debris. Supernatants were removed and used to measure IFN-γ release using a commercial ELISA kit as described in “Materials and Methods.” Figures shown are the means of duplicate values of IFN-γ. Similar results were obtained in repeated experiments for both patients. Left, Patient 6 (a responder to the MART vaccine): the first pair of columns from the left shows the cytokine release response to FLU restimulated PBMCs pre-vaccination (Pre-vaccine Flu) by either FLU-pulsed T2 targets (□) or MART-127–35-pulsed targets (▪). *, zero level of cytokine release. The cytokine release response to MART-127–35-restimulated PBMCs for the two targets is shown in the second group (Pre-vaccine MART-1), and for FLU or MART-127–35-restimulated PBMCs post-vaccine (Post-vaccine Flu or Post-vaccine MART-1) in which a FLU-specific response was detected pre- and post-vaccine, as shown, and where a MART-1-specific response was detected post- but not pre-vaccine Similar data are shown for MART-1 nonresponding patient 11 (right), in which a FLU-specific response was detected pre- and post-vaccine, as shown, but no response to MART-127–35.

Close modal
Table 1

MART-1 vaccine trial demographics

Total number of patients 25 
Male 12 
Female 13 
Median age (yr) 52 
Median time since primary diagnosis (yr) 2.5 
Stage of disease  
 IIB 
 III 15 
 IV 
Prior therapy  
 Surgery 25 
 Chemotherapy 
 Biologic therapy 10 
 Radiation therapy 
Anergy to skin test reagents pre-vaccine 12/25 
Post-vaccine MART-1 DTH skin test 15/25a 
Patients who developed positive MART-1  skin test who were anergic pre-vaccine 7/13 
Patients with positive MART-1 ELISA assay  who had positive MART-1 skin test 6/11 
Total number of patients 25 
Male 12 
Female 13 
Median age (yr) 52 
Median time since primary diagnosis (yr) 2.5 
Stage of disease  
 IIB 
 III 15 
 IV 
Prior therapy  
 Surgery 25 
 Chemotherapy 
 Biologic therapy 10 
 Radiation therapy 
Anergy to skin test reagents pre-vaccine 12/25 
Post-vaccine MART-1 DTH skin test 15/25a 
Patients who developed positive MART-1  skin test who were anergic pre-vaccine 7/13 
Patients with positive MART-1 ELISA assay  who had positive MART-1 skin test 6/11 
a

Three of 25 patients had positive MART-1 skin tests pre-vaccine; one became negative, and the other two, who remained positive, are included in the total of 15 positive.

Table 2

MART-1 toxicitiesa

Level 1(n = 4)Level 2(n = 10)Level 3(n = 8)Level 4(n = 3)
1/2b3/4b1/2b3/4b1/2b3/4b1/2b3/4b
Allergy       
Fever       
Granulocytopenia       
Arthralgia     
Fatigue     
Headache      
Thrombocytopenia        
Nausea    
Diarrhea      
SGOT        
SGPT        
Granuloma     
Dermatologic      
Local pain     
Ocular         
Vitiligo         
Autoimmune         
Level 1(n = 4)Level 2(n = 10)Level 3(n = 8)Level 4(n = 3)
1/2b3/4b1/2b3/4b1/2b3/4b1/2b3/4b
Allergy       
Fever       
Granulocytopenia       
Arthralgia     
Fatigue     
Headache      
Thrombocytopenia        
Nausea    
Diarrhea      
SGOT        
SGPT        
Granuloma     
Dermatologic      
Local pain     
Ocular         
Vitiligo         
Autoimmune         
a

Level 1, 300 μg MART-1 + IFA every 3 weeks × 4 doses; Level 2, 1000 μg MART-1 + IFA every 3 weeks × 4 doses; Level 3, 2000 μg MART-1 + IFA every 3 weeks × 4 doses; and Level 4, 300 μg MART-1 + CRL 1005 every 3 weeks × 4 doses.

b

Toxicity/grade.

Table 3

Proliferation of PBMC pre- and post-vaccine

Frozen PBMCs were thawed and added to 96-well plates as described in “Materials and Methods,” and either complete medium, PHA at 1 μg/ml, MART-127–35 at 1 and 10 μg/ml, or CASTA at 5 μg/ml were added for 5 days. One microcurie of tritiated thymidine was added to each well for 18 h, and the wells were harvested and counted using a beta scintillation counter. Incorporation of tritiated thymidine is indicated. PMBCs from six healthy volunteers were used to establish the normal values (not shown).
Pre-vaccineaPost-vaccinea
Complete medium 6252 ± 1957 7140 ± 2451 
PHA 84,163 ± 20,610 89,634 ± 15,901 
MART-1 μg/ml 7047 ± 2367 7892 ± 2491 
MART-10 μg/ml 6567 ± 2011 8188 ± 2536 
ACTG-5 μg/ml 37,835 ± 13,265 45,411 ± 10,883 
Frozen PBMCs were thawed and added to 96-well plates as described in “Materials and Methods,” and either complete medium, PHA at 1 μg/ml, MART-127–35 at 1 and 10 μg/ml, or CASTA at 5 μg/ml were added for 5 days. One microcurie of tritiated thymidine was added to each well for 18 h, and the wells were harvested and counted using a beta scintillation counter. Incorporation of tritiated thymidine is indicated. PMBCs from six healthy volunteers were used to establish the normal values (not shown).
Pre-vaccineaPost-vaccinea
Complete medium 6252 ± 1957 7140 ± 2451 
PHA 84,163 ± 20,610 89,634 ± 15,901 
MART-1 μg/ml 7047 ± 2367 7892 ± 2491 
MART-10 μg/ml 6567 ± 2011 8188 ± 2536 
ACTG-5 μg/ml 37,835 ± 13,265 45,411 ± 10,883 
a

Data are means of quintuplicate counts per minute of incorporated tritiated thymidine ± SD.

Table 4

Immune response to MART-1 vaccination: Release of IFN-γ by peptide-stimulated effector cells pre- and post-vaccination

Effector cells were prepared as described in “Materials and Methods” by restimulation of PBMCs in the presence of MART-127–35 peptide-pulsed antigen-presenting cells. 100,000 resulting effector cells were plated after the fourth restimulation in vitro with 100,000 targets consisting of either HLA-A2+ T2 cells or T2 cells pulsed with MART-127–35 peptide (peptide at a concentration of 10 μg/ml) in a 24 well plate in complete medium for 18 h in a volume of 1 ml. The supernatant was harvested and then spun in a microcentrifuge at 14,000 × g for 30 s to pellet cells and debris. Supernatants were removed and used to measure IFN-γ release using a commercial ELISA kit as described in “Materials and Methods.” Figures shown are the means of duplicate values of IFN-γ. Similar results were obtained in repeated experiments for each patient.
DoseLevelPatient no.T2 unpulsed targetsaT2 MART-1 pulsed targetsa624-mela
300 Pre 390 1650 1260 
 Post  3300b 2025b 
300 Pre 160 35 
 Post  165 35 250b 
300 Pre 
 Post  
1000 Pre 168 342 210 
 Post  180 2880b 1800b 
1000 Pre 96 16 96 
 Post  215 23 670 
1000 Pre 117 170 
 Post  912b 468b 
2000 Pre 80 245 
 Post  10 118b 160 
2000 Pre 10 10 
 Post  
2000 Pre 
 Post  90 
2000 Pre 10 21 40 
 Post  416b 299b 
2000 Pre 11 15 10 
 Post  10 
2000 Pre 12 212 88 251 
 Post  233 1583b 908b 
2000 Pre 13 80 46 
 Post  53 24 50 
2000 Pre 14 2350 779 
 Post  205 1065 616 
2000 Pre 15 19 
 Post  
2000 Pre 16 1060 
 Post  865 
2000 Pre 17 
 Post  2580b 273b 
2000 Pre 18 52 21 336 
 Post  22 40 
2000 Pre 19 43 
 Post  442 688b 737b 
2000 Pre 20c 
 Post  10 50 
2000 Pre 21c 350 300 250 
 Post  450 90 230 
2000 Pre 22c 96 24 62 
 Post  50 163b 
Effector cells were prepared as described in “Materials and Methods” by restimulation of PBMCs in the presence of MART-127–35 peptide-pulsed antigen-presenting cells. 100,000 resulting effector cells were plated after the fourth restimulation in vitro with 100,000 targets consisting of either HLA-A2+ T2 cells or T2 cells pulsed with MART-127–35 peptide (peptide at a concentration of 10 μg/ml) in a 24 well plate in complete medium for 18 h in a volume of 1 ml. The supernatant was harvested and then spun in a microcentrifuge at 14,000 × g for 30 s to pellet cells and debris. Supernatants were removed and used to measure IFN-γ release using a commercial ELISA kit as described in “Materials and Methods.” Figures shown are the means of duplicate values of IFN-γ. Similar results were obtained in repeated experiments for each patient.
DoseLevelPatient no.T2 unpulsed targetsaT2 MART-1 pulsed targetsa624-mela
300 Pre 390 1650 1260 
 Post  3300b 2025b 
300 Pre 160 35 
 Post  165 35 250b 
300 Pre 
 Post  
1000 Pre 168 342 210 
 Post  180 2880b 1800b 
1000 Pre 96 16 96 
 Post  215 23 670 
1000 Pre 117 170 
 Post  912b 468b 
2000 Pre 80 245 
 Post  10 118b 160 
2000 Pre 10 10 
 Post  
2000 Pre 
 Post  90 
2000 Pre 10 21 40 
 Post  416b 299b 
2000 Pre 11 15 10 
 Post  10 
2000 Pre 12 212 88 251 
 Post  233 1583b 908b 
2000 Pre 13 80 46 
 Post  53 24 50 
2000 Pre 14 2350 779 
 Post  205 1065 616 
2000 Pre 15 19 
 Post  
2000 Pre 16 1060 
 Post  865 
2000 Pre 17 
 Post  2580b 273b 
2000 Pre 18 52 21 336 
 Post  22 40 
2000 Pre 19 43 
 Post  442 688b 737b 
2000 Pre 20c 
 Post  10 50 
2000 Pre 21c 350 300 250 
 Post  450 90 230 
2000 Pre 22c 96 24 62 
 Post  50 163b 
a

Data shown are pg of IFN-γ secreted per 105 cells in 24 h.

b

Vaccine responses.

c

Patients who received CRL 1005 block copolymer.

Table 5

Immune response by ELISPOT to MART-1 vaccination

Effector cells were prepared as described in “Materials and Methods” by restimulation of PBMCs in the presence of MART-127–35 peptide-pulsed stimulator cells. After the second restimulation in vitro, 10,000 or 30,000 resulting effector cells were plated with 10,000 HLA-A2+ T2 cells, T2 cells pulsed with a HLA-A2 restricted FLU peptide, or T2 cells pulsed with the HLA-A2 restricted MART-127–35 peptide on nitrocellulose filters as described in “Materials and Methods,” and an ELISPOT assay was performed. The mean number of spots enumerated by a computerized digital imaging system that automatically counted spots per well in triplicate for each number of effectors per 10,000 targets is shown for paired samples pre- and postvaccine.
DoseLevelPatient no.No peptideaMART-1aFLUa
3 × 10e410e43 × 10e410e43 × 10e410e4
300 Pre 186 110 282 174 222 160 
 Post  230 145 220 155 223 133 
300 Pre 57 40 171 84 40 14 
 Post  122 53 303b 147b 76 52 
1000 Pre 90 60 111 103 138 89 
 Post  188 199 223b 157b 101 85 
1000 Pre 200 119 105 55 291 94 
 Post  210 172 85 76 446 211 
2000 Pre 94 47 268 92 219 78 
 Post  160 62 69 40 64 17 
2000 Pre 81 29 217 52 266 42 
 Post  137 28 12 13 
2000 Pre 69 42 197 67 NDc ND 
 Post  164 81 156 82 ND ND 
2000 Pre 10 79 65 78 114 221 138 
 Post  160 96 >500b 454b 105 77 
2000 Pre 11 82 88 88 86 84 57 
 Post  258 58 223b 157b 101 85 
2000 Pre 12 52 17 225 145 18 11 
 Post  40 287b 298b 50 23 
2000 Pre 13 98 41 230 92 127 157 
 Post  132 41 318b 202b 41 25 
2000 Pre 14 106 56 346 184 173 97 
 Post  207 117 391b 244b 129 64 
2000 Pre 15 95 61 253 149 227 106 
 Post  222 108 246b 322b 86 41 
2000 Pre 16 128 50 13 
 Post  54 10 296b 118b 11 
2000 Pre 17 103 66 248 192 152 103 
 Post  167 105 329 222 124 108 
2000 Pre 18 153 117 136 80 170 186 
 Post  191 179 306b 190b 249 190 
2000 Pre 19 34 85 34 43 20 
 Post  91 13 63 27 17 
2000 Pre 20d 59 74 122 56 137 58 
 Post  103 71 122 68 94 85 
2000 Pre 21d 64 94 141 82 111 72 
 Post  112 90 224b 149b 123 70 
2000 Pre 22d 146 97 197 96 307 96 
 Post  214 75 188 99 146 83 
Effector cells were prepared as described in “Materials and Methods” by restimulation of PBMCs in the presence of MART-127–35 peptide-pulsed stimulator cells. After the second restimulation in vitro, 10,000 or 30,000 resulting effector cells were plated with 10,000 HLA-A2+ T2 cells, T2 cells pulsed with a HLA-A2 restricted FLU peptide, or T2 cells pulsed with the HLA-A2 restricted MART-127–35 peptide on nitrocellulose filters as described in “Materials and Methods,” and an ELISPOT assay was performed. The mean number of spots enumerated by a computerized digital imaging system that automatically counted spots per well in triplicate for each number of effectors per 10,000 targets is shown for paired samples pre- and postvaccine.
DoseLevelPatient no.No peptideaMART-1aFLUa
3 × 10e410e43 × 10e410e43 × 10e410e4
300 Pre 186 110 282 174 222 160 
 Post  230 145 220 155 223 133 
300 Pre 57 40 171 84 40 14 
 Post  122 53 303b 147b 76 52 
1000 Pre 90 60 111 103 138 89 
 Post  188 199 223b 157b 101 85 
1000 Pre 200 119 105 55 291 94 
 Post  210 172 85 76 446 211 
2000 Pre 94 47 268 92 219 78 
 Post  160 62 69 40 64 17 
2000 Pre 81 29 217 52 266 42 
 Post  137 28 12 13 
2000 Pre 69 42 197 67 NDc ND 
 Post  164 81 156 82 ND ND 
2000 Pre 10 79 65 78 114 221 138 
 Post  160 96 >500b 454b 105 77 
2000 Pre 11 82 88 88 86 84 57 
 Post  258 58 223b 157b 101 85 
2000 Pre 12 52 17 225 145 18 11 
 Post  40 287b 298b 50 23 
2000 Pre 13 98 41 230 92 127 157 
 Post  132 41 318b 202b 41 25 
2000 Pre 14 106 56 346 184 173 97 
 Post  207 117 391b 244b 129 64 
2000 Pre 15 95 61 253 149 227 106 
 Post  222 108 246b 322b 86 41 
2000 Pre 16 128 50 13 
 Post  54 10 296b 118b 11 
2000 Pre 17 103 66 248 192 152 103 
 Post  167 105 329 222 124 108 
2000 Pre 18 153 117 136 80 170 186 
 Post  191 179 306b 190b 249 190 
2000 Pre 19 34 85 34 43 20 
 Post  91 13 63 27 17 
2000 Pre 20d 59 74 122 56 137 58 
 Post  103 71 122 68 94 85 
2000 Pre 21d 64 94 141 82 111 72 
 Post  112 90 224b 149b 123 70 
2000 Pre 22d 146 97 197 96 307 96 
 Post  214 75 188 99 146 83 
a

Data shown are number of spots counted by a digital imager per number of effector cells indicated at top.

b

Vaccine response.

c

ND, not done.

d

Patients who received CRL 1005 block copolymer.

We thank Drs. Mario Sznol, Jay Greenblatt, and Jan Morgan and the staff of the Cancer Therapy Evaluation Program for assistance with obtaining MART-127–35 peptide and IFA for the clinical trial described herein. Franco Marincola was generous with time for discussion of the manuscript and reagents. Kathy Pfeiffer rendered superb secretarial assistance.

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