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Clinical Trials |
University of Southern California/Norris Comprehensive Cancer Center and the Division of Medical Oncology, Department of Medicine, University of Southern California School of Medicine, Los Angeles, California 90033
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 
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 x 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 x g to pellet cells and debris, and frozen at -80°C until assays were done. IFN- was detected in supernatants using an antihuman IFN- 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- 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- 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- or the difference of post-vaccine minus pre-vaccine levels of IFN- released as continuous variables. Kaplan-Meier plots were constructed, and the log-rank test was used to calculate Ps.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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-
, 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.
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. 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.
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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.
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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- 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.
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after two restimulations with peptide-pulsed stimulators ex vivo. This assay did not directly measure antigen-specific IFN--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.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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; -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 V 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- 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- 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- 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- 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. 
2 To whom requests for reprints should be addressed, at USC/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Room 6428, Los Angeles, CA 90033. Phone: (323) 865-3919; Fax: (323) 865-0061; E-mail: jweber{at}hsc.usc.edu
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.
Received 12/31/98; revised 7/13/99; accepted 7/26/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin gene encodes a melanoma specific antigen recognized by tumor-infiltrating lymphocytes. J. Exp. Med., 183: 1185-1192, 1996.-chain variable region TCRBV14 in HLA-A2 matched primary human melanoma. Cancer Res., 55: 3374-3379, 1995. chain sequence used by an HLA-A2 restricted and melanocyte lineage-specific CTL clone. J. Exp. Med., 178: 1231-1248, 1993.This article has been cited by other articles:
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 M. Barve, J. Bender, N. Senzer, C. Cunningham, F. A. Greco, D. McCune, R. Steis, H. Khong, D. Richards, J. Stephenson, et al. Induction of Immune Responses and Clinical Efficacy in a Phase II Trial of IDM-2101, a 10-Epitope Cytotoxic T-Lymphocyte Vaccine, in Metastatic Non-Small-Cell Lung Cancer J. Clin. Oncol., September 20, 2008; 26(27): 4418 - 4425. [Abstract] [Full Text] [PDF]
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S. A. Rosenberg, R. M. Sherry, K. E. Morton, W. J. Scharfman, J. C. Yang, S. L. Topalian, R. E. Royal, U. Kammula, N. P. Restifo, M. S. Hughes, et al. Tumor Progression Can Occur despite the Induction of Very High Levels of Self/Tumor Antigen-Specific CD8+ T Cells in Patients with Melanoma J. Immunol., November 1, 2005; 175(9): 6169 - 6176. [Abstract] [Full Text] [PDF]
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Y.-P. Liao, C.-C. Wang, L. H. Butterfield, J. S. Economou, A. Ribas, W. S. Meng, K. S. Iwamoto, and W. H. McBride Ionizing Radiation Affects Human MART-1 Melanoma Antigen Processing and Presentation by Dendritic Cells J. Immunol., August 15, 2004; 173(4): 2462 - 2469. [Abstract] [Full Text] [PDF]
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G. J. Ullenhag, J.-E. Frodin, M. Jeddi-Tehrani, K. Strigard, E. Eriksson, A. Samanci, A. Choudhury, B. Nilsson, E. D. Rossmann, S. Mosolits, et al. Durable Carcinoembryonic Antigen (CEA)-Specific Humoral and Cellular Immune Responses in Colorectal Carcinoma Patients Vaccinated with Recombinant CEA and Granulocyte/Macrophage Colony-Stimulating Factor Clin. Cancer Res., May 15, 2004; 10(10): 3273 - 3281. [Abstract] [Full Text] [PDF]
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R. Soiffer, F. S. Hodi, F. Haluska, K. Jung, S. Gillessen, S. Singer, K. Tanabe, R. Duda, S. Mentzer, M. Jaklitsch, et al. Vaccination With Irradiated, Autologous Melanoma Cells Engineered to Secrete Granulocyte-Macrophage Colony-Stimulating Factor by Adenoviral-Mediated Gene Transfer Augments Antitumor Immunity in Patients With Metastatic Melanoma J. Clin. Oncol., September 1, 2003; 21(17): 3343 - 3350. [Abstract] [Full Text] [PDF]
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M. Ayyoub, A. Zippelius, M. J. Pittet, D. Rimoldi, D. Valmori, J.-C. Cerottini, P. Romero, F. Lejeune, D. Lienard, and D. E. Speiser Activation of Human Melanoma Reactive CD8+ T Cells by Vaccination with an Immunogenic Peptide Analog Derived from Melan-A/Melanoma Antigen Recognized by T Cells-1 Clin. Cancer Res., February 1, 2003; 9(2): 669 - 677. [Abstract] [Full Text] [PDF]
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V. Monsurro, D. Nagorsen, E. Wang, M. Provenzano, M. E. Dudley, S. A. Rosenberg, and F. M. Marincola Functional Heterogeneity of Vaccine-Induced CD8+ T Cells J. Immunol., June 1, 2002; 168(11): 5933 - 5942. [Abstract] [Full Text] [PDF]
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S. G. Schaed, V. M. Klimek, K. S. Panageas, C. M. Musselli, L. Butterworth, W.-J. Hwu, P. O. Livingston, L. Williams, J. J. Lewis, A. N. Houghton, et al. T-Cell Responses against Tyrosinase 368-376(370D) Peptide in HLA*A0201+ Melanoma Patients: Randomized Trial Comparing Incomplete Freund's Adjuvant, Granulocyte Macrophage Colony-stimulating Factor, and QS-21 as Immunological Adjuvants Clin. Cancer Res., May 1, 2002; 8(5): 967 - 972. [Abstract] [Full Text] [PDF]
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Y. Miyagi, N. Imai, T. Sasatomi, A. Yamada, T. Mine, K. Katagiri, M. Nakagawa, A. Muto, S. Okouchi, H. Isomoto, et al. Induction of Cellular Immune Responses to Tumor Cells and Peptides in Colorectal Cancer Patients by Vaccination with SART3 Peptides Clin. Cancer Res., December 1, 2001; 7(12): 3950 - 3962. [Abstract] [Full Text] [PDF]
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P. Lee, F. Wang, J. Kuniyoshi, V. Rubio, T. Stuges, S. Groshen, C. Gee, R. Lau, G. Jeffery, K. Margolin, et al. Effects of Interleukin-12 on the Immune Response to a Multipeptide Vaccine for Resected Metastatic Melanoma J. Clin. Oncol., September 15, 2001; 19(18): 3836 - 3847. [Abstract] [Full Text] [PDF]
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S. Weijzen, S. C. Meredith, M. P. Velders, A. G. Elmishad, H. Schreiber, and W. M. Kast Pharmacokinetic Differences Between a T Cell-Tolerizing and a T Cell-Activating Peptide J. Immunol., June 15, 2001; 166(12): 7151 - 7157. [Abstract] [Full Text] [PDF]
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T. Todo, R. L. Martuza, S. D. Rabkin, and P. A. Johnson Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing PNAS, May 22, 2001; 98(11): 6396 - 6401. [Abstract] [Full Text] [PDF]
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) 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.