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Journal of Clinical Oncology, Vol 19, Issue 18 (September), 2001: 3836-3847
© 2001 American Society for Clinical Oncology

Effects of Interleukin-12 on the Immune Response to a Multipeptide Vaccine for Resected Metastatic Melanoma

By P. Lee, F. Wang, J. Kuniyoshi, V. Rubio, T. Stuges, S. Groshen, C. Gee, R. Lau, G. Jeffery, K. Margolin, V. Marty, J. Weber

From the Departments of Medicine, Division of Medical Oncology, and Department of Preventive Medicine, Keck/University of Southern California School of Medicine, Los Angeles; Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford; and Department of Experimental Therapeutics, City of Hope National Medical Center, Duarte, CA.

Address reprint requests to Jeffrey Weber, MD, PhD, University of Southern California Norris Comprehensive Cancer Center, Rm 6428, 1441 Eastlake Ave, Los Angeles, CA 90089; email: jweber{at}hsc.usc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: Forty-eight patients with high-risk re-sected stage III or IV melanoma were immunized with two tumor antigen epitope peptides derived from gp100209-217(210M) (IMDQVPSFV) and tyrosin-ase368-376(370D) (YMDGTMSQV) emulsified with incomplete Freund’s adjuvant (IFA). Patients received peptides/IFA with or without interleukin (IL)-12 30 ng/kg to evaluate the toxicities and immune responses in either arm with time to relapse and survival as secondary end points.

PATIENTS AND METHODS: Immunizations were administered every 2 weeks for 8 weeks, then every 4 weeks for 12 weeks, and then once 8 weeks later. A leukapheresis to obtain peripheral-blood mononuclear cells for immune analyses was done before and after vaccination. Skin testing with peptides and recall reagents was performed before and after vaccinations.

RESULTS: Local pain and granuloma formation, fever, and lethargy of grade 1 or 2 were observed. Transient vaccine-related grade 3—but no grade 4—toxicity was observed. Thirty-four of 40 patients developed a positive skin test response to the gp100 peptide but none to tyrosinase. Immune responses were measured by release of gamma-interferon in an enzyme-linked immunosorbent assay (ELISA) by effector cells in the presence of peptide-pulsed antigen-presenting cells or by an antigen-specific tetramer flow cytometry assay. Thirty-three of 38 patients demonstrated an immune response by ELISA after vaccination, as did 37 of 42 patients by tetramer assay. Twenty-four of 48 patients relapsed with a median follow-up of 20 months, and 10 patients in this high-risk group have died.

CONCLUSION: These data suggest a significant proportion of patients with resected melanoma mount an antigen-specific immune response against a peptide vaccine and indicate that IL-12 may increase the immune response and supporting further development of IL-12 as a vaccine adjuvant.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EUKARYOTIC CELLS express thousands of proteins which are degraded intracellularly into peptides via the multisubunit proteasome, enter the endoplasmic reticulum via the adenosine triphosphate–binding cassette transporter for antigen processing, and are subsequently presented on the surface of antigen-presenting cells and other cells as antigens for recognition by T cells complexed with class I and II major histocompatibility complex (MHC) molecules.1-3 The generation and detection of tumor antigen-specific immune responses in humans have been enormously simplified by the discovery that peptide/MHC complexes are the ultimate target for T cells.4,5 Clinical trials of peptide vaccines with and without adjuvants in patients with metastatic and resected cancer have been facilitated by the identification of T-cell epitopes from several classes of tumor-associated and tumor-specific antigens on melanomas and breast, gastrointestinal, cervix, and lung cancers recognized by CD8+ lymphocytes in association with frequently expressed HLA class I alleles.6-8

Several groups have defined members of a group of melanoma differentiation antigens that comprise melanosome-related "neoantigens" derived from gene products produced in normal cells. Antigen pMel17/gp100, a transmembrane glycoprotein of 100 kd, was defined via recognition by cytolytic T-cell (CTL) clones from melanoma patient peripheral blood and by tumor-infiltrating lymphocytes (TILs).9-11 Multiple peptides derived from gp100 that fit the consensus motif for binding to HLA-A2 antigen were recognized by TILs from melanoma patients, including gp100 209 to 217 (ILDQVPSFV), gp100 154 to 162 (TKTWGQYWQV), as well as gp100 457 to 466 (LLDGTAATLRL).12 TILs specific for gp100 have been reported to induce regression of metastatic melanoma lesions in patients receiving TIL and interleukin (IL)-2 therapy,13 which indicates that gp100 may be a promising target for immunotherapy using peptide vaccination strategies.

Tyrosinase is a membrane-bound protein involved in the melanin synthesis pathway that is expressed by virtually all primary melanoma lesions and by up to 90% of metastatic lesions.14 It encodes several epitope peptides that are presented by HLA-A2 antigen to CTLs reactive with human melanoma cells.15 A peptide derived from tyrosinase, amino acid 368 to 376, YMNGTMSQV, was shown to be posttranslationally modified by deamidation of asparagine to aspartic acid, resulting in a sequence recognized by human CTLs, YMDGTMSQV, known as tyrosinase 368 to 376 (370D).16

A number of small pilot studies have been conducted in which patients with metastatic melanoma received multiple subcutaneous injections of a single tumor antigen peptide emulsified with incomplete Freund’s adjuvant (IFA) at 3-week intervals. MART-1 27 to 35, gp100 209 to 217, gp100 154 to 162, and gp100 280 to 288 as well as MAGE-3 peptides have been used in these trials.17-22 A variety of cytokine adjuvants have been tested in melanoma peptide vaccine trials to boost immune responses and overcome likely immune suppressive influences in tumor-bearing patients. Jaeger et al23 showed enhanced immune responses to peptides encoded by melanoma-differentiation antigens after use of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) in three patients, and all three had objective clinical responses. In contrast, Rosenberg et al24 did not find clinical antitumor responses and in fact observed decreased levels of T-cell precursors after GM-CSF and after IL-12 administration in conjunction with the gp100209-217(210M) peptide/IFA.

In the current trial, the hypothesis that a low-dose of IL-12 at the injection site would be capable of boosting peptide-specific immune CTL responses as was shown in murine experiments was tested in a small randomized trial of melanoma peptides/IFA with or without IL-12. The primary end points were immunologic, and relapse-free and overall survival were secondary end points.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Trial Eligibility
All patients had resected stage III or IV melanoma by the 1988 modified American Joint Commission on Cancer staging system and were made free of disease surgically. They were required to have a magnetic resonance imaging or computed tomographic scan of the head and computed tomographic scans of the chest, abdomen, and pelvis showing no indication of disease within 4 weeks of therapy to verify that they were clinically free of melanoma. Eligibility criteria included age 18 or greater, creatinine of less than 2.0 mg/dL, bilirubin of less than 2.0 mg/dL, platelet count of 100,000/µL or more, hemoglobin of 9 g/dL or more, and total WBC of 3,000/µL or greater. Human immunodeficiency virus, hepatitis C antibody, and hepatitis B surface antigen were required to be negative, and all patients were HLA-A2 antigen–positive by a microcytotoxicity assay. All patients were required to comprehend and sign an informed consent form approved by the National Cancer Institute (NCI) and the Los Angeles County/University of Southern California Institutional Review Board.

Clinical-Grade Peptides
The peptide vaccine was administered as outpatient therapy. Peptides gp100209-217(210M) and tyrosinase368-376(370D) were produced by solid-phase synthesis using 9-fluoenylmethoxy carbonyl chemistry and reverse-phase high-pressure liquid-phase chromatography purification and supplied by Ben Venue Laboratories, Inc (Bedford, OH). The chemical identity was verified by mass spectrometry, and the finished injectable dosage form was manufactured by the Monoclonal Antibody/Recombinant Protein Production Facility, NCI (NCI/SMARP), Frederick, MD. Peptide was provided by the Cancer Therapy Evaluation Program of the NCI, in Bethesda, MD under investigational new drug application BB 6123 held by the NCI. The vials of peptide contained no preservative.

The tyrosinase368-376(370D) peptide (National Services Center no. 699048) and gp100209-217(210M) peptide (National Services Center no. 68347) are HLA-A2–restricted, nine–amino acid epitope peptides and have the amino acid sequences YMDGTMSQV and IMDQVPFSV, respectively. The peptides were supplied in vials containing 1 mL of a sterile 1-mg/mL solution for injection with 0.1 N HCl added to adjust the pH.

Adjuvant
Montanide ISA-51 (also known as IFA) was manufactured by Seppic, Inc (Franklin Lakes, NJ), and supplied in glass ampoules containing 3 mL of sterile adjuvant solution without preservative.

Vaccine Preparation and Administration
One milliliter of gp100209-217(210M) or tyrosinase368-376(370D) peptide with sterile saline was added in a 1:1 volume to Montanide ISA 51 then mixed in a Vortex mixer (Fisher, Inc, Alameda, CA) for 10 minutes at room temperature. The resulting emulsion was injected deeply subcutaneously in the lateral thigh in a volume of 2 mL using a glass syringe. Subcutaneous 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 eight injections which were done over 26 weeks. The intervals between injections were 2 weeks for the first four injections, 4 weeks for the next three injections, and 8 weeks between the seventh and eighth injections. Forty-eight patients had a leukapheresis with an exchange of approximately 5 L of blood volume performed within 2 weeks before beginning vaccinations, and 38 patients had a pheresis within 3 weeks after the final vaccination to collect peripheral-blood mononuclear cells (PBMCs), which were frozen for future analysis. Four patients could not have a pheresis performed due to poor venous access, and six progressed before their pheresis could be performed. Skin tests were performed using 50 µg of the gp100209-217(210M) or tyrosinase368-376(370D) peptide injected intradermally in a volume of 100 µL using a tuberculin syringe and a 27-gauge needle.25,26 Candida extract, mumps, and trichophyton provided a positive control and saline was a negative control for assessment of delayed-type hypersensitivity (DTH). At least 5 mm of induration or erythema read 48 hours after intradermal injection was required to score a gp100 or tyrosinase skin test as positive.

IL-12
Recombinant human (rhu) IL-12 was produced by recombinant DNA techniques in Escherichia coli to Good Manufacturing Practices grade and was obtained from Genetics Institute (Cambridge, MA). It is a heterodimeric 70-kd glycoprotein of 503 amino acids composed of unrelated disulfide-linked subunits of 35 kd (p35) and 40 kd (p40). Co-expression of the two subunits is necessary for efficient production of a biologically active molecule. The rhuIL-12 was supplied as a lyophilized powder in 5-mL vials containing 50 µg of drug. The rhuIL-12 was administered intradermally at a dose of 30 ng/kg with a 1-mL syringe and 27-gauge needle split evenly at each peptide/IFA injection site.

Screening for Vitiligo and Eye Changes
All patients underwent a complete skin examination before therapy and at each visit for vaccination to screen for vitiligo. Slit lamp examinations and iris photos were performed by an ophthalmologist before the start of therapy in all patients, and retinal and iris examinations with a hand-held ophthalmoscope were performed at each vaccination visit to assess ocular toxicity. No patient had evidence of ocular toxicity, and only one patient who received peptides/IFA alone developed vitiligo.

Preparation of PBMC Specimens
Pheresis samples were processed to purify PBMCs by sedimentation on a Ficoll-Hypaque cushion (Pharmacia, Alameda, CA) and extensive washing in Hanks’ balanced salt solution. Cells were frozen in 40% human AB serum (Gemini Bioproducts, Calabasas, CA), 50% RPMI 1640 (GIBCO, Grand Island, NY), and 10% dimethyl sulfoxide (Sigma, St Louis, MO) and stored in a liquid nitrogen freezer at -168°C until use.

Peptides
Peptides used for in vitro studies were synthesized at the University of Southern California 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 gp100209-217(210M), gp100209-217(WT), tyrosinase368-376(370D), or FLU-MI peptide-pulsed dendritic cells that were irradiated with 6,000 rad at a 1:3 ratio in wells of a 24-well plate (Corning, Oneonta, NY). Cells were plated in Iscove’s minimal essential 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 to 4 days. After 10 days, the T cells were harvested for a 10-day assay or restimulated with thawed autologous PBMCs pulsed with 10 µg/mL of the above peptides at 37°C for 2 hours and irradiated with 3,000 rad. IL-2 was again added 48 hours later at 50 IU/mL. T cells were restimulated with peptide-pulsed PBMCs for another 7 days and were then harvested for day 17 immune assays. For the cytokine release assay, 105 peptide-stimulated T cells were harvested at day 10 or 17 and incubated with 105 T2 cells pulsed with 10 µg/mL gp100209-217(210M), tyrosinase368-376(370D), or FLU-MI peptide or 624 mel cells as targets in a total volume of 1 mL of RPMI medium without serum for 18 hours 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 performed. Gamma-interferon was detected in supernatants using an antihuman gamma-interferon Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems, Minneapolis, MN).

Tetramer Assays
The tetramers containing the gp100209-217(210M) and tyrosinase368-376(370D) peptides were produced following the approach of Altman et al27 and Lee et al.28 Briefly, the extracellular domain on the HLA-A 02*01 heavy chain was fused to a biotinylation site and cloned into an expression plasmid. Full-length human beta-2 microglobulin was cloned into an expression vector, and recombinant proteins were obtained after induction of bacteria with isopropyl-beta-D-thiogalactopyranoside and further purified. Insoluble HLA-A 0201 and beta-2 microglobulin were dissolved in 8 M urea and refolded in the presence of 100 µg/mL melanoma peptides purified by high-pressure liquid-phase chromatography with protease inhibitors. The product was purified by gel filtration and in vitro biotinylated for 1 hour at 37°C in the presence of 15 µg of Birk (Avidity, Boulder, CO), 80 µmol/L biotin, 10 mmol/L adenosine triphosphate, 10 mmol/L MgOAc, 20 mmol/L bicine, and 10 mmol/L Tris HCl (pH 8.3). To remove free biotin, monomeric complexes were again purified by gel filtration, tested for biotinylation, and tetramerized by addition of phycoerythrin (PE)-labeled streptavidin (Molecular Probes, Eugene, OR) at a 4:1 ratio. Tetramers were purified by gel filtration and stored at 5 mg/mL at 4°C. Tetrameric assessment of CTLs was accomplished by three-color staining using fluorescein isothiocyanate–labeled anti-CD8, peridinin chlorophyll protein–labeled anti-CD69, and PE-labeled melanoma peptide or irrelevant control tetramer. One million CD8+ and CD69+ cells were gated and used for PE labeling with tetramers using a FACSstar (Becton Dickinson, Mountain View, CA). The proportion of CD8+ cells that stained with tetramer was measured before and after vaccination.

Immunohistochemical Staining for gp100 and Tyrosinase
The T311 antibody for tyrosinase (Novocastra Laboratories, Newcastle-on-Tyne, United Kingdom) and HMB-45 antibody for gp100 (Ventana Medical Systems, Tucson, AZ) were used for immunohistochemical staining of paraffin-embedded sections on glass slides by the Vectastain technique (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Appropriate negative and positive control sections were included with each assay.

Statistics
The association between postvaccine ELISA cytokine release or tetramer staining and time to relapse was calculated using the postvaccine level of gamma-interferon or tetramer staining as well as the difference of postvaccine minus prevaccine levels of gamma-interferon released as continuous variables. Kaplan-Meier plots were constructed and the log-rank test was used to calculate P values. Values for cytokine release and for the products of the largest perpendicular dimensions of the DTH erythema site were expressed as geometric means with 95% confidence intervals (CIs).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Demographics
Forty-eight patients with stage III or IV resected melanoma were treated on this trial. The demographic details of this high-risk group are listed in Table 1. The median age of the 24 men and 24 women was 50 years. Twenty-six patients had resected stage III disease, eight with lymph node or subcutaneous recurrences after adjuvant alpha-interferon therapy, and 22 had resected stage IV disease. Forty-four had cutaneous melanoma, and four had ocular melanoma. The median time since diagnosis of the primary lesion for the whole group was 2.5 years, and time since diagnosis of primary melanoma was 21.5 months. In 13 of the patients, previous alpha-interferon therapy had failed, and in five patients, a cellular vaccine had failed. Six patients did not undergo pheresis because of disease recurrence before they finished the series of vaccinations, and four could not be pheresed because of inadequate venous access. This left 38 patients with leukapheresis samples collected for evaluation both before and after vaccination. All four patients who could not have a pheresis due to venous access problems had 80 mL of whole blood collected for use in tetramer analyses (described below).


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  		Table 1.  Demographics Tyrosinase/gp100 With or Without IL-12
 
Toxicities
The overall toxicities of the gp100209-217(210M)/tyrosinase368-376(370D) vaccine for all 48 patients are listed in Table 2. The vaccine with or without IL-12 was generally well tolerated, but most patients (42 of 48) had grade 1 or 2 local tenderness, granuloma, and pain at the injection sites. Thirty-three patients developed granulomata at the injection sites, although none needed to undergo resection because of symptoms; small ulcerations that healed spontaneously developed in three patients, two of whom were in the peptides/IFA group. The only grade 3 toxicities believed to be vaccine-related were local pain requiring acetaminophen with codeine in one patient in the peptides/IFA arm and nausea in another patient; local pain requiring acetaminophen with codeine or hydrocodone and acetaminophen occurred in two patients, and headache requiring hydrocodone and acetaminophen occurred in another patient; all three patients were in the peptides/IFA/IL-12 group. Nausea was observed in nine patients, and transient fevers (invariable low grade) were seen in 16 patients. Sixteen patients experienced headaches, and 19 had arthralgia. No appreciable differences in overall toxicity were noted between the two arms of the trial, as indicated in Table 2. Patients were screened for vitiligo and ocular toxicity as indicated in Patients and Methods, and vitiligo was observed in only one patient who did not receive IL-12. In conclusion, the toxicity of the gp100209-217(210M)/tyrosinase368-376(370D)/IFA vaccine with or without IL-12 was modest, transient, and not related to the administration of IL-12


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  		Table 2.  Tyrosinase/gp100 With or Without IL-12: Toxicities
 
DTH Skin Test Results
Skin test reactivity to recall antigens (Candida albicans or mumps) was assessed before the first and after the final vaccine. Only three of 48 patients were anergic to recall antigens before vaccination. DTH skin test results for Candida (Fig 1A) were measured as the product of the perpendicular diameters of erythema (geometric means with 95% CIs are shown). There were no significant differences between pre- and postvaccine readings for the non–IL-12 group (164 mm2 [95% CI, 113 to 240 mm2] v 309 mm2 [95% CI, 195 to 487 mm2]) and for the IL-12 group (80 mm2 (95% CI, 245 to 140 mm2] v 338 mm2 [95% CI, 224 to 511 mm2]) (P = .88), although the IL-12 group seemed to have a greater increase in reactivity (P = .012). DTH to the gp100209-217(210M)/tyrosinase368-376(370D) peptides was also assessed by intradermal skin testing, and two of 48 patients reacted to the gp100209-217(210M) peptide before vaccination. Forty-two patients were tested after vaccination for DTH to the gp100209-217(210M) and tyrosinase368-376(370D) peptides, and 36 of 42 (or 85%) were positive for gp100 reactivity, ie, they developed gp100 antigen-specific DTH reactivity after vaccination (defined as at least 5 mm of induration and/or erythema), including two patients who were anergic to the panel of recall antigens before and after vaccination. In the group that received IL-12 plus peptides/IFA, there was a significantly greater gp100-specific DTH compared with the group that did not receive IL-12. Values for gp100-specific erythema before and after vaccination are indicated in Fig 1B, with a mean 30 mm2 (95% CI, 17 to 55 mm2) for peptides/IFA versus 156 mm2 (95% CI, 97 to 248 mm2) for peptides/IFA/IL-12 (P = .039). Interestingly, no DTH reactivity to the tyrosinase368-376(370D) peptide was seen before or after vaccination, suggesting that it was not strongly immunogenic. As shown below, 32 of 34 patients with an immune response by the 10-day gamma-interferon ELISA assay also had a positive result with the gp100209-217(210M) skin test.



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  		Fig 1. DTH to (A) Candida albicans and (B) gp100209-217(210M). The product of the diameters of erythema measured with calipers 48 hours after intradermal injection of 100 µg of skin test reagent is shown on the ordinate as a bar graph, before and after vaccination.

 
Cytokine Release Immune Assays in gp100/Tyrosinase–Vaccinated Patients
Peptide-specific immunity was measured in 38 patients who had pre- and postvaccination pheresis samples available by measuring antigen-specific release of gamma-interferon by ELISA from effector cells restimulated once (analyzed at day 10) or twice (analyzed at day 17) with peptide-pulsed irradiated PBMC stimulators. Effectors were incubated for 18 hours with control HLA-A2 antigen-positive T2 cells, gp100209-217(210M), gp100209-217(WT), or tyrosinase368-376(370D) peptide-pulsed T2 cells or 624-mel, a HLA-A2 antigen-positive gp100/tyrosinase-positive melanoma cell line, as described in Patients and Methods. A total of 34 of 38 patients who received or did not receive IL-12 showed evidence of increased reactivity to gp100209-217(210M), gp100209-217(WT), and tyrosinase368-376(370D) peptide-pulsed T2 targets with release of gamma-interferon secreted per 105 cells/mL that ranged from 100 to 19,000 pg/mL. The cytokine release had to be at least 100 pg/mL above the T2 unpulsed control to be scored as positive; this value represented two SDs from the mean of the T2 unpulsed controls. The results of pre- and postvaccine cytokine release assays to gp100209-217(210M) for those 38 patients are shown in Fig 2. After one restimulation at day 10, mean (geometric) gp100209-217(210M)–specific cytokine release for the peptides/IFA patients was 15 pg/mL/24 hours (95% CI, 7 to 31 pg/mL/24 hours) before vaccination and 816 pg/mL/24 hours (95% CI, 583 to 1,024 pg/mL/24 hours) after vaccination, as shown in the left bar graph with error bars. For the peptides/IFA/IL-12 group, mean gp100209-217(210M)–specific cytokine release was 44 pg/mL/24 hours (95% CI, 21 to 90 pg/mL/24 hours) before vaccination and 2,171 pg/mL/24 hours (95% CI, 1,751 to 2,691 pg/mL/24 hours) after vaccination, as shown in the right bar graph. There was a significant increase in the reactivity after vaccination in both groups compared with before vaccination (both P = .001), and for the peptides/IFA/IL-12 group compared with the peptides/IFA group (P = .020). For tyrosinase, the corresponding figures for gamma-interferon release were 20 pg/mL/24 hours (95% CI, 9 to 43 pg/mL/24 hours) before vaccination and 349 pg/mL/24 hours (95% CI, 210 to 581 pg/mL/24 hours) after vaccination in the peptides/IFA group. In the peptides/IFA/IL-12 group, it was 40 pg/mL/24 hours (95% CI, 20 to 83 pg/mL/24 hours) before vaccination, and 283 pg/mL/24 hours (95% CI, 166 to 484 pg/mL/24 hours) after vaccination. There was a significant difference between the pre- and postvaccine values for both groups (P < .02), but there were no differences between the peptides/IFA and peptides/IFA/IL-12 groups (P = .78). Only four patients, including two responders by ELISA, had detectable gp100 reactivity before vaccination.



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  		Fig 2. Cytokine release assay shows T-cell immune response to gp100209-217(210M) after 10 days, before and after vaccination. The release of gamma-interferon at 18 hours stimulated with peptide-pulsed stimulators at a 1:1 ratio is shown.

 
All of the cytokine release assays were repeated at day 17, with similar results for gp100209-217(210M) (data not shown). After two restimulations at day 17, mean (geometric) gp100209-217(210M)–specific cytokine release for the peptides/IFA patients was 40 pg/mL/24 hours (95% CI, 17 to 95 pg/mL/24 hours) before vaccination and 725 pg/mL/24 hours (95% CI, 352 to 1,491 pg/mL/24 hours) after vaccination. For the peptides/IFA/IL-12 group, mean (geometric) gp100209-217(210M)–specific cytokine release was 90 pg/mL/24 hours (95% CI, 45 to 182 pg/mL/24 hours) before vaccination and 2,532 (95% CI, 1,988 to 3,224 pg/mL/24 hours) after vaccination. Postvaccine values in both groups were significantly higher than prevaccine values (P = .001 for both), although gp100-specific cytokine release was not significantly higher in the peptides/IFA/IL-12 group than in the peptides/IFA group (P = .11), in contrast to the 10-day assays.

For tyrosinase at day 17, the corresponding figures were 74 pg/mL/24 hours (95% CI, 38 to 145 pg/mL/24 hours) before vaccination and 316 pg/mL/24 hours (95% CI, 160 to 621 pg/mL/24 hours) after vaccination in the peptides/IFA group and 24 pg/mL/24 hours (95% CI, 10 to 58 pg/mL/24 hours) before vaccination and 776 pg/mL/24 hours (95% CI, 478 to 1,261 pg/mL/24 hours) after vaccination in the peptides/IFA/IL-12 group (P = .30; no significant difference between the post–IL-12 and no IL-12 groups, but P = .01 for both groups before compared with after vaccination), as shown in the dot plot in Fig 3. In contrast to the tyrosinase-specific assays at day 10, multiple restimulations at day 17 revealed an increase in reactivity to the weak immunogen tyrosinase in the peptides/IFA/IL-12 group compared with the group receiving peptides/IFA alone, although it did not quite reach statistical significance.



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  		Fig 3. Cytokine release assay shows T-cell immune response to tyrosinase after 17 days, before and after vaccination. The release of gamma-interferon at 18 hours stimulated with peptide-pulsed stimulators at a 1:1 ratio is shown as a dot plot.

 
For cytokine release assays, background release of unpulsed targets incubated with effectors ranged from 0% to 60% of gp100209-217(210M)/tyrosinase368-376(370D) peptide-pulsed targets incubated with effectors. The observation that reactivity was seen with gp100209-217(210M)/gp100209-217(WT)/tyrosinase368-376(370D) peptide-pulsed T2 cell targets as well as the HLA-A2 antigen–positive gp100209-217(210M)/tyrosinase368-376(370D)–expressing cell line 624-mel (data not shown) suggested that the increased immune effectors detected in the peripheral blood could recognize naturally processed gp100209-217(WT)/tyrosinase368-376(370D) peptide on the surface of that tumor cell line. The gp100209-217(210M)–specific cytokine release at day 10 was higher in the IL-12 group by greater than a factor of two, and the difference was statistically significant.

To verify that increased gp100209-217(210M)/tyrosinase368-376(370D)–specific reactivity measured by specific cytokine release after vaccination represented antigen-specific lytic activity by PBMC effector cells, a patient with a high degree of cytokine release was chosen, and the thawed PBMCs were used in a chromium release assay with T2 cells pulsed with gp100 and tyrosinase peptides as well as 624 mel cells as targets. In a chromium release assay, 25% antigen-specific lysis was seen at an effector-to-target ratio of 50:1 with unstimulated effectors from fresh PBMC after vaccination in multiple assays (data not shown), compared with activity before vaccination, confirming that postvaccine cytokine release was a surrogate marker for lytic activity.

In the experiments described in Figs 2 and 3, the presence of an HLA-A2 antigen– restricted influenza virus matrix protein response was monitored as a positive control for immune integrity in pre- and postvaccine cytokine release assays. The HLA-A2 antigen–restricted FLU peptide was pulsed onto irradiated PBMC stimulators in parallel with the cytokine release assays and then used as effectors in a cytokine release assay with T2 target cells pulsed with FLU peptide. Significant FLU-specific cytokine release was expected for FLU-stimulated effector cells both before and after vaccination for all patients, and equivalent FLU-specific cytokine release was observed both before and after vaccination in 36 of 38 patients. FLU-specific cytokine release for the peptides/IFA patients was 524 pg/mL/24 hours (95% CI, 305 to 900 pg/mL/24 hours) before vaccination and 634 pg/mL/24 hours (95% CI, 355 to 1131 pg/mL/24 hours) after vaccination (P = .5). For the peptides/IFA/IL-12 patients, FLU-specific cytokine release was 675 pg/mL/24 hours (95% CI, 381 to 1,196 pg/mL/24 hours) before vaccination and 1,045 pg/mL/24 hours (95% CI, 813 to 1,343 pg/mL/24 hours) after vaccination (P = .2), as indicated in Fig 4. These results are in contrast to the significant increase in gp100209-217(210M)–specific reactivity that was seen only after vaccination, as shown in Fig 2.



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  		Fig 4. Cytokine release assay shows T-cell immune response to FLU-M1 after 10 days, before and after vaccination. The release of gamma-interferon at 18 hours stimulated with peptide-pulsed stimulators at a 1:1 ratio is shown.

 
Cytokine Release to Wild-Type gp100 209 to 217
All assays of cytokine release described above were performed using both gp100-modified 209 to 217 (210M) and wild-type peptide 209 to 217 (WT)-pulsed T2 targets. For the peptides/IFA group, mean (geometric) gp100 (WT)-specific cytokine release was 22 pg/mL/24 hours (95% CI, 10 to 47 pg/mL/24 hours) before vaccination and 569 pg/mL/24 hours (95% CI, 333 to 971 pg/mL/24 hours) after vaccination. For the peptides/IFA/IL-12 group, mean (geometric) gp100 (WT)-specific cytokine release was 14 pg/mL/24 hours (95% CI, 5 to 35 pg/mL/24 hours) before vaccination and 1,707 pg/mL/24 hours (95% CI, 1,315 to 2,217 pg/mL/24 hours) after vaccination. The results of the 10-day stimulations with gp100209-217(WT) before and after vaccination for the peptides/IFA group (Fig 5, left bars) and the peptides/IFA/IL-12 group (Fig 5, right bars) indicate that there was a similar increase in cytokine release for both groups after vaccination as well as an augmentation with the addition of IL-12 to the peptides/IFA treatment (significant difference between before v after vaccination for both groups, P = .001, and borderline difference between the peptides/IFA and peptides/IFA/IL-12 groups, P = .07). These data indicate that the naturally expressed epitope as well as the substituted peptide used in the vaccine could be recognized on target cells by PBMC effectors.



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  		Fig 5. Cytokine release assay shows T-cell immune response to gp100209-217(WT) before and after vaccination. The release of gamma-interferon at 18 hours stimulated with peptide-pulsed stimulators at a 1:1 ratio is shown.

 
Tetramer Immune Assays in gp100/Tyrosinase-Vaccinated Patients
Flow cytometry assays using tetramers constructed with the gp100209-217(210M) and tyrosinase368-376(370D) peptides were performed to enumerate antigen-specific T cells from fresh blood samples. Positive cells were defined as gated fresh CD8+ CD3+ cells staining with PE-labeled peptide tetramer. The limit of detection was defined as 0.01% or 1:10,000 CD8+ cells. With this limit of detection, there was no evidence of any pre-existing gp100- or tyrosinase-specific immune cells before vaccination in any of the 42 patients analyzed. Of the 42 patients analyzed, 37 had greater than 0.01% tetramer-positive cells after vaccination. The level of positivity ranged from 0.01% to 2.5%. For fresh CD8+ cells, the mean (geometric) gp100-specific tetramer staining after vaccination was 0.03% for the peptides/IFA patients, compared with less than 0.01% before vaccination. For the peptides/IFA/IL-12 group, the mean (geometric) gp100-specific CD8+ staining after vaccination was 0.08%, compared with less than 0.01% before vaccination. The data comparing the level of staining in the peptides/IFA and the peptides/IFA/IL-12 group are plotted in Fig 6 as a bar graph with error bars showing that there was a three-fold but borderline significant increase in the mean (geometric) staining (P = .08). Minimal increases were seen above background after vaccination for staining with the tyrosinase tetramers in six patients, four who received IL-12 and two who did not (data not shown), indicating again that it was a weak immunogen. The results for gp100 tetramer staining and 10-day cytokine release assays were correlated with a calculated coefficient of correlation r = .46, which indicated that a significant degree of correlation existed between the two assays.



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  		Fig 6. Tetramer assay on fresh blood shows T-cell response to gp100209-217(210M) before and after vaccination. The percentage of tetramer-positive cells by flow cytometry is shown. Cell preparation and flow cytometry are described in Patients and Methods.

 
Tetramer Kinetic Assays in gp100/Tyrosinase-Vaccinated Patients
Six patients had blood drawn at multiple time points during the 6-month duration of vaccination, three who received IL-12 and three who did not. For each sample, a gp100209-217(210M) tetramer flow cytometry assay was performed to enumerate gp100-specific T cells in fresh blood. These data are plotted in Fig 7 and indicate that significant numbers of antigen-specific cells in fresh blood above 1:10,000 were not detected until after the third month or the fifth of eight vaccines, supporting the notion that repeated vaccination with tumor antigen peptide with adjuvant is necessary to generate high levels of immunity in patients with resected melanoma.



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  		Fig 7. Kinetic analysis of tetramer assays on fresh blood shows T-cell immune response to gp100209-217(210M) over time. Percentage of tetramer-positive cells by flow cytometry is shown for patients from the no IL-12 (n = 3) and IL-12 (n = 3) groups. Each line represents one patient (solid line, IL-12 treatment; dashed line, no IL-12).

 
Tetramer Plots in gp100/Tyrosinase-Vaccinated Patients
The actual flow cytometry plots from selected vaccine patients before and after vaccination are shown in Fig 8A. Infrequent events consisting of as few as 0.02% tetramer-positive circulating CD8+ cells can be seen in the upper right quadrant of the plot. Figure 8B shows the flow cytometry profiles from a patient whose gp100 tetramer-positive responses are displayed in Fig 7.



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  		Fig 8. Representative FACS plots of G209-2M–specific T cells. (A) Pre- and postvaccine samples from two representative patients from the no IL-12 and IL-12 arms. (B) Serial samples from one representative patient from the IL-12 arm demonstrate a gradual, consistent increase in G209-2M–specific T cells after each vaccination.

 
Expression of gp100 on Tumors From Relapsed Patients After Vaccine Treatment
A total of 24 of 48 patients had relapsed by a median follow-up of 20 months. Eight patients relapsed during the treatment regimen, and 16 relapsed after finishing the 6-month vaccination regimen. Sixteen samples were available for immunohistochemical staining for gp100 with the HMB-45 antibody, as well as for tyrosinase. In 15 cases, detectable HMB-45 positivity was observed, ranging from 10% to 70% positivity; one test result was negative. These data suggest that little or no immunoselection directed against the gp100 antigen took place after vaccination, or that antigen-positive clones had grown after vaccination at a time when the level of immune reactivity had declined to low or ineffective levels. These data did not confirm prior results from other groups which suggested that antigen loss induced by immunization was a common mechanism for generation of escape variants from vaccine control.29-31

Clinical Results
The patients on this trial were followed for a median of 24 months since initiation of treatment. Twenty-four patients have relapsed and 10 have died. Median survival has not been reached; the median time to relapse was 20 months. Of the 24 patients who relapsed, five have been rendered free of disease by surgery and have no evidence of disease. The time-to-relapse curve of the IL-12 group is identical to that of the peptides/IFA group, as shown in Fig 9.



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  		Fig 9. Time-to-relapse survival curve of all patients who received either peptides/IFA or peptides/IFA/IL-12.

 
A key question is whether the immunologic assays correlated with any clinical parameters, so correlation was drawn between gp100 DTH results, 10-day (one restimulation) and 17-day (two restimulation) ELISA, percent tetramer staining, and time to relapse. The Cox proportional hazards model was used to analyze the association between recurrence and the following variables: gp100 DTH erythema product (postvaccination), gp100 210M ELISA gamma-interferon release at days 10 and 17 (postvaccination), and gp100 tetramer percentage (postvaccination). No significant association was found. Further analysis by Kaplan-Meier plots and log-rank tests (comparing high v low groups, as defined by the median) corroborated the conclusion that there is no significant association.

Relapse-free and overall survival for the group of 48 patients at 20 months’ median time to relapse compares favorable for any group of similar stage III or IV patients analyzed in vaccine trials,32 although the small numbers and noncontrolled nature of this pilot study preclude any further conclusions about the clinical efficacy of the peptide vaccine with or without IL-12.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-12 is a 70-kd heterodimeric cytokine that is a growth factor for activated T and natural killer cells. The action of this enhancer of T and natural killer cytolytic activity results in gamma-interferon production by those cells.33-36 IL-12 also shifts the differentiation of T helper cells toward Th1 and away from the Th2 phenotype.37 It plays a pivotal role in cell-mediated and humoral immunity and has been shown to be an effective adjuvant for tumor peptide and tumor cDNA vaccines in mice.38-40 IL-12–transfected tumor cells have been shown to be more immunogenic than nontransfected tumor cells in mice.41,42 Moreover, IL-12 induces upregulation of the B7-1 molecule on dendritic cells, inhibits anergy induction, and reverses unresponsiveness to tumor peptides. Co-administration of IL-12 resulted in strong proliferative responses by CD8+ T cells and development of lytic effector function.43 Recent studies indicated that IL-12 acted as a third signal, along with TcR and costimulatory molecules, to reverse antigen-induced tolerance and expand antigen-specific CD8+ T cells.44-46 IL-12 may further activate dendritic cells in an autocrine fashion and contribute to the activation of CD8+ T cells recognizing MHC class I-restricted epitopes on dendritic cells.47-49 These strong preclinical data support the use of IL-12 as an immunologic adjuvant in vaccine trials.

In human clinical trials, IL-12 has been shown to have modest clinical activity against melanoma and renal cell carcinoma, and it has been well tolerated at a dose of 50 ng/kg administered subcutaneously daily for 5 days.50 Given the extensive preclinical testing in mice showing activity as a vaccine adjuvant, and preliminary clinical data suggesting antitumor activity51 when used as an adjuvant for peptide-pulsed antigen-presenting cells, we assessed whether IL-12 would be a promising reagent to boost the immunologic activity of a melanoma peptide vaccine. In one study, escalating doses of the gp100 209 (ITDQVPSFY), 280 (YLEPGPVTA), and 154 (KTWGQYWQV) peptides at doses from 1 to 10 mg were administered subcutaneously every 3 weeks with IFA.18 Ninety percent to 100% of patients had strong evidence of boosted immune reactivity after vaccination, as shown by a sensitive cytokine release gamma-interferon assay. Higher release of cytokine was seen after four immunizations than after two in most patients, and a greater level of reactivity was observed when substituted peptides were used in assays in contrast with "native" peptides. Boosted cytokine release was shown to correlate with cytolytic responses. When the amino acid–substituted gp100109-217(210M) peptide was used to immunize patients with metastatic melanoma, it was shown to be more effective in stimulating an immune response in vivo than the wild-type epitope.52 On the basis of these preliminary results, a randomized trial was designed to assess the potential role of IL-12 as an adjuvant, with immune response as the primary end point. The trial included patients with resected high-risk melanoma treated with multiple substituted epitope peptides from gp100 and tyrosinase with IFA with or without IL-12. Patients with resected melanoma were chosen because of the likelihood that they would survive at least 6 months without progressive disease and be able to receive multiple vaccinations. Their selection was also based on the hypothesis that they might be less immune suppressed than patients with metastatic disease.

The results of this clinical trial were that IL-12 seemed to augment peptide-specific DTH reactivity to the gp100 antigen and boosted the gp100-specific and tyrosinase-specific immune response measured by release of gamma-interferon from 10-day (one restimulation) or 17-day (two restimulations) peripheral-blood T cells. Immune response to gp100 measured by MHC-peptide gp100 tetramer assay on fresh CD8+ T cells was also augmented in the majority of patients (37 of 42, or 88%) who received the vaccine with or without IL-12. The addition of IL-12 at a single dose of 30 ng/kg with each vaccination augmented the number of gp100-specific CTLs measured in the blood by tetramer assay. Patients who received the gp100 and tyrosinase peptides/IFA demonstrated DTH reactivity to gp100 but not tyrosinase after peptide vaccination. They also displayed significant increases in the aforementioned immune assays directed against both gp100 and tyrosinase. Immune reactivity against gp100 was clearly higher than that seen against tyrosinase whether IL-12 was administered or not. The increases in DTH reactivity, cytokine release by ELISA after one stimulation in vitro, and the tetramer assays for gp100 CTLs all seemed to correlate with one another and were present in patients who received peptide IFA with or without IL-12, but the increases seemed to be stronger in the IL-12 group. By every measure of immunity, in this trial the tyrosinase368-376(370D) epitope seemed to be less immunogenic than gp100209-217(210M).

In contrast to trials in which IL-12 was administered intravenously at higher repeated doses,50 there was no evidence of severe or life-threatening side effects from IL-12 given once intradermally at 30 ng/kg with each vaccination. There was no difference overall in side effects when the group that received peptides/IFA was compared with the group that received peptides/IFA with IL-12, indicating that the combination was well tolerated. Of the three patients with ulceration of their vaccine-induced granulomata, two received no IL-12.

A prior trial of a gp100 peptide with IL-12 has been published indicating that higher multiple doses of IL-12 given intravenously as an adjuvant resulted in a decreased antigen-specific immune response compared with peptide/IFA alone,24 in contradistinction to our results. Dose, route, and scheduling are likely to play a role in the adjuvant effect of IL-12, and available murine data do not suggest that higher repetitive dosing is superior to single-dose administration at the injection site. Gajewski et al51 treated 15 metastatic melanoma patients with subcutaneous Melan-A/MART-1 peptide-pulsed peripheral-blood cells with rhuIL-12 in escalating doses subcutaneously every other day, with three doses after each cell injection. A partial response, two mixed responses, and three patients with stable disease were observed, with minimal toxicity. Antigen-specific immune reactivity directed against Melan-A/MART-1 was augmented in eight of 13 patients, supporting the idea that IL-12 could function as an effective vaccine adjuvant in patients.

A kinetic analysis on tetramer staining for gp100-specific CTLs was performed in six patients. The data indicated that tetramer-specific CTL responses did not increase to a plateau until at least six vaccinations were given over 3 months, and that even after four vaccinations over 2 months, responses were weak. These data support a prolonged schedule of peptide vaccinations in cancer patients over a period of at least 3 to 6 months, and suggest that maximal vaccine-related immune responses may not be easily achieved in patients with rapidly progressive metastatic cancer with limited survival times of less than 6 months.

Of the 24 patients who relapsed, 16 had biopsy specimens available for staining to detect the gp100 antigen. Fifteen specimens revealed evidence of positive staining which was usually heterogenous but which did not support the widespread emergence of antigen-negative clones of tumor cells induced by immune selection after repeated vaccination with a gp100 peptide. Although 37 of 42 patients (88%) had detectable staining of circulating fresh CD8 T cells with the gp100 tetramer, some as high as 2.5%, a serious concern is that those circulating T-cell clones may not have received a proper activation signal and may not be able to destroy tumor cells in situ.53-56 When peripheral-blood cells from a patient with 2.5% circulating gp100 tetramer-specific CD8+ T cells were used in a chromium release assay, significant lysis of gp100209-217 peptide-expressing targets above background was seen, which indicates that these T cells are functional and capable of destroying antigen-expressing cells (data not shown). Whether the CTLs induced by peptide immunization with or without IL-12 are memory cells or whether they indeed have the phenotype of activated or memory T cells, or whether T cells from IL-12–treated patients can be distinguished functionally or phenotypically from non–IL-12–treated patients, is a subject of active investigation. In future studies, we will characterize the phenotype and functional activity of tetramer-positive CD8+ T cells from patients who have been vaccinated with gp100 and tyrosinase peptides.

The clinical data from this study indicate that for all 48 patients, median time to relapse was 20 months. This is similar to results from a recently published study of adjuvant GM-CSF in patients with resected stage III/IV melanoma.32,57

On the basis of these data, in an upcoming trial we plan to explore the optimal dose and pharmacokinetic use of IL-12 as an adjuvant added to a melanoma peptide vaccine regimen with three peptides. Our goal is to establish the contribution of prolonged pharmacokinetics and T-cell help for the enhancement of melanoma antigen-specific immunity.


   ACKNOWLEDGMENTS
 
Supported by grant no. RPG-CCE-89038 from the American Cancer Society and in part by Children’s Cancer Study Group grant no. 5P30-CA14089 from the National Cancer Institute.

We acknowledge the outstanding administrative and secretarial assistance of Kathy Pfeiffer and are grateful to Bridgit O’Toole, PhD, of Genetics Institute for helpful discussions and encouragement.


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Submitted March 20, 2001; accepted June 12, 2001.




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