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Clinical Cancer Research 13, 215, January 1, 2007. doi: 10.1158/1078-0432.CCR-06-1450
© 2007 American Association for Cancer Research
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Cancer Therapy: Clinical

Alum with Interleukin-12 Augments Immunity to a Melanoma Peptide Vaccine: Correlation with Time to Relapse in Patients with Resected High-Risk Disease

Omid Hamid1, Jolie C. Solomon2, Ronald Scotland2, Marile Garcia2, Shirley Sian1, Wei Ye3, Susan L. Groshen3 and Jeff S. Weber2

Authors' Affiliations: 1 The Angeles Clinic and Research Institute, Santa Monica; 2 Department of Medicine, University of Southern California/Norris Comprehensive Cancer Center; and 3 Department of Preventive Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California

Requests for reprints: Jeffrey S. Weber, University of Southern California/Norris Comprehensive Cancer Center, Suite 3440, 1441 Eastlake Avenue, Los Angeles, CA 90089. Phone: 323-865-3962; Fax: 323-865-0061; E-mail: jweber{at}usc.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We attempted to augment immunity to melanoma antigens using interleukin-12 (IL-12) with aluminum hydroxide (alum) for sustained release or granulocyte macrophage colony-stimulating factor (GM-CSF) added to a multipeptide vaccine.

Experimental Design: Sixty patients with high-risk resected melanoma were randomized to receive melanoma peptides gp100209-217 (210M), MART-126-35 (27L), and tyrosinase368-376 (370D) with adjuvant Montanide ISA 51 and either IL-12 at 30 ng/kg with alum (group A), IL-12 at 100 ng/kg with alum (group B), or IL-12 at 30 ng/kg with 250 µg GM-CSF (group C).

Results: Three patients had stage IIC (5%), 50 had stage III (83%), and 7 had stage IV (12%) melanoma. Most toxicities were grade 1/2 and resolved rapidly. Significant toxicity included grade 3 colitis and visual changes and grade 3 headache resolving after stopping IL-12 but continuing peptide vaccine. A higher rate of post-vaccine 6-month immune response to gp100 and MART-1 was observed in group A (15 of 19) or B (19 of 20) that received IL-12 plus alum versus group C with IL-12/GM-CSF (4 of 21; P < 0.001). Post-vaccine enzyme-linked immunospot response rates to peptide analogues in group B were higher than group A (P = 0.031 for gp100 and P = 0.010 for MART-1); both were higher than group C (P < 0.001 for gp100 and P < 0.026 for MART-1). With a median of 24 months of follow-up, 23 patients have relapsed. Post-vaccine immune response to MART-1 was associated with relapse-free survival (P = 0.012).

Conclusions: IL-12 with alum augmented an immune response to melanoma antigens compared with IL-12 with GM-CSF. Immune response was associated with time to relapse.

Recent data from cancer vaccine trials have shown that immune responses can be generated against self-antigens encoded by tumor cells, but evidence of clinical benefit and correlation of immune response with clinical benefit has been infrequent (1, 2). Hurdles to successful cancer vaccination are both quantitative and qualitative. Precursor frequencies have been low, and the generation of high-avidity T cells has been problematic. T cells in cancer patients recognize self-antigens, such as differentiation molecules, or cancer-testis antigens to which central and peripheral tolerance exists (3–5). A number of candidate vaccines have been developed for melanoma using whole-cell, adenoviral, vaccinia, fowlpox, DNA plasmid, protein, and peptide approaches (6–12). Peptide vaccine trials in patients with metastatic and resected melanoma confirmed that immune responses are frequently seen at high levels in those patients, but immune responses generally correlated poorly with clinical effect (13, 14). Recently, in trials of peptides with adjuvant Montanide ISA 51 in small numbers of patients, enzyme-linked immunospot (ELISPOT) immune responses were shown to correlate with time to progression (15–17). To augment the generation of antigen-specific cells in patients with high-risk resected melanoma, we have added aluminum hydroxide (alum) for sustained release to the immune stimulatory cytokine interleukin-12 (IL-12) for a multipeptide vaccine.

IL-12 is a heterodimeric p70 molecule consisting of a p35 and a p40 subunit. It promotes Th1 cell differentiation and stimulates the production of IFN-{gamma} from Th1 CD4 and CD8 T cells as well as natural killer cells (17–19), bridging innate and adaptive immunity. IFN-{gamma} and other downstream cytokines elicited by IL-12 inhibit tumor cells directly, augment immune cell function, and activate potent antiangiogenic mechanisms. IL-12 in soluble form and delivered by plasmid has been shown to have potent adjuvant activity in murine infectious and tumor vaccine models (20–22).

Melanoma peptides, including those in the current trial, are immunogenic in vivo (23–25). In a prior trial, i.d. soluble IL-12 added to peptides and adjuvant was shown to augment T-cell reactivity to melanoma antigen gp100 (26). In that trial, there was no correlation between immune response assays using peripheral blood and time to relapse or overall survival. In the current trial, we did a randomized phase II trial in 60 human leucocyte antigen HLA-A2–positive patients with resected stage IIB/C, III, and IV melanoma who were vaccinated with a multipeptide vaccine given s.c. Groups received melanoma peptide vaccine/Montanide either with 30 ng/kg IL-12 with alum with each vaccination (group A), with 100 ng/kg IL-12 with alum given after each vaccination (group B), or with 30 ng/kg IL-12 at the vaccine site with granulocyte macrophage colony-stimulating factor (GM-CSF) at 83 µg per dose (total of 250 µg) emulsified with each of the three peptides/Montanide (group C). We hypothesized that higher or more sustained levels of IL-12 at the injection site might augment immunity and be associated with superior relapse-free survival. The principal end points of the study were the toxicities and tolerability of the regimens, immune response to the antigen peptides, and a quantitative comparison of immune responses in each group. We show for the first time that alum given with IL-12 to sustain its release added to a multipeptide vaccine resulted in superior levels of immunity by a functional antigen-specific assay, and that immune responses were associated with prolonged relapse-free survival.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Trial eligibility. Patients had stage IIC, III, and IV melanoma by the 2001 modified American Joint Commission on Cancer staging system and were rendered 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 disease within 4 weeks of initiating therapy. Eligibility criteria included age ≥18 years, creatinine <2.0 mg/dL, bilirubin <2.0 mg/dL, platelets ≥100,000 per mm3, hemoglobin ≥9 g/dL, and total WBC ≥3,000 per mm3. Negative HIV, hepatitis C antibody, and hepatitis B surface antigen assays were required. All patients were HLA-A*0201 antigen positive by a DNA PCR assay done at the University of California at Los Angeles Immunogenetics Laboratory. All patients were required to comprehend and sign an informed consent form approved by the National Cancer Institute and the Los Angeles County/University of Southern California Institutional Review Board.

Delayed-type hypersensitivity skin tests. Skin tests were done as previously described (26).

ELISPOT assay. Peripheral blood mononuclear cells were thawed and cultured overnight then tested in an ELISPOT assay previously published (26). Pre-wetted ELISPOT IP plates (Millipore, Bedford, MA) were exposed to anti–human IFN-{gamma} monoclonal antibody (1-D1K, Mabtech, Malmo, Sweden) capture antibody. Uncultured, cryopreserved pre- and post-vaccine peripheral blood mononuclear cells were thawed, washed, and then added to ELISPOT plates (105 per 50 µL per well in triplicates). After cells settled onto the membrane, relevant or irrelevant peptides were added at a final concentration of 10 µg/mL, and the plates were incubated (48 h) at 37°C/5% CO2. Cells were then removed by washing six times with PBS/0.05% Tween 20 (PBS/T; Fisher Scientific, Pittsburgh, PA) on an automated plate washer (Skatron, Lier, Norway). Captured cytokine was detected at sites of secretion by incubation (2 h at 37°C/5% CO2) with biotinylated anti–human IFN-{gamma} monoclonal antibody (7-B6-1; Mabtech) in PBS with 0.5% bovine serum albumin (Sigma, St. Louis, MO). Avidin-peroxidase complex (diluted 1:100; Vectastain Elite kit; Vector, Burlingame, CA) in PBS/T was added for 1 h at room temperature. Unbound complex was removed by successive washings. Color development was done with 3-amino-9-ethyl-carbazole (Sigma). After processing, ELISPOT plates were read on a KS Elispot reader (Carl Zeiss, Thornwood, NY). Values were normalized to spots per 100,000 CD8 T cells. HIV reverse transcriptase A2.1 peptide (ILKEPVHGV) was used as a negative control, whereas pokeweed mitogen (Sigma) was used as a nonspecific positive control.

Clinical-grade peptides. The tyrosinase368-376 (370D) [NSC# 699048], MART-126-35 (27L) [NSC# 709401], and gp100209-217 (210M) peptides [NSC #683472] restricted to HLA-A*0201 were prepared to Good Manufacturing Practices and given as previously described (24). They were supplied by Ben Venue Laboratories, Inc. (Bedford, OH) and provided by the Cancer Therapy Evaluation Program of the National Cancer Institute (Bethesda, MD) under an Investigational New Drug application BB 6123 held by the National Cancer Institute.

Adjuvant. Montanide ISA-51 (incomplete Freund's adjuvant) was manufactured by Seppic, Inc. (Franklin Lakes, N.J) and supplied by Cancer Therapy Evaluation Program of the National Cancer Institute under Investigational New Drug application BB 6123 in glass ampoules containing 3 mL of sterile adjuvant solution without preservative.

Rehydragel. Alum (Rehydragel HPA) was purchased from Reheis, Inc. (Berkeley Heights, NJ). It was supplied as a suspension in a 500-mL sterile bottle that was aliquoted into 10-mL sterile vials, wrapped in aluminum foil to protect it from light, and stored at 4°C for <6 months. IL-12 in aqueous solution was mixed with Rehydragel HPA in a final volume of <1.1 mL for s.c. injection.

Vaccine preparation and administration. The peptide vaccine was given as outpatient therapy as previously described (26). Alternating thighs were used for a total of nine injections given over 52 weeks. IL-12 and vaccine were given simultaneously, within 5 cm of each other. Intervals between injections were 2 weeks for the first four injections, 4 weeks for the next three injections, 8 weeks between the seventh and eighth injection, then after 26 weeks for a ninth injection.

Screening for vitiligo and eye changes. All patients had a complete skin examination before therapy and at each visit for vaccination to screen for vitiligo. Ocular toxicity was determined as previously described (26).

IL-12. Recombinant human IL-12 was produced by recombinant DNA techniques in Escherichia coli to GMP grade and was obtained from Genetics Institute (Cambridge, MA). The recombinant human IL-12 was supplied as a lyophilized powder in 5-mL vials containing 50 µg of drug. The recombinant human IL-12 adsorbed to alum (Rehydragel HPA, Reheis) was given at a dose of 30 or 100 ng/kg s.c. with a 1-mL syringe and 27-gauge needle just proximal to the peptide/incomplete Freund's adjuvant injection sites, or given s.c. in the same location at a dose of 30 ng/kg in aqueous solution.

Peptides. Peptides used for in vitro studies were synthesized at the University of Southern California/Norris Cancer Center Core Peptide Facility.

Definition of end points. For each peptide, an ELISPOT immune response at 6 months was defined as ≥10 spots per 105 effectors that were >3 SDs above the baseline value and a 3-fold increase over baseline. A baseline ELISPOT response was defined as ≥10 spots per 105 effectors before starting the trial. Time to recurrence was calculated from the date of first vaccination to the date of recurrence; patients without recurrence were censored at the date of last follow-up.

Statistical methods. This randomized phase II trial was designed to enroll 60 patients and randomly assign them to one of three treatment groups using a permuted block design. To select a regimen for further study, 20 patients per group would ensure a >75% chance that the regimen with the greatest immunologic effect would be observed to have the greatest number of immunologic responders: when the best regimen exceeded the others by at least 30%. To compare the three treatment groups for baseline patient characteristics and ELISPOT responses (responder versus nonresponder), contingency tables and Pearson's {chi}2 test or Fisher's exact test (if the numbers were small) were used. The geometric means of ELISPOT responses were also compared among the three treatment groups using ANOVA after the log transformation of the data. The least significant difference method was used for the pairwise comparisons, once the overall F test was significant at the 0.05 level. The 95% confidence interval (95% CI) associated with the geometric means was calculated for each treatment group. Products of the largest perpendicular dimensions of the delayed-type hypersensitivity (DTH) erythema site were used to assess skin test responses. The comparisons of the skin test responses between the treatment groups were done by Kruskal-Wallis test. Kaplan-Meier plots and the log-rank test were used to compare the risk of relapsing among different groups of patients. To compare the risk of relapsing as a function of the post-vaccine immune response, the landmark method was used: time to relapse was calculated from 6 months after start of treatment (when the immune response was assessed), and the analysis included only those patients who were still at risk of relapsing at 6 months after the treatment started. The data from those who did not have a relapse were censored at the date of last follow-up.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Demographics. Sixty patients with stage IIC, III, and IV resected melanoma were randomized. The demographic details of this high-risk cohort are shown by group (Table 1 ). Median age of the 35 men and 25 women was 57 years. Three patients had resected stage IIC, 50 patients had resected stage III, and seven had resected stage IV cutaneous melanoma. Prior therapy consisted of chemotherapy in three patients, radiation in three patients, and chemoradiation in one patient. Four patients failed IFN-{alpha} in the adjuvant setting (two in group B and two in group C). One patient received prior chemo-biotherapy. No patient had received prior vaccine therapy. Median time from diagnosis of the primary lesion to entering the study for the whole group was 5.6 months (range, 1.4-69.9 months). There was no evidence of imbalance in terms of age, gender, race, and stage among the three treatment arms. At 6 months, four patients could not be apheresed due to inadequate venous access, leaving 56 patients with apheresis samples both before and after vaccination. All four patients who could not have an apheresis due to venous access problems had 80 mL of whole blood collected.


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  		Table 1. Patient characteristics by treatment group

 
Toxicities. Toxicities (by group and grade) of the gp100209-217 (210M)/MART-126-35 (27L)/tyrosinase 368-376 (370D) vaccine for all 60 patients are shown in Table 2 . The majority of patients had constitutional or flu-like symptoms (36 of 60), injection site reactions consisting of erythema and granulomata formation (55 of 60), and rigors/chills (31 of 60) that were directly associated with therapy and of grade 1 or 2. The grade 3/4 toxicities included one episode of grade 3 bloody diarrhea with a colonoscopy showing lymphocytic colitis in group B that resolved completely. Transient grade 3 headache, nausea, visual disturbance, and injection site reaction were also observed in individual patients. No appreciable differences in toxicity were noted among the three groups (Table 2). Treatment was generally felt to be well tolerated. One episode of ocular toxicity was observed. Vitiligo was observed in one patient in group C.


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  		Table 2. Treatment toxicity

 
DTH skin test results. DTH to the gp100209-217 (210M)/MART-126-35 (27L)/tyrosinase 368-376 (370D) peptides and recall antigen Candida albicans was assessed by i.d. skin testing before the first and after the eighth vaccine at month 6. Results were measured as the product of the perpendicular diameters of erythema, and a positive was scored if the dimensions were at least 5 x 5 mm. One of 46 patients reacted to the gp100209-217 (210M) peptide before vaccination. Seventeen of 46 patients tested were anergic to recall antigens before vaccination. Twenty-two of 46 or 48% developed gp100 and/or MART-1 antigen-specific DTH reactivity defined above after vaccination. Of the patients anergic at baseline, 12 of 17 had an immune response to at least one peptide at 6 months. There was no association between anergy at baseline and time to relapse (P = 0.94). In the high dose IL-12/alum plus peptides/Montanide group B, there was greater gp100-specific DTH after vaccination compared with group C (median = 135 versus 0 mm2, P = 0.002). The difference in gp100-specific DTH after vaccination between groups B and A (median = 36 mm2) was not statistically significant (P = 0.40). Interestingly, no DTH reactivity was seen to the tyrosinase368-376 (370D) peptide before or after vaccination, suggesting that it was not strongly immunogenic. Both groups A and B had a significantly higher change in gp100 (P = 0.006 for group A versus group C and P = 0.002 for group B versus group C) and MART-1 (P = 0.038 for group A versus group C and P = 0.009 for group B versus group C) reactivity than group C (Fig. 1A ), although the changes at 6 months in groups A and B did not significantly differ (P = 0.48 for gp100 and P = 0.35 for MART-1).


Figure 1
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  		Fig. 1. A, percentages of patients with positive DTH to the gp100209-217 (210M)/MART-1126-35 (27L) and their 95% CI. Ordinate, percentages; abscissa, three treatment groups. B, geometric means and 95% CI of measurements of CD8+ T-cell reactivity against gp100209-217 (210M) and MART-1126-35 (27L) in uncultured peripheral blood mononuclear cells by IFN-{gamma} ELISPOT. Ordinate, spots per 100,000 CD8+ effectors; abscissa, three treatment groups.

 
ELISPOT assays. All 60 patients were analyzed for reactivity to the wild-type gp100209-217 and MART-126-35 as well as the analogue gp100209-217 (210M) and MART-126-35 (27L) peptides by {gamma} IFN ELISPOT at baseline and after vaccine at month 6. None of the four baseline ELISPOT values were associated with treatment group (all P ≥ 0.20). Neither the four baseline peptide ELISPOT values (all P > 0.40) nor the number of responses at baseline (P = 0.16) were associated with time to recurrence.

A post-vaccine immune response was defined in Materials and Methods. By that criterion, 18 of 20 or 95% of patients in group B had an immune response to gp100 or MART-1 analogue epitopes (100 ng/kg IL-12/peptides/alum), 14 of 19 or 79% in group A (30 ng/kg IL-12/peptides/alum), and only 2 of 21 or 19% in group C (30 ng/kg IL-12 + GM-CSF). For the wild-type epitopes, 19 of 20 patients had an immune response to gp100 or MART-1 in group B, 14 of 19 in group A, and 4 of 21 in group C. The geometric mean of the 6-month analogue MART-126-35 (27L) ELISPOT response was higher in group B (63 spots) than either group A (22 spots, P = 0.004) or group C (12 spots, P < 0.001). For gp100 209-217 (210M), group B (55 spots) was also higher than group C (8 spots, P < 0.001) but not significantly higher than group A (42 spots, P = 0.42; Fig. 1B). The ELISPOT response rate after vaccination for all four peptides was higher in groups A and B than group C (all overall P < 0.001) as seen in Table 3 . The ELISPOT response rate to the analogue peptides was higher in group B than either group A [P = 0.031 for gp100209-217 (210M) and P = 0.010 for MART-126-35 (27L)] or group C [P < 0.001 for both gp100209-217 (210M) and MART-126-35 (27L); Table 3]. These data indicate that alum with IL-12 augments immunity to MART-1 and gp100, and that group B had the highest overall immune response to the analogue peptides. In contrast, there were no detectable ELISPOT post-vaccine responses to tyrosinase, again indicating its low immunogenicity.


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  		Table 3. ELISPOT response rate by treatment group

 
ELISPOT assays over time. Five patients each in groups A, B, and C had peripheral blood samples available for analyses over time of ELISPOT reactivity to native MART-1 26-35 wild-type and gp100 209-217 wild-type peptides at months 0, 3, 6, and 9. The data for those three sets of patients are shown in Fig. 2A (MART-1) and Fig. 2B (gp100), indicating that there seemed to be an earlier increase of reactivity at month 3 and a more prolonged decay of reactivity at months 6 and 9 in patients in group B (100 ng/kg IL-12 with alum) than those in group A (30 ng/kg IL-12 with alum). Similar data were observed for the analogue gp100 and MART-1 epitopes (data not shown). These results are hypothesis generating, rather than definitive, because a small numbers of patients were examined, but the data suggest that 100 ng/kg IL-12 with alum (group B) may accelerate the onset of immune response to gp100 and MART-1 and maintain the response over time.


Figure 2
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  		Fig. 2. Measurement of CD8+ T-cell reactivity against MART-126-35 wild-type (A) and gp100 209-217 wild-type (B) in uncultured peripheral blood mononuclear cells by IFN-{gamma} ELISPOT. Ordinate, spots per 100,000 CD8+ effectors; abscissa, three treatment groups.

 
Clinical data. Of the 23 relapsed patients, 14 had no immune response, and 9 had immune response. Twenty-eight of the 37 nonrelapsing patients had an immune response. Ten relapses were in the low-dose IL-12/GM-CSF group, seven were in the low-dose IL-12/alum group, and six were in the high-dose IL-12/alum group. There was no significant difference in the risk of relapsing between groups (P = 0.40). Fifty patients were still at risk at 6 months after the treatment started. By the landmark analysis, the risk of relapsing for patients who had ELISPOT response to MART-1 analogue epitope was only 25% (95% CI, 8-81%) of that for those who did not have ELISPOT response to MART-1 analogue epitope (P = 0.012; Fig. 3A ). However, the association between an ELISPOT response to gp100 analogue epitope and time to relapse was not statistically significant (relative hazard ratio, 0.50; 95% CI, 0.17-1.49 for responders versus nonresponders; P = 0.20; Fig. 3B). The patients who had an immune response to any of the four peptides had a lower risk of relapsing than those who did not have an immune response to any of the four peptides (relative hazard ratio, 0.34; 95% CI, 0.11-1.02; P = 0.042; Fig. 4 ).


Figure 3
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  		Fig. 3. Relapse-free survival of patients with or without an immune response to MART-126-35 27L (A) and gp100 209-217 210M (B). Ordinate, percentage of patients free of disease; abscissa, time in months. Hash marks, patients without relapse.

 

Figure 4
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  		Fig. 4. Association of response to any peptide with recurrence-free survival.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Preclinical and clinical data suggested that IL-12 was a promising vaccine adjuvant, although it showed toxicity and only modest response rates in metastatic melanoma or renal cell cancer trials (27, 28). In mice, IL-12 with alum for sustained release or encoded within viral vectors or DNA plasmids showed activity as a vaccine adjuvant (29–31). Codelivery of soluble IL-12 or plasmid-encoded IL-12 with DNA vaccines encoding HIV and bacterial and tumor antigens augmented Th1 immunity characterized by strong CD8+ CTL priming and generation of memory (32). IL-12 increased the survival of memory CD8 T cells and augmented IFN-{gamma} and proinflammatory cytokine secretion by effectors (33, 34).

In the current trial, we asked whether IL-12 with alum was a superior adjuvant to augment immunity to melanoma antigens compared with soluble IL-12 with GM-CSF. Three groups of patients each received three melanoma peptides with Montanide ISA 51 and one of two doses of IL-12 with alum, or soluble IL-12 with additional GM-CSF emulsified with the peptides. The regimens were all well tolerated, with colitis as the only dose-limiting toxicity in one patient. Immune responses by functional IFN-{gamma} ELISPOT assay using fresh unstimulated peripheral blood cells were statistically significantly higher in the two groups receiving IL-12 with alum than in the soluble IL-12/GM-CSF group. Immune responses for both wild-type and modified MART-1 and gp100 peptides were higher both in magnitude and proportion of patients in the 100 ng/kg IL-12/alum group than the group receiving 30 ng/kg IL-12/alum, or the IL-12/GM-CSF group, suggesting a dose response. The risks of recurrence was lowest for group B (1.0; reference group), intermediate for group A (1.24; 95% CI, 0.42-3.70), and highest for group C (1.92; 95% CI, 0.69-5.30), although they were not statistically significantly different (P = 0.40). There was a significant association of an ELISPOT response to MART-1 for all patients with recurrence-free survival (P = 0.012) but not for gp100 (P = 0.20). A significant association of immune response to any of the four peptides and relapse-free survival was observed with a median time to relapse not yet reached but projected at 4 years (P = 0.042). ELISPOT analyses over time in selected patients indicated that both an earlier increase in immunity and a slower decay back to baseline occurred in the higher-dose IL-12/alum group than the lower-dose group, even when the peak responses were similar. Therefore, the addition of alum to IL-12 as an adjuvant to a peptide vaccine resulted in T cells with superior immune activity against well-defined tumor antigens compared with soluble IL-12 with GM-CSF, and MART-1 immune responses seemed to be associated with better clinical outcome.

In trials of IL-12 with peptide-pulsed peripheral blood mononuclear cells and with peptide vaccines, there was a correlation between the magnitude of immunity and clinical response (35, 36). In patients with resected high-risk melanoma treated with multiple melanoma peptides with Montanide ISA 51 with or without IL-12 (30 ng/kg), immune responses in the IL-12 group were greater than without IL-12 by DTH skin test or MHC/peptide tetramer assays (P < 0.05; ref. 26). The data from the current trial expands upon those results to indicate that a vaccine including a sustained release preparation of IL-12 with alum generated higher levels of immunity and more frequent immune responses than vaccine with soluble IL-12 and GM-CSF. Although the addition of GM-CSF to the soluble IL-12 plus vaccine in group C may have suppressed immunity, there are no preclinical or clinical data to support that idea. Alum with IL-12 induced sustained immune responses and the generation of functional IFN-{gamma}–secreting CD8 T cells. IL-12 may have directly augmented the activity of CD8 T cells and/or may have polarized type 1 helper T cells at the vaccine site to generate a more robust and long-lived cytolytic T-cell response, and alum may have played an important role due to its sustained release effect.

The best approach for future cancer vaccine development is unclear, given the discouraging results from recent cellular vaccine trials. Only through continued clinical investigation of immune activation and suppression mechanisms in cancer can we hope to define promising adjuvants and vaccines. For example, recently, Slingluff et al. (37, 38) have found that vaccination with multiple peptides in an emulsion of GM-CSF in adjuvant was immunogenic. We conclude from the current study of 60 patients that alum augments immune responses to a peptide vaccine with IL-12, and that immune responses to the MART-1 antigen are associated with relapse free survival. Although a response to MART-1, independent of adjuvant used, was associated with better recurrence-free survival, this may not reflect the value of vaccination and may be simply a measurement of the strength of the immune system. Immune response and outcome has been positively correlated in previous phase II adjuvant trials where antibody response has been associated with clinical outcome. Unfortunately, subsequent phase III testing failed to confirm this benefit (39). Although the combination of IL-12 with alum seems promising based on immune data, that combination was not shown to be clinically superior to IL-12/GM-CSF, the patient numbers in this preliminary evaluation were insufficient to show statistical power needed to discern clinical efficacy. Based on the DTH and ELISPOT data showing stronger immune response, we conclude that the regimen of 100 ng/kg IL-12 with alum used in group B should be further developed as an adjuvant in cancer vaccine strategies.


   Acknowledgments
 
We thank Kathy Pfeiffer for outstanding administrative and secretarial assistance; the dedicated members of the Cancer Therapy Evaluation Program, National Cancer Institute, including Howard Streicher, James Zweibel, and Jay Greenblatt, without whose efforts this trial would not have occurred; and the inspiring and selfless patients that took part in this clinical protocol.


   Footnotes
 
Grant support: Grant CA90751-01 and National Cancer Institute/Cancer Center Support grant 5P30-CA14089.

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.

Note: These data were presented in part at the 2005 American Society of Clinical Oncology meeting in Orlando, Florida.

Received 6/15/06; revised 9/15/06; accepted 10/ 6/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rosenberg SA. Shedding light on immunotherapy for cancer. N Engl J Med 2004;350:1461–3.[Free Full Text]
  2. Pardoll DM. Spinning molecular immunology into successful immunotherapy. Nat Rev Immunol 2002;2:227–38.[CrossRef][Medline]
  3. Rosenberg SA. Identification of cancer antigens: impact on development of cancer immunotherapies. Cancer J 2000;6 Suppl 3:S200–7.
  4. Van Der Bruggen P, Zhang Y, Chaux P, et al. Tumor-specific shared antigenic peptides recognized by human T cells. Immunol Rev 2002;188:51–64.[CrossRef][Medline]
  5. Scanlan MJ, Simpson AJ, Old LJ. The cancer/testis genes: review, standardization, and commentary. Cancer Immun 2004;4:1.[Medline]
  6. Soiffer R, Hodi FS, Haluska F, 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 2003;21:3343–50.[Abstract/Free Full Text]
  7. Rosenberg SA, Zhai Y, Yang JC, et al. Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens. J Natl Cancer Inst 1998;90:1894–900.[Abstract/Free Full Text]
  8. Belli F, Testori A, Rivoltini L, et al. Vaccination of metastatic melanoma patients with autologous tumor-derived heat shock protein gp96-peptide complexes: clinical and immunologic findings. Clin Oncol 2002;20:4169–80.
  9. Zajac P, Oertli D, Marti W, et al. Phase I/II clinical trial of a nonreplicative vaccinia virus expressing multiple HLA-A0201-restricted tumor-associated epitopes and costimulatory molecules in metastatic melanoma patients. Hum Gene Ther 2003;14:1497–510.[CrossRef][Medline]
  10. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Recombinant fowlpox viruses encoding the anchor-modified gp100 melanoma antigen can generate antitumor immune responses in patients with metastatic melanoma. Clin Cancer Res 2003;9:2973–80.[Abstract/Free Full Text]
  11. Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol 2005;175:633–9.[Abstract/Free Full Text]
  12. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 1998;4:321–7.[CrossRef][Medline]
  13. Lee KH, Wang E, Nielson MB, et al. Increased vaccine-specific T cell frequency after peptide-based vaccination correlates with increased susceptibility to in vitro stimulation but does not lead to tumor regression. J Immunol 1993;163:6292.
  14. Marchand M, Van Baren N, Weynants P, et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer 1999;80:219–30.[CrossRef][Medline]
  15. Slingluff CL, Jr., Petroni GR, Yamshchikov GV, et al. Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J Clin Oncol 2003;21:4016–26.[Abstract/Free Full Text]
  16. Oka Y, Tsuboi A, Taguchi T, et al. Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression. Proc Natl Acad Sci U S A 2004;101:13885–90.[Abstract/Free Full Text]
  17. Letsch A, Keilholz U, Fluck M, et al. Peptide vaccination after repeated resection of metastases can induce a prolonged relapse-free interval in melanoma patients. Int J Cancer 2005;114:936–41.[CrossRef][Medline]
  18. Kobayashi M, Fitz L, Ryan M, et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989;170:827–45.[Abstract/Free Full Text]
  19. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of TH1 CD4+ T cells through IL-12 produced by listeria-induced macrophages. Science 1993;260:547–9.[Abstract/Free Full Text]
  20. Macatonia SE, Hosken NA, Litton M, et al. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 1995;154:5071–9.[Abstract]
  21. Boyer JD, Cohen AD, Ugen KE, et al. Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids. AIDS 2000;14:1515–22.[CrossRef][Medline]
  22. Salem ML, Kadima AN, Zhou Y, et al. Paracrine release of IL-12 stimulates IFN-{gamma} production and dramatically enhances the antigen-specific T cell response after vaccination with a novel peptide-based cancer vaccine. J Immunol 2004;172:5159–67.[Abstract/Free Full Text]
  23. Nanni P, Nicoletti G, De Giovanni C, et al. Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice. J Exp Med 2001;194:1195–205.[Abstract/Free Full Text]
  24. Cormier JN, Salgaller ML, Prevette T, et al. Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART1/Melan-A. Cancer J Sci Am 1997;3:37.[Medline]
  25. Pullarkat V, Scotland R, Rubio V, et al. Phase I trial of SD-9427 (Progenipoietin) with a multipeptide vaccine for resected metastatic melanoma. Clin Cancer Res 2003;9:1301–12.[Abstract/Free Full Text]
  26. Lee P, Wang F, Kuniyoshi J, et al. Effects of interleukin-12 on the immune response to a multipeptide vaccine for resected metastatic melanoma. J Clin Oncol 2001;19:3836–44.[Abstract/Free Full Text]
  27. Atkins MB, Robertson MJ, Gordon M, et al. Phase I evaluation of intravenous recombinant human interleukin-12 in patients with advanced malignancies. Clin Cancer Res 1997;3:409–17.[Abstract]
  28. Gollob JA, Mier JW, Veenstra K, et al. Phase I trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-{gamma} induction is associated with clinical response. Clin Cancer Res 2000;6:1678–92.[Abstract/Free Full Text]
  29. Jankovic D, Caspar P, Zweig M, et al. Adsorption to aluminum hydroxide promotes the activity of IL-12 as an adjuvant for antibody as well as type 1 cytokine responses to HIV-1 gp120. J Immunol 1997;159:2409–17.[Abstract/Free Full Text]
  30. Kenney RT, Sacks DL, Sypek JP, Vilela L, Gam AA, Evans-Davis K. Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis. J Immunol 1999;163:4481–8.[Abstract/Free Full Text]
  31. Egan MA, Chong SY, Megati S, et al. Priming with plasmid DNAs expressing interleukin-12 and simian immunodeficiency virus gag enhances the immunogenicity and efficacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus. AIDS Res Hum Retroviruses 2005;21:629–43.[CrossRef][Medline]
  32. Stobie L, Gurunathan S, Prussin C, et al. The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge. Proc Natl Acad Sci U S A 2000;97:8427–32.[Abstract/Free Full Text]
  33. Chang J, Cho JH, Lee SW, Choi SY, Ha SJ, Sung YC. IL-12 priming during in vitro antigenic stimulation changes properties of CD8 T cells and increases generation of effector and memory cells. J Immunol 2004;172:2818–26.[Abstract/Free Full Text]
  34. Portielje JE, Kruit WH, Eerenberg AJ, et al. Subcutaneous injection of interleukin 12 induces systemic inflammatory responses in humans: implications for the use of IL-12 as vaccine adjuvant. Cancer Immunol Immunother 2005;54:37–43.[CrossRef][Medline]
  35. Peterson AC, Harlin H, Gajewski TF. Immunization with Melan-A peptide-pulsed peripheral blood mononuclear cells plus recombinant human interleukin-12 induces clinical activity and T-cell responses in advanced melanoma. J Clin Oncol 2003;21:2342–8.[Abstract/Free Full Text]
  36. Cebon J, Jager E, Shackleton MJ, et al. Two phase I studies of low dose recombinant human IL-12 with Melan-A and influenza peptides in subjects with advanced malignant melanoma. Cancer Immun 2003;3:7.[Medline]
  37. Slingluff CL, Petroni GR, Yamshchikov GV, et al. Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J Clin Oncol 2003;21:4016–26.[Abstract/Free Full Text]
  38. Slingluff CL, Jr., Petroni GR, Yamshchikov GV, et al. Immunologic and clinical outcomes of vaccination with a multiepitope melanoma peptide vaccine plus low-dose interleukin-2 administered either concurrently or on a delayed schedule. J Clin Oncol 2004;22:4474–85.[Abstract/Free Full Text]
  39. Kirkwood JM, Ibrahim JG, Sosman JA, et al. High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIB-III melanoma: results of intergroup trial E1694/S9512/C509801. J Clin Oncol 2001;19:2370–80.[Abstract/Free Full Text]



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