|
Advertisement |
|
|
||
 |
|
|||||
|
|||||
|
 |
||||||
|
||||||
|
||||||
|
Journal of Clinical Oncology, Vol 22, No 22 (November 15), 2004: pp. 4474-4485 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.10.212 Immunologic and Clinical Outcomes of Vaccination With a Multiepitope Melanoma Peptide Vaccine Plus Low-Dose Interleukin-2 Administered Either Concurrently or on a Delayed ScheduleFrom the Department of Surgery/Division of Surgical Oncology, Department of Health Evaluation Sciences, Cancer Center, Department of Medicine/Division of Hematology-Oncology, Department of Pathology, Department of Radiology, University of Virginia Health System, Charlottesville, VA Address reprint requests to Craig L. Slingluff Jr, MD, Department of Surgery, Human Immune Therapy Center, University of Virginia, 1352 Jordan Hall, PO Box 801457, Charlottesville, VA 22908; e-mail: cls8h{at}virginia.edu
PURPOSE: A phase II trial was performed to test whether systemic low-dose interleukin-2 (IL-2) augments T-cell immune responses to a multipeptide melanoma vaccine. Forty patients with resected stage IIB-IV melanoma were randomly assigned to vaccination with four gp100- and tyrosinase-derived peptides restricted by human leukocyte antigen (HLA) -A1, HLA-A2, and HLA-A3, and a tetanus helper peptide plus IL-2 administered daily either beginning day 7 (group 1), or beginning day 28 (group 2). PATIENTS AND METHODS: T-cell responses were assessed by an interferon gamma ELIspot assay in peripheral blood lymphocytes (PBL) and in a lymph node draining a vaccination site (sentinel immunized node [SIN]). Patients were followed for disease-free and overall survival. RESULTS: T-cell responses to the melanoma peptides were observed in 37% of PBL and 38% of SINs in group 1, and in 53% of PBL and 83% of SINs in group 2. The magnitude of T-cell response was higher in group 2. The tyrosinase peptides DAEKSDICTDEY and YMDGTMSQV were more immunogenic than the gp100 peptides YLEPGPVTA and ALLAVGATK. T-cell responses were detected in the SINs more frequently, and with higher magnitude, than responses in the PBL. Disease-free survival estimates at 2 years were 39% (95% CI, 18% to 61%) for group 1, and 50% (95% CI, 28% to 72%) for group 2 (P = .32). CONCLUSION: The results of this study support the safety and immunogenicity of a vaccine composed of four peptides derived from gp100 and tyrosinase. The low-dose IL-2 regimen used for group 1 paradoxically diminishes the magnitude and frequency of cytotoxic T lymphocyte responses to these peptides.
Cytokines administered with cancer vaccines may affect immunogenicity and therapeutic impact by local or systemic effects. We have incorporated interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) as systemic and local adjuvants, respectively, for peptide vaccines administered in incomplete Freund's adjuvant (Montanide ISA-51; Seppic Inc, Paris, France). This approach has resulted in evidence of immunogenicity in 80% of patients with advanced stage III (unresectable) and stage IV melanoma.1,2 The rationale for inclusion of GM-CSF is based on substantial data from human and murine systems in which local GM-CSF augments antitumor immunity through effects on antigen-presenting cells.3-5 When administered in an emulsion with Montanide ISA-51 adjuvant and peptides, GM-CSF is an effective local adjuvant in murine models.6 In murine models, low-dose IL-2 can increase immunogenicity of tumor vaccines.7 In humans, low-dose IL-2 can augment delayed-type hypersensitivity recall responses and may expand ongoing immune responses.8,9 However, in one study,10 addition of high-dose IL-2 to a peptide vaccine was associated with decreased detection of T cells reactive to antigen, as measured in the peripheral blood. Several possible explanations exist, ranging from activation-induced cell death of activated cytotoxic T lymphocytes (CTLs) to changes in T-cell trafficking. Thus, no consensus exists on the value of IL-2 as an adjuvant to specific immunotherapy with vaccines, despite substantial preclinical and clinical experience with IL-2 to date. An essential step in the development of effective cancer vaccines is identification of the immune response parameters defining protective immunity. We have found evaluation of immune responses in a lymph node draining a vaccine site (sentinel immunized node [SIN]) provides a more sensitive measure of immunogenicity than evaluation of immune responses only in the peripheral blood in patients with measurable advanced melanoma.1 In the present trial, we incorporated the evaluation of the SIN to augment measures of immunogenicity in the adjuvant setting. The randomized phase II trial reported here was designed to select the treatment regimen (upfront IL-2 or delayed IL-2) most likely to enhance the immunologic efficacy of a vaccine comprising melanoma peptides plus GM-CSF in-adjuvant, in patients with resected high-risk melanoma. The administration of IL-2 was delayed in group 2; therefore it was possible to assess peptide-specific immune responses with IL-2 (group 1) or without IL-2 (group 2) in the SIN. Thus, in addition to providing insight about the immune response to vaccination in patients with minimal residual disease, this trial was also designed to assess the value of low-dose IL-2 as a systemic vaccine adjuvant.
Patients Patients with resected AJCC stage IIB, III, or IV melanoma, who were serologically typed as human leukocyte antigen (HLA) -A1, -A2, or -A3+, and whose melanoma cells expressed gp100 and/or tyrosinase as determined by immunohistochemistry, were studied following informed consent, and with institutional review board (HIC#8515) and US Food and Drug Administration (BB-IND #7593) approval.
Inclusion criteria included ages 18 to 85 years, Eastern Cooperative Oncology Group performance status 0 to 1, adequate liver and renal function, and completion of surgical therapy within the preceding 6 months. Exclusion criteria included pregnancy; cytotoxic chemotherapy, interferon gamma (IFN-
Clinical Trial Design
End Points for the Study
Vaccine Composition
Systemic Low-Dose IL-2
Toxicity Assessment and Stopping Rules Toxicities were recorded by each patient using a daily toxicity diary and were reviewed by interview with a study physician or nurse each week. Toxicities were graded in accordance with the National Cancer Institute Common Toxicity Criteria version 2.0. In patients with nondermatologic grade 3 or greater toxicities, IL-2 was delayed until the toxicity resolved to grade 1 or 0. At that time, IL-2 was resumed with a 25% dose reduction. Up to three such dose reductions were permitted, after which any serious toxicity was followed by permanent cessation of IL-2. Patients with hyperthyroidism consistent with autoimmune thyroiditis, whether symptomatic or not, had IL-2 discontinued, but were continued on vaccines. Patients with unequivocal disease progression were removed from treatment.
Harvest of the SIN
Cell Lines Used
Peptides
ELIspot Assays Assessment of immunologic response was based on a fold increase. For the SIN, the fold increase was measured as the ratio of T cells responding to the peptide over maximum negative control. For PBL responses over time, the fold increase was measured as the ratio compared with the prevaccine ratio. Observed fold increases less than 1 were converted to 1 to indicate no response. When the prevaccine PBL value was zero, this value was converted to 1 to avoid dividing by zero. Evaluation of T-cell responses was based on the following definitions: Nvax, number of T cells responding to peptide in the vaccine; Nneg, number of T cells responding to negative control (maximum of two negative controls: [a] C1R-A1, T2, or C1R-A3, alone, or [b] C1R-A1, T2, or C1R-A3 pulsed with an irrelevant peptide); and Rvax, ratio of Nvax/Nneg.
For evaluations of PBL, a patient is considered to have a T-cell response to vaccination only if all of the following criteria have been met: (1) Nvax exceeds Nneg by at least 30 cells per 100,000 (corresponds to
Since prevaccination lymph node samples were not routinely evaluated in this study, criterion 4 of the previous paragraph was not applied to the SIN evaluation. Differences in proportions (ie, response rates) between the two groups were assessed by using the
Summary of Clinical Toxicities Forty patients were randomly assigned to one of two groups. Details of the patient population are listed in Table 1. Patients experienced mild to moderate toxicities on this regimen, mostly attributable to IL-2. These toxicities are listed in Table 2. Additional details, including changes in clinical laboratory blood values over time, are reported separately,19 and these data further support the assignment of cause predominantly to IL-2.
Two patients had grade 4 toxicities. Patient 1 (group 1) was hospitalized for correction of hyperglycemia (> 500 mg/dL) unrelated to the treatment regimen. IL-2 was held for 2 days, and subsequent therapy was well tolerated. Patient 29 (group 1) developed symptoms of motor weakness and signs of multiple brain lesions. It remains unclear whether these lesions were tumor deposits or intracerebral hemorrhages, and whether they were related to the treatment regimen or to tumor progression. Patient 24 (group 2) developed an atypical skin reaction of indeterminate origin, predominantly on her legs, before receiving any IL-2; she was taken off protocol. Patient 34 (group 2) developed an intense local reaction at the arm vaccination site, accompanied by a persistent high fever that was suspicious for cellulitis. Although all cultures were negative for infection, she was treated with antibiotics, and ultimately, her symptoms resolved. Five patients developed hyperthyroidism consistent with autoimmune thyroiditis. IL-2 was discontinued if hyperthyroidism was detected before completion of the IL-2 course. Patients 26 (group 2) and 30 (group 1) were removed from the study due to disease progression. Patient 26 developed visceral metastases (stage IV) before the initiation of therapy. His treatment is considered to have failed in the clinical assessments, but he was excluded from immunologic assessments. Patient 30 was withdrawn from the study after five vaccines.
Immune Response Data in PBL and SIN In many cases, the T-cell response to vaccination peaked after the third vaccine, then declined (Fig 2B and C). Patterns of T-cell response over time varied among patients, and varied among responses to different peptides for the same patient (Fig 2). We defined these patterns of response as falling into five categories: absent (YLEPGPVTA, Figs 2B and D), transient (YMDGTMSQV, Fig 2B; and ALLAVGATK, Fig 2C), persistent (DAEKSDICTDEY, Fig 2C), intermittent (DAEKSDICTDEY, Fig 2D), and increasing (Fig 2A; YMDGTMSQV, Fig 2D). Considering the entire patient population, T-cell responses were observed in 69% of patients, with 45% of PBL positive, and 61% of SIN positive (Table 3). Among those patients with T-cell responses detected in PBL, the best response was transient in 10 patients (53%), persistent in four patients (21%), increasing in three patients (16%), and intermittent in two patients (11%; data not shown).
Fifty-three percent of patients with T-cell responses detected in the SIN had T-cell responses detected in PBL (Table 4). However, 71% of patients with responses detected in PBL also had responses detected in the SIN. The magnitude of T-cell response was compared in PBL and in the SIN for all patients with any evidence of immunogenicity in PBL or SIN. The magnitude of the T-cell response was higher in the SIN than in the comparable PBL from the same patients (Table 5) .
When we first introduced the concept of evaluating the SIN as a measure of vaccine immunogenicity, we did not have access to "nonsentinel" nodes as negative controls. In the present study, we were able to collect such nodes and to evaluate T-cell responses to peptides in these "random" nodes in comparison to responses in the SIN. When there was a response in the SIN, the nonsentinel node was either negative or had a much lower T-cell response than in the SIN (data not shown, manuscript in preparation). These data suggest that evaluation of the SIN is usually a more sensitive measure of immunogenicity than evaluation of the PBL. However, these data also support evaluation of both the PBL and the SIN as a more complete assessment of immunogenicity than evaluation of either compartment alone.
T-Cell Responses to Individual Peptides
The magnitude of CTL responses measured by ELIspot has been quantified, for formal statistical analyses, based on the fold-increase over background values. The magnitude of these ELIspot measures is also presented as the number of spots counted per 100,000 cells plated, subtracting the maximum of two negative controls (Nvax – Nneg; Fig 4). The highest values are for the SIN in group 2, and the most immunogenic peptides are DAEKSDICTDEY and YMDGTMSQV.
T-Cell Responses for the Two Treatment Groups In assessing the T-cell responses to peptides between the two treatment groups, it is most illustrative to assess responses in the SIN 1 week after the third vaccine, when IL-2 had been administered for 3 weeks to group 1 and had not yet been administered to group 2. We hypothesized that the addition of IL-2 would substantially increase the magnitude of the measured T-cell response in group 1 patients as compared with group 2 patients. Overall, responses were present in either SIN or PBL less commonly in group 1 (55%, 11 of 20) than in group 2 (84%, 16 of 19; P = .048; Table 3). The primary end point for comparison of the two groups was in the SIN, in which responses were less frequent in group 1 (38% v 83%; P = .008; Table 3). Similarly, responses in PBL were less frequent in group 1 (37% v 53%; P = .33; Table 3). In assessing the magnitude of response between treatment groups (Fig 4), responses were of a higher magnitude in group 2 in the SIN (P = .015) and SIN or PBL (P = .006), but not in the PBL alone (P = .18). The data were also analyzed using a more stringent criterion for positivity, for which a response must be at least 3-fold the prevaccine level, rather than 2-fold. This accounts, in part, for differences in both frequency and magnitude of response. In this assessment, the proportion of overall positive responses in group 2 decreases from 84% to 79%, and in group 1 decreases from 55% overall to 45% (P values for differences between the two treatment arms for PBL and SIN are .044 and .024, respectively; Table 3). When the responses are assessed for each of the four class I-restricted peptides, the responses to the two tyrosinase peptides were more common in group 2, but the few observed responses to the gp100 peptides were seen only in the patients with upfront IL-2 (Fig 3). The median response magnitudes in the SIN were 2.7 times and 19.2 times higher in group 2 for DAEKSDICTDEY and YMDGTMSQV, respectively, revealing that most of the responses observed in the group 1 patients were of very low magnitude (data not shown).
Clinical Outcomes
We also determined whether clinical failure correlated with failure to develop cellular immune responses to vaccination. As presented in Table 6, at the time of follow-up, 50% of patients without T-cell responses remained alive, and 42% remained clinically free of disease, but those values were slightly higher at 74% and 70%, respectively, for patients with T-cell responses. For those patients with T-cell responses detected at two or more different time points, those values were 78% and 78%, respectively.
Of those patients who had disease recurrence and who remained alive with disease or who died, 46% had a positive T-cell response in the SIN. In contrast, of those patients who had disease recurrence and were rendered clinically free of disease by surgery and remain with no evidence of disease, 83% had CTL responses in the SIN (Table 7).
In the present study, we report the immunologic results after vaccination with a mixture of four peptides from the melanocytic differentiation proteins gp100 and tyrosinase. Immune responses were detected against all four peptides, confirming their immunogenicity in vivo. However, the tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY) and HLA-A2 (YMDGTMSQV) were more immunogenic than the gp100 peptides restricted by HLA-A2 (YLEPGPVTA) and by HLA-A3 (ALLAVGATK). The low immunogenicity of the gp100280-288 (YLEPGPVTA) peptide has been observed in prior studies1,2 and may be attributable in part to relatively low affinity for the HLA-A2 molecule.13 In a smaller study, there was a higher rate of immune responses to the ALLAVGATK peptide,2 and the present study included only eight HLA-A3+ patients; so the confidence intervals of the low response rate detected are wide. Regardless, both of the tyrosinase peptides were immunogenic in a majority of patients; in HLA-A1 or -A2+ patients, T-cell responses were observed in the PBL or the SIN in approximately 75% of patients.
Slightly more than one-third of responses detected by the combined evaluation of PBL and SIN would have been missed if T-cell responses had been assessed only in the PBL (Table 3). Thus, addition of the SIN evaluation increases the detectable immunogenicity approximately 1.5-fold. The responses in the SIN were also higher in magnitude than responses in the PBL (Table 4). We defined positive responses as those in which the IFN- The principal end point for the immunologic analyses for this study was based on ELIspot assays performed after a single in vitro sensitization. In other studies, we have detected T-cell responses directly ex vivo by ELIspot assay and by tetramer analysis1,2 after peptide vaccination (data not shown). However, tetramers for the tyrosinase240-251S (DAEKSDICTDEY) peptide, as provided from the National Institute of Allergy and Infectious Diseases core facility, were not stable. Thus, it was not possible to evaluate the entire patient population with tetramers. Direct ex vivo ELIspot assays have lower sensitivity than ELIspot assays performed on sensitized lymphocytes. Thus, we have focused the present study on ELIspot assays performed after one in vitro sensitization. Since PBLs were almost always negative prevaccine, it can be inferred that the in vitro sensitization alone was inadequate to induce a positive ELIspot assay. Thus, positive responses are the result of in vivo exposure to peptides in the vaccine, amplified ex vivo. A major aim of the present study was to assess the value of low-dose IL-2 as a systemic adjuvant to a peptide vaccine for melanoma. Because of strong murine data for the adjuvant effect of low-dose IL-2, we designed the current trial to permit administration of low-dose IL-2 to all patients, but to randomize patients to receive it either concurrently (weeks 1 to 7) with the vaccines or on a delayed schedule (weeks 4 to 10). The primary end point of this design was the response in the SIN. Patients in group 1 (upfront IL-2) had received IL-2 daily for 2 weeks (14 doses) before harvest of the SIN; whereas patients in group 2 (delayed IL-2) had not received IL-2. Thus, the comparison of ELIspot data in the SIN represents a comparison of the effect of IL-2. We hypothesized that the addition of IL-2 would increase the CTL response detected by ELIspot by at least 40%, and designed the trial to detect this difference. However, the findings are that T-cell responses were detected in the SIN of 83% of patients in group 2, but in only 38% in group 1. This is a 2-fold decrease in the patients who had received IL-2 (P = .008; Table 3). This included a decrease in the response rate to DAEKSDICTDEY (100% v 75%), and a decrease in the response rate to YMDGTMSQV (88% v 30%; Fig 3A). No responses to YLEPGPVTA or ALLAVGATK were detected in the SIN in either group. Furthermore, the magnitude of the T-cell response in the SIN was lower in group 1 (P = .015, Figs 4C and D). In addition to comparing T-cell responses in the SIN between the two treatment groups, we have the ability to compare T-cell responses in the PBL between groups. If responses in the PBL were discordant with the response in the SIN and were increased by the low-dose IL-2, then one could hypothesize that effects on trafficking may be beneficial to the immune response. However, responses in the PBL were also lower in group 1 than in group 2 (37% v 53%; Table 3 and Fig 3). These patients were clinically free of disease, so it is unlikely that trafficking of antigen-specific T cells to tumor deposits could significantly affect the T-cell responses detected in the periphery or in the SIN. Thus, a comprehensive assessment of T-cell responses to this peptide vaccine has been accomplished, and suggests that low-dose IL-2 at 3 MU/m2/d is not a useful adjuvant for increasing T-cell responses to a peptide vaccine when administered on the schedule used in group 1. Potential mechanisms for decreasing T-cell responses to vaccination by this low-dose IL-2 regimen include activation-induced cell death, alteration in the immunologic milieu by secondary cytokine cascades, or downregulation of IL-2 receptor expression before full T-cell activation. Studies to evaluate these possible mechanisms are currently underway. The delayed regimen may have advantages by allowing initiation of the T-cell responses to the vaccine before addition of systemic IL-2. The improved clinical outcome in patients receiving low-dose IL-2 on a delayed schedule may be due either to beneficial effects of IL-2 administered after immune responses are initiated, or to the absence of negative effects of early IL-2. Future trials without IL-2 or with IL-2 in other dose regimens are warranted. An important conclusion is that there is substantial interpatient variability in immune responses to each peptide. There are also intrapatient variations in the response to different peptides. Thus, it is unlikely that a vaccine incorporating only a single peptide for a given MHC molecule will be universally immunogenic. Considering this fact, plus the heterogeneity of antigen expression in tumor deposits, it is likely that vaccination with multiple different peptides for each MHC restriction will be more likely to provide tumor protection in a large proportion of patients. However, the findings about vaccine efficacy derived from studies like the present one will aid in optimizing vaccine delivery strategies and adjuvants. As shown in Figure 2, T-cell responses in blood varied over time, often peaking after vaccine 3, then decreasing after vaccine 6 (Fig 2B). In some cases, there was simply a gradual increase throughout the sequence of vaccines (Fig 2A). On average, the assays after vaccine 6 were performed 25 days after that vaccine, when the patient returned for follow-up. It is noteworthy that many patients on the vaccine trial who developed T-cell responses after the third vaccine still had positive responses almost a month after the last vaccine. This evidence that the effects of vaccination may persist beyond the period of vaccination is encouraging, and supports the vaccination approach used in this study. We also have collected blood from multiple time points, up to 2 years from the first vaccine. Evaluation of T-cell responses in these PBL samples, and in any sites of recurrent tumor that may arise, will be helpful in determining the longevity of responses against each of the peptides in this vaccine preparation, and may provide a basis for proposing a schedule for booster vaccinations to enable even more long-lived responses. In summary, the present study supports the safety and immunogenicity of four melanoma peptides derived from gp100 and tyrosinase, and demonstrates the greater immunogenicity of the two tyrosinase peptides. A major end point of this study was to assess the effect of low-dose IL-2 on the immunogenicity of this peptide mixture. The hypothesis that low-dose IL-2 would increase T-cell response is not supported by the data. Instead, it seems that the low-dose IL-2 regimen used for group 1 in this study paradoxically diminishes the magnitude and frequency of CTL responses to these peptides. The data from this study support the value of measuring T-cell responses in the SIN, in addition to PBL.
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Received more than $2,000 a year from a company for either of the last 2 years: Craig L. Slingluff Jr, Chiron Corp, Immunex/Berlex; William W. Grosh, Chiron Corp.
The authors would like to thank Carol Block for her generous assistance in quality assurance studies for peptide lot release, Dr Kyo Chu for performing one of the sentinel immunized node biopsies, and Melanie Mayer for secretarial and administrative assistance.
Supported by the Chiron Corp and by National Institutes of Health/National Cancer Institute grant R01 CA57653 (C.L.S.). Support was also provided by the Cancer Research Institute by provision of infrastructure support for the University of Virginia Human Immune Therapy Center; the University of Virginia Cancer Center Support Grant (NIH/NCI P30 CA44579, Clinical Trials Office, Tissue Procurement Facility, Biomolecular Core Facility); the University of Virginia General Clinical Research Center (NIH M01 RR00847); and the Pratt Fund at the University of Virginia. Authors' disclosures of potential conflicts of interest are found at the end of this article.
1. Yamshchikov GV, Barnd DL, Eastham S, et al: Evaluation of peptide vaccine immunogenicity in draining lymph nodes and blood of melanoma patients. Int J Cancer 92:703-711, 2001[CrossRef][Medline]
2. Slingluff CL Jr, Yamshchikov GY, Barnd DL, et al: Clinical and immunologic results of a randomized phase II trial of vaccination utilizing four melanoma peptides either administered in GMCSF-in-adjuvant or pulsed on dendritic cells. J Clin Oncol 21:4016-4026, 2003
3. Spitler LE, Grossbard ML, Ernstoff MS, et al: Adjuvant therapy of stage III and IV malignant melanoma using granulocyte-macrophage colony-stimulating factor. J Clin Oncol 18:1614-1621, 2000 4. Scheibenbogen C, Schmittel A, Keilholz U, et al: Phase 2 trial of vaccination with tyrosinase peptides and granulocyte-macrophage colony-stimulating factor in patients with metastatic melanoma [published erratum appears in J Immunother 2000 Jul-Aug;23(4):509]. J Immunother 23:275-281, 2000 5. Jager E, Ringhoffer M, Dienes HP, et al: Granulocyte-macrophage-colony-stimulating factor enhances immune responses to melanoma-associated peptides in vivo. Int J Cancer 67:54-62, 1996[CrossRef][Medline] 6. Ahlers JD, Dunlop N, Alling DW, et al: Cytokine-in-adjuvant steering of the immune response phenotype to HIV-1 vaccine constructs: Granulocyte-macrophage colony-stimulating factor and TNF-alpha synergize with IL-12 to enhance induction of cytotoxic T lymphocytes. J Immunol 158:3947-3958, 1997[Abstract] 7. Stidham KR, Ricci WM, Vervaert C, et al: Modulation of specific active immunization against murine melanoma using recombinant cytokines. Surg Oncol 5:221-229, 1996[Medline] 8. Farace F, Angevin E, Dietrich PY, et al: Low-dose IL-2 treatment: Activation of discrete T- and NK-cell sub-populations in vivo. Int J Cancer 62:523-528, 1995[Medline]
9. Jacobson EL, Pilaro F, Smith KA: Rational interleukin 2 therapy for HIV positive individuals: Daily low doses enhance immune function without toxicity. Proc Natl Acad Sci U S A 93:10405-10410, 1996 10. 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 [see comments]. Nat Med 4:321-327, 1998[CrossRef][Medline]
11. Kittlesen DJ, Thompson LW, Gulden PH, et al: Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: Implications for tumor vaccine development [published erratum appears in J Immunol 1999 Mar 1;162(5):3106]. J Immunol 160:2099-2106, 1998
12. Skipper JC, Hendrickson RC, Gulden PH, et al: An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp Med 183:527-534, 1996
13. Cox AL, Skipper J, Chen Y, et al: Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines. Science 264:716-719, 1994 14. Skipper JC, Kittlesen DJ, Hendrickson RC, et al: Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp100. J Immunol 157:5027-5033, 1996[Abstract]
15. Slingluff CL Jr, Yamshchikov GV, Neese P, et al: Phase I trial of a melanoma vaccine with gp100 280-288 and tetanus helper peptide in adjuvant: Immunologic and clinical outcomes. Clin Cancer Res 7:3012-3014, 2001 16. Storkus WJ, Salter RD, Cresswell P, et al: Peptide-induced modulation of target cell sensitivity to natural killing. J Immunol 149:1185-1190, 1992[Abstract] 17. Anderson KS, Alexander J, Wei M, et al: Intracellular transport of class I MHC molecules in antigen processing mutant cell lines. J Immunol 151:3407-3419, 1993[Abstract] 18. Blum-Tirouvanziam U, Servis C, Habluetzel A, et al: Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum. J Immunol 154:3922-3931, 1995[Abstract] 19. Woodson EMH, Chianese-Bullock KA, Wiernasz CJ, et al: Assessment of the toxicities of systemic low-dose IL-2 administered in conjunction with a melanoma peptide vaccine. J Immunother 27:380-388, 2004 Submitted October 31, 2003; accepted August 25, 2004. This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
INTRODUCTION
), or radiation administered within the preceding 1 month; known or suspected allergies to vaccine components; multiple brain metastases; use of steroids; class II, III or IV heart disease; hyperthyroidism; active connective tissue disease requiring medication; or other severe autoimmune disease. 
0.15% of CD8+ cells); (2) Rvax more than 2; (3) (Nvax – 1 standard deviation) 
(Nneg + 1 standard deviation); and (4) Rvax after vaccination 
2 test, and differences in the magnitude of response were assessed using the Wilcoxon rank sum test. 
