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Adjuvant Immunization of HLA-A2–Positive Melanoma Patients With a Modified gp100 Peptide Induces Peptide-Specific CD8⁺ T-Cell Responses

John W. Smith, II, Edwin B. Walker, Bernard A. Fox, Daniel Haley, Ketura P. Wisner, Teri Doran, Brenda Fisher, Lisa Justice, William Wood, John Vetto, Holden Maecker, Annemiek Dols, Sybren Meijer, Hong-Ming Hu, Pedro Romero, W. Gregory Alvord, Walter J. Urba

From the Providence Portland Medical Center, Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, and Oregon Health Sciences University, Portland, OR; Ludwig Institute for Cancer Research-Division of Oncology-Immunology, Lausanne, Switzerland; Data Management Services, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD; and Becton-Dickinson Biosciences, San Jose, CA.

Address reprint requests to John W. Smith II, MD, Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, 4805 NE Glisan St, 5F40, Portland, OR 97213-2967; email: josmith{at}providence.org.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: To measure the CD8⁺ T-cell response to a melanoma peptide vaccine and to compare an every-2-weeks with an every-3-weeks vaccination schedule.

Patients and Methods: Thirty HLA-A2–positive patients with resected stage I to III melanoma were randomly assigned to receive vaccinations every 2 weeks (13 vaccines) or every 3 weeks (nine vaccines) for 6 months. The synthetic, modified gp100 peptide, g209–2M, and a control peptide, HPV16 E7, were mixed in incomplete Freund’s adjuvant and injected subcutaneously. Peripheral blood mononuclear cells obtained before and after vaccination by leukapheresis were analyzed using a fluorescence-based HLA/peptide-tetramer binding assay and cytokine flow cytometry.

Results: Vaccination induced an increase in peptide-specific T cells in 28 of 29 patients. The median frequency of CD8⁺ T cells specific for the g209–2M peptide increased markedly from 0.02% before to 0.34% after vaccination (P < .0001). Eight patients (28%) exhibited peptide-specific CD8⁺ T-cell frequencies greater than 1%, including two patients with frequencies of 4.96% and 8.86%, respectively. Interferon alfa-2b–treated patients also had significant increases in tetramer-binding cells (P < .0001). No difference was observed between the every-2-weeks and the every-3-weeks vaccination schedules (P = .59).

Conclusion: Flow cytometric analysis of HLA/peptide-tetramer binding cells was a reliable means of quantifying the CD8⁺ T-cell response to peptide immunization. This assay may be suitable for use in future trials to optimize different vaccination strategies. Concurrent interferon treatment did not inhibit the development of a peptide-specific immune response and vaccination every 2 weeks, and every 3 weeks produced similar results.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
MANY PATIENTS with cutaneous malignant melanoma remain at significant risk of distant recurrence and death following primary surgical excision. For patients at the highest risk of recurrence (eg, > 4 mm primary lesions or node-positive disease) interferon alfa-2b adjuvant therapy has been shown to improve disease-free survival.¹ For patients with melanomas 1 to 4 mm thick, this risk is lower but still significant at 15% to 40%.² These patients have been enrolled in clinical trials to assess the benefit of therapeutic tumor vaccines, which are attractive because of their low toxicity and the responsiveness of melanoma to many immunologically based treatments. A recently reported Southwest Oncology Group study of one such vaccine composed of a lysate of two melanoma cell lines (Melacine; Corixa Corp, Seattle, WA) failed to decrease relapse rates in all enrolled patients; however, a subgroup of patients who were positive for HLA-A2 and/or HLA-C3 may have benefited from the vaccine.³ Interest in the development of therapeutic vaccines for this group of patients remains high.

Patients in the trial reported herein were vaccinated with the synthetic modified gp100 peptide, gp100:209–217(210M) (hereafter referred to as g209–2M), which was created by altering the anchoring amino acid (methionine in place of threonine) at position 2 of the native gp100:209–217 peptide (hereafter referred to as g209).⁴ This modification enhanced the peptide’s affinity for HLA-A2 molecules and increased its immunogenicity in vitro⁴ and in vivo.⁵ Native gp100 is a nonmutated protein differentiation antigen expressed by cells of the melanocytic lineage including melanomas, normal melanocyte, and pigmented retinal cells, but not by other normal tissues. T-cell responses to the native gp100 antigen have been noted in patients with metastatic melanoma who experienced tumor regression following adoptive therapy with tumor-infiltrating lymphocytes (TIL) and interleukin 2 (IL-2).⁶ When the g209–2M peptide was administered with incomplete Freund’s adjuvant (IFA) to 11 patients with metastatic melanoma, CD8⁺ T-cell responses to both the native and modified peptides were observed in 10 patients.⁷ Although none of the patients experienced an objective response, three patients exhibited mixed responses with regression of individual lesions. When vaccination was followed by high-dose IL-2 treatment in another group of 31 patients, the response rate was 42%—much higher than the historic response rate of IL-2 alone.⁷ The use of the g209–2M peptide (combined with a tyrosinase peptide) vaccine in the adjuvant setting was studied by Lee et al,⁸ who administered a total of eight vaccines over 26 weeks to 48 completely resected stage III or IV melanoma patients. They demonstrated minimal toxicity to the repetitive vaccinations and reported delayed-type hypersensitivity (DTH) skin test responses as well as antigen-specific T-cell responses in nearly all the patients.

The detection of peptide-specific T cells in the peripheral blood has been greatly simplified by the development of the fluorescence-based tetramer-binding assay.⁹ Tetramers are quaternary molecular constructs of four biotin-avidin linked sets of one synthetic HLA heavy chain and a single beta-2 microglobulin light chain folded with noncovalently bound peptide antigens containing eight to 10 amino acids.^9,10 After conjugation of fluorescent labels, tetramers can be used to specifically label T-cell receptor complexes of defined HLA and peptide specificity. Flow cytometry has been performed using peptide-antigen-specific, HLA class I–restricted, fluorescenated tetramers to enumerate circulating antigen-specific memory/effector CD8⁺ T cells in human peripheral blood without prior in vitro stimulation and expansion of T cells.^11–14 Tetramer staining and flow cytometry analysis have been used successfully to quantitate memory/effector T cells specific for cytomegalovirus, human immunodeficiency virus (HIV), and other viral peptides^15–17 and for the detection of CD8⁺ T cells specific for tumor-associated peptides in cancer patients.^18–20 The assay has a reported detection sensitivity of 1 in 10,000 cells or 0.01%.²¹ Because this fluorescence-based tetramer-binding assay had the potential to be more reproducible and quantitative than DTH skin testing, and because Weber⁸ had already shown that the g209–2M peptide induced a positive skin test in almost all patients, we omitted DTH testing from our study.

This report describes a pilot study in which patients with stage I to III melanoma were vaccinated with two HLA-A2–binding synthetic peptides (a modified-self gp100 and nonself human papilloma virus (HPV) 16E7 peptide). We present the clinical results of this study and a detailed description of the immunological monitoring performed with HLA-A2/peptide tetramers for both immunogens.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Eligibility Criteria
Patients with cutaneous malignant melanoma 1 mm thick were eligible for this study if they had no evidence of distant metastatic disease and were HLA-A2–positive. Patients determined to be at high risk for recurrence (lesion > 4 mm in thickness or presence of lymph node metastases) were offered interferon alfa (IFN) along with their vaccinations. Patients had to have a Karnofsky performance status of 80, good organ function, and no requirement for treatment with systemic corticosteroids.

Pretreatment Testing
Before enrollment, patients had the following blood tests: complete blood count, complete metabolic panel including lactate dehydrogenase, and HLA-A2 testing. Testing for HIV and screening for hepatitis B and C were performed before leukapheresis. Peripheral blood mononuclear cells were collected at 1 mL/min and less than 3% colorgram during a 3-hour leukapheresis performed before vaccine therapy by the Red Cross to obtain sufficient peripheral blood mononuclear cells for immunologic assays. Imaging studies were done if distant metastatic disease was suspected, but they were not required. In addition to the standard history and physical examination, a baseline ophthalmologic exam was performed.

Treatment
Patients were vaccinated with subcutaneous injections of g209–2M peptide and a control peptide HPV16E7:12–20. The g209–2M peptide (National Security Code no. 683472) is an HLA-A2–restricted nine–amino acid epitope derived from melanoma antigen gp100 and has the amino acid sequence IMDQVPFSV. The peptide, which was supplied in vials containing l mL of a sterile l mg/mL solution for injection with 0.1 N HCL added to adjust the pH, was provided by the Cancer Therapy Evaluation Program (CTEP) under a National Cancer Institute (NCI) investigational new drug (IND) (BB 6123). HPV16E7:12–20 peptide (NSC no. 673925) is an HLA-A2–restricted nine–amino acid peptide of the HPV16E7 protein with the amino acid sequence MLDLQPETT. It was supplied as a 1 mg/mL concentration in vials containing 1 mL of the solution. The peptide was dissolved in sterile water for injection (United States Pharmacopeia). It was supplied by CTEP under a National Cancer Institute IND (BB 6349). Montanide ISA 51 (NSC no. 675756) is an oil-based adjuvant product similar to IFA, which, when mixed with a water-based solution, forms a water-in-oil emulsion. The product is manufactured by Seppic, Inc (Fairfield, NJ), and was provided by CTEP, NCI.

Each vaccine was prepared from frozen vials of the gp209–2M peptide that were thawed at room temperature. Once thawed, 1.2 mL of Montanide ISA 51 was added to the 1.2 mL of peptide solution and emulsified by vortexing for 12 minutes. The HPV16E7 peptide was prepared the same way except that it was initially refrigerated at 4°C and did not need to be thawed.

After the solution was prepared, 1 mL (containing 0.5 mg peptide) was withdrawn from the gp209–2M peptide/adjuvant emulsion and 1 mL (containing 0.5 mg peptide) was withdrawn from the HPV16E7 peptide/adjuvant emulsion and administered into the subcutaneous tissue of two separate sites near each other on one extremity. Another two 1-mL injections of the peptide emulsions were administered in the subcutaneous tissue of two separate sites near each other on a different extremity. Therefore, each time a patient was vaccinated, he or she received a total of 1 mg of each peptide and a total of 2 mL of Montanide ISA 51. In patients who were vaccinated before their sentinel lymph node (SLN) biopsy, one of the sites for the first two immunizations was the site of the primary melanoma. After SLN biopsy, vaccinations were rotated among all extremities except the limb that had undergone lymph node surgery. The abdomen and the upper buttock region were also used as injection sites. Patients were observed in the clinic for a minimum of 2 hours after the initial immunization and for 15 minutes after each subsequent vaccination.

Patients were randomly assigned to two different vaccination schedules: group A received vaccinations every 2 weeks for 6 months (13 total injections), and group B received vaccinations every 3 weeks for 6 months (nine total vaccinations). In patients in whom it had not already been performed, SLN biopsy was done approximately 10 days after the second vaccination in both groups. A wide local excision was done at the same time if one had not previously been performed. Patients whose lymph nodes contained metastatic melanoma also underwent a completion lymphadenectomy. Patients with positive lymph nodes or primary lesions more than 4 mm thick were permitted to receive adjuvant high-dose IFN at the discretion of the treating physician at the standard dose and schedule according to the package insert.

All patients underwent a leukapheresis similar to the pretreatment leukapheresis 2 to 4 weeks after the completion of vaccine therapy. Patients were seen every 3 months for the remainder of the first year, every 4 months in the second year, and every 6 months thereafter. This protocol was reviewed by CTEP, NCI, and approved by the Providence Health System institutional review board. All patients voluntarily gave their written informed consent before they were screened for eligibility.

Tetramer-Binding Assay
The tetramer-binding assessment of CD8⁺ T cells was performed using four-color flow cytometry analysis. Cell surface staining was carried out using fluorescein isothiocyanate (FITC) anti-CD56, peridinin chlorophyll protein (PerCP)–conjugated anti-CD3, allophycocyanin (APC)-conjugated anti-CD8, phycoerythrin (PE)-labeled gp100 (209–2M), and HPV 16E7:12 to 20 peptide or HIV (polymerase [pol]) HLA-A2–restricted tetramer reagents. The gp209–2M tetramer reagent and a correlated HIV (pol)-negative control tetramer were provided by Dr. Pedro Romero (Ludwig Institute-Lausanne, Switzerland); the HPV tetramer reagent and its associated HIV (pol) control tetramer were purchased from Beckman-Coulter Immunomics (San Diego, CA). Tetramer staining analysis data were collected on 5 x 10⁴ to 1 x 10⁵ gated CD8⁺/CD3⁺ T lymphocytes and are expressed in the data tables and figures as the percentage of total CD8⁺ T cells that are positive for tetramer fluorescence for each patient. Tetramer analysis was performed on cryopreserved peripheral-blood mononuclear cells (PBMCs) from the leukapheresis collections before and after vaccination. All tetramer data are expressed as the net specific HPV or gp209–2M tetramer staining after the nonspecific HIV (pol)-mediated tetramer staining is subtracted from each sample.

Cytokine (IFN) Flow Cytometry Assay
Cryopreserved PBMCs from vaccinated patients were stimulated directly ex vivo with recall g209–2M peptide antigen or the native g209 peptide to determine the number of functionally responsive CD8⁺ T cells as measured by the expression of intracellular IFN. Briefly, cells were thawed and washed twice with cold medium (RPMI 1640 containing 10% heat-inactivated fetal calf serum). PBMCs were plated at 1 x 10⁶ cells in 170 µL of medium per well in a 96-well V-bottom tissue culture plate and allowed to rest overnight at 37°C in a humidified CO₂ incubator (5% CO₂). g209–2M peptide or the native g209 peptide at 2 µg/well and brefeldin A at 2 µg/well (10 µg/mL) were added together in a 30-µL volume to give a final well volume of 200 µL. Cells were incubated (37°C and 5% CO₂) for an additional 5 hours. Before collection and analysis, EDTA was added to each well (20 µL of 20 mmol/L EDTA) and incubated at room temperature for 10 minutes to promote recovery of adherent T cells. Cells were centrifuged in the 96-well plate for 5 minutes at 400 x g and supernatants were aspirated. Cells were than resuspended in 200 µL/well of 1x BD FACS permeabilization buffer (BD Biosciences, San Jose, CA) and incubated for 10 minutes at room temperature. Cells were washed twice (200 µL fluorescence-activated cell sorting [FACS] wash buffer/centrifugation at 500 x g for 5 minutes) and resuspended in 200 µL of FACS wash buffer before monoclonal antibody staining. Cells were stained in the dark at room temperature for 30 minutes using a cocktail of fluorochrome-conjugated mouse antihuman antibodies for IFN-FITC (25723.11, immunoglobulin G [IgG_2b]), CD69-PE (L78, IgG₁), CD8-PerCp-Cy5.5 (SK1, IgG₁), and CD3-APC (SK7, IgG₁). A separate cocktail of isotype-matched controls IgG_2a-FITC (X39), IgG₁-PE (X40), IgG₁-CD8-PerCp-Cy5.5 (SK1), and IgG₁-CD3-APC (SK7) were run in parallel with every test sample. Stained cells were washed in flow wash buffer and fixed in 1% paraformaldehyde for 1 hour at 4°C before acquisition on the same day. Cell acquisition and analysis were performed on the FACSCalibur instrument using CellQuest Pro software (BD Biosciences). Acquisition was performed in list mode by gating on 20,000 CD8⁺-positive T cells within the typical viable lymphocyte region assigned using the forward and side scatter plot.

Statistical Methods
Pre- and postimmunization T-cell immunity to g209–2M peptide, to HPV16E7 peptide, and to a negative control HLA-A2 HIV peptide (pol) were assessed using HLA-A2/peptide tetramer-specific binding analysis. Within-subject analyses were performed to determine differences between pre- and postimmunization responses to the g209–2M and HPV peptides and to the negative control HIV peptide after completion of 6 months of vaccination. Because of heterogeneity in the frequency of postimmunization peptide-specific T cells, we report probabilities obtained from nonparametric Wilcoxon signed rank tests for within-subject pre- versus postimmunization responses. The study was designed with a planned comparison of the responses of group A (vaccination every 2 weeks) to the responses of group B (vaccination every 3 weeks). Pre- versus postimmunization response differences were used as criterion measures in between-group (among subjects) analyses. Because of heterogeneity in difference scores, we report probabilities from Wilcoxon rank sum tests for between-group differences. Several unplanned exploratory comparisons were also made; for example, the responses of patients also receiving interferon versus those not receiving interferon, and the responses of node-positive patients versus node-negative patients, elderly patients versus younger patients, and men versus women. As a general strategy to control for possible compounding type I errors in hypothesis tests, we report probabilities less than .01 as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Demographics
Thirty eligible patients were enrolled on this study beginning in May 1999 and ending in May 2001. All patients were evaluable for toxicity and recurrence. There were 17 men and 13 women with a median age of 51 years and a median Karnofsky performance status of 100. Eighteen of the 30 patients (60%) had stage III (node-positive) disease. The patient characteristics are summarized in Table 1 and were similar in both groups A and B. The main differences were the younger median age and the predominance of men in group A.

Clinical Outcome
As of August 1, 2002, the median time on study of the entire patient population is 23 months. Seven patients (all originally stage III) have suffered recurrence of their disease; four patients from group A and three patients from group B. Four patients have died from progressive melanoma (two patients in each group). Three patients are alive with recurrent disease. One patient had an isolated lung nodule resected, which stained positive for gp100, and later developed liver metastases. He is currently receiving biochemotherapy. Another patient had an isolated liver metastasis treated with radiofrequency ablation and later developed brain metastases. The third patient recurred in a regional lymph node basin that was treated with radiation and surgery. The other 23 patients (77%) remain free of recurrent disease.

Immunologic Responses
All but one patient underwent pretreatment leukapheresis, and 23 patients underwent posttreatment leukapheresis. PBMC obtained from peripheral blood draws or leukaphereses performed before vaccination and 2 to 4 weeks after the final vaccine were analyzed for the presence of CD8⁺ T cells capable of binding the g209–2M and HPV16E7 peptide HLA-A2/tetramers. The cryopreserved cells of one patient in group A were not viable after repeated attempts at thawing; therefore, this patient was excluded from the immunologic analysis. Figure 1A depicts the tetramer staining of PBMCs from patient EA8, who exhibited the best response. Neither the pre- nor postvaccination CD8⁺ T cells from this patient exhibited increased binding to the control HIV (pol) tetramers. After nine vaccinations, 8.86% of this patient’s circulating CD8⁺ T cells bound g209–2M peptide tetramers. EA8 also mounted a significant response to HPV, with 2.06% of the CD8⁺ T cells binding the HPV tetramer. Two sets of HIV (pol) controls were included because the tetramers for g209–2M and HPV16E7 were obtained from different sources and therefore might be expected to exhibit different levels of background staining. Figure 1B depicts the results from patient EA10, who exhibited a less pronounced response. Again, there was little binding to the control HIV (pol) peptide either before or after vaccination, but there was a significant expansion of g209–2M- and HPV-specific CD8⁺ T cells after vaccination. Note that even though the frequency of g209–2M tetramer–positive CD8⁺ T cells was low (0.78%) after vaccination, the tetramer (PE)-specific staining was bright and well resolved in this histogram depicting events from 50,000 gated CD8⁺ T cells, and the frequency is significantly greater than the frequency of tetramer-binding cells present before vaccination. This level of discrimination was typical of the responses seen in other patients and permitted the detection of responses at levels of 1 tetramer-positive cell/1,000 CD8⁺ T cells.

View larger version (73K): [in this window] [in a new window]   Fig 1. Tetramer-binding of pre- and postvaccination unstimulated peripheral-blood mononuclear cells from patients (A) EA8 and (B) EA10. The ordinate depicts CD8 and the abscissa depicts binding of the indicated peptide tetramers. Human immunodeficiency virus control appears twice because g209-2M and human papilloma virus tetramers were obtained from two different sources (see Methods).

View larger version (36K): [in this window] [in a new window]   Fig 2. The net percentage of CD8+ T cells positive for the g209-2M peptide-tetramer before and after vaccination for patients from (A) group A (2-week schedule) and (B) group B (3-week schedule). The net percentage is the percentage of g209-2M peptide-tetramers minus the percentage of human immunodeficiency virus (polymerase) peptide-tetramers.

View larger version (37K): [in this window] [in a new window]   Fig 3. The net percentage of CD8+ T cells positive for the g209-2M peptide-tetramer before and after vaccination for patients (A) who did not and (B) who did receive concurrent adjuvant interferon alfa. The net percentage is the percentage of g209-2M peptide-tetramers minus the percentage of human immunodeficiency virus (polymerase) peptide-tetramers.

In addition to the modified self-gp100 peptide, all patients were vaccinated with an A2-binding foreign peptide from HPV. Figure 4 presents a comparison of the tetramer staining for the foreign HPV16E7 peptide along with the g209–2M peptide postvaccination for 22 patients from groups A and B who were tested for responses to both peptides. All 22 patients responded to the g209–2M peptide and 20 of 22 patients made an immune response to the HPV peptide vaccine after immunization (P < .0001). Patients EA19 and EA30 responded to the gp100 epitope but not to HPV. The single patient who failed to respond to g209–2M was not tested for HPV response because of insufficient cells. Overall, there was no difference between the median responses to the g209–2M peptide and HPV peptides (P = .92). However, the response to the g209–2M peptide and the HPV peptide within an individual was not always the same; nine patients made a stronger immune response to the g209–2M peptide than to the HPV peptide, three patients made essentially the same response, and 10 patients made a stronger response to the HPV peptide. If one focuses on the group of 11 patients that had a g209–2M peptide tetramer response of less than 0.5%, five patients had a greater response to HPV, four patients had an equal response, and two patients had a greater response to g209–2M peptide.

View larger version (44K): [in this window] [in a new window]   Fig 4. The net percentage of CD8+ T cells positive for the g209-2M peptide-tetramer and the HPV peptide-tetramer before and after vaccination for selected patients from (A) group A and (B) group B. The net percentage is the percentage of g209-2M or HPV peptide-tetramers minus the percentage of human immunodeficiency virus tetramers.

View larger version (48K): [in this window] [in a new window]   Fig 5. The top panel shows the g209-2M peptide-tetramer and CD8+ staining from four patients for postvaccination peripheral-blood mononuclear cells that were also thawed and stimulated in vitro for 5 hours with the g209-2M peptide and stained to detect the presence of CD8+ and intracellular interferon gamma (bottom panel).

View larger version (30K): [in this window] [in a new window]   Fig 6. The net percentage of CD8+ interferon gamma-positive T cells in response to a 5-hour stimulation in vitro with the g209-2M peptide and with the native g209 peptide at 4 µg/well for nine patients after vaccination.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

One concern about immunization with a synthetically modified peptide was that the g209–2M peptide-specific T cells might not respond to the native g209 peptide. The nine patients we tested for intracellular IFN production by cytokine flow cytometry (CFC) responded to both the native and modified peptide, albeit to a lesser degree with the native peptide. This result parallels the findings of Marincola,²⁴ who demonstrated by tetramer binding analysis the postimmunization induction of T cells specific for the native g209 peptide as well as the modified g209–2M peptide. We did not have a native g209 peptide tetramer reagent available to run in parallel with the g209–2M peptide tetramer, so we were unable to determine the differences in g209 and g209–2M tetramer binding cells. In addition, we have been able to show that the tetramer-binding CD8⁺ T cells induced by peptide vaccination produced IFN in response to stimulation with A2-positive/gp100-positive melanoma cell lines, demonstrating that T cells produced in response to vaccination with the modified peptide recognized naturally processed antigen on the surface of tumor cells.²⁵

In previous studies, when the g209–2M peptide in IFA was used to immunize patients with metastatic melanoma, the majority responded to both the native and the modified g209–2M peptide.⁷ However, immune responses were not observed in freshly isolated uncultured lymphocytes; they were detected only after restimulation with the immunizing peptide in vitro for at least 4 days. The in vitro stimulation and expansion of T cells with antigenic peptides and cytokines can alter the frequency and functional characteristics of these cells. Therefore, immune function and phenotype assays using in vitro stimulation–expanded T cells may not always reflect the fidelity of the in vivo antitumor immune response. Direct ex vivo measurement with peptide-HLA tetramer complexes provides a more accurate representation of the true frequency of circulating peptide-specific T cells in vivo.

Using HLA/g209–2M peptide tetramer analysis, Marincola²⁴ reported that all seven metastatic melanoma patients immunized with g209–2M peptide in IFA alone demonstrated significant increases in peptide-specific T-cell precursor frequencies after vaccination. The responses ranged from 0.2% to 2.4%, with two of the seven patients showing a response greater than 1%. After vaccination, all seven patients also exhibited an increased frequency of circulating CD8⁺ T cells that recognized the native g209 peptide, but at a lower frequency than for the modified peptide. In this study, similar increases, which ranged from 0.05% to 8.86%, were observed after vaccination; in eight of 29 patients (28%), more than 1% of the CD8⁺ T cells recognized the g209–2M peptide, including in two patients with the exceptionally high frequencies of 4.96% and 8.86%, respectively. Lee et al⁸ reported g209–2M peptide-specific CD8⁺ T-cell frequencies that ranged between 0% and 2.5% (mean, 0.03%) in 37 resected stage III or IV melanoma patients who received eight g209–2M peptide vaccines in IFA during 6 months. The higher frequency of peptide-specific T cells observed in our study compared with the study by Lee et al⁸ might be caused by the difference in patient population (there were no stage IV patients in our study) or by differences in the technique used in the tetramer assay used to detect the g209–2M peptide-specific CD8⁺ T-cells.

Although there was good correlation between the levels of CFC (IFN) response and tetramer staining for most of the patients studied (Fig 5), not all tetramer-binding cells in every patient will be functional. This is illustrated by the response of patient EA8, in whom less than 50% of the tetramer-binding cells produced IFN (Fig 5). They may produce other cytokines or could conceivably have become anergic, as reported by Lee et al²⁰ for circulating tetramer-binding CD8 cells in patients with metastatic melanoma. Because HLA-restricted peptide-specific tetramer staining permits the quantitative comparison of different vaccination strategies in different patient groups monitored by different laboratories, it could serve as at least one of the measurements by which an optimal vaccination strategy may be chosen.

Vaccination with the nonself HPV16E7 peptide was performed as a control to observe the immune response in T cells that would not have been subject to negative selection in the thymus. The fact that 20 of 22 patients (91%) studied responded to this peptide indicates that it could serve as a positive control in trials of other new peptide vaccines.

Previous clinical trials of vaccination with the g209–2M peptide showed that concomitant administration of IL-2, IL-12, or granulocyte-macrophage colony-stimulating factor actually decreased the circulating peptide-specific T-cell precursor frequency.²⁶ We were interested in whether the patients in our study who received IFN in addition to their vaccines would demonstrate a similar effect. Overall, no significant difference was seen in the two groups, indicating that IFN did not diminish the effectiveness of the g209–2M peptide in IFA vaccine, a finding that complements the observations of Kirkwood et al,²⁷ who showed that high-dose IFN did not diminish the antibody response to the GM2 ganglioside. In fact, we observed a trend toward enhancement of the vaccine by IFN, indicated by the observation that seven of 13 IFN-treated patients had more than 0.5% tetramer-positive cells versus five of 16 patients who did not receive IFN. It should be noted that patients were not randomly assigned to receive IFN and that this population could have been skewed because it generally excluded older, less medically fit patients who might also have had less response to immunization. However, we believe that the data support inclusion of IFN treatment in clinical trials of other immune strategies in patients with stage III melanoma.

The lack of tumor regression in patients with metastatic melanoma immunized with the g209–2M peptide alone may not have been a result of peripheral tolerance, but it could have been because the overall quantitative immune response was too low. The circulating T-cell response in viral infections or autoimmune disease models measured by epitope/HLA tetramers is much higher. In HIV patients, an inverse correlation has been reported between HIV-specific cytotoxic T lymphocyte frequency and the viral RNA load, and in recent studies,^15,16 patients whose frequency is approximately 2% remain asymptomatic. Furthermore, animal models indicate that the ability to clear tumor correlates with the intensity of the vaccine-elicited response.²⁸ The target for an immunization strategy in cancer patients is presently unknown, but it seems reasonable to attempt to achieve numbers of circulating tumor-specific memory/effector T cells in the range seen with infections. Despite the fact that our patient population had a low tumor burden and received nine to 13 vaccinations over 6 months, peptide-specific T-cell frequencies more than 1% and more than 2% were achieved in only 28% and 10% of patients, respectively. This indicates that better immunization strategies are necessary. Our study indicates that enumeration of vaccine-elicited T cells by HLA/peptide tetramers is an excellent way to evaluate improvements in vaccine development and immunization strategies. However, for optimal results, a functional assay such as the enzyme-linked immunospot assay or CFC should also be used.²²

	NOTES

 
Supported by NIH grant 1R21-CS 82614-01 (W.J.U.), the M.J. Murdock Charitable Trust, and the Chiles Foundation.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted September 4, 2002; accepted January 10, 2003.

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