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© 2003 American Society for Clinical Oncology 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
From the University of Chicago, Departments of Pathology and Medicine, Section of Hematology/Oncology, and the Ben May Institute for Cancer Research, Chicago, IL. Address reprint requests to Thomas F. Gajewski, MD, PhD, University of Chicago, 5841 S. Maryland Ave., MC2115, Chicago, IL 60637; email: tgajewsk{at}medicine.bsd.uchicago.edu.
Purpose: Preclinical studies showed that immunization with peripheral blood mononuclear cells (PBMC) loaded with tumor antigen peptides plus interleukin-12 (IL-12) induced CD8+ T-cell responses and tumor rejection. We recently determined that recombinant human (rh) IL-12 at 30 to 100 ng/kg is effective as a vaccine adjuvant in patients. A phase II study of immunization with Melan-A peptide-pulsed PBMC + rhIL-12 was conducted in 20 patients with advanced melanoma. Patients and Methods: Patients were HLA-A2–positive and had documented Melan-A expression. Immunization was performed every 3 weeks with clinical re-evaluation every three cycles. Immune responses were measured by ELISpot assay before and after treatment and through the first three cycles, and were correlated with clinical outcome. Results: Most patients had received prior therapy and had visceral metastases. Nonetheless, two patients achieved a complete response, five patients achieved a minor or mixed response, and four patients had stable disease. The median survival was 12.25 months for all patients and was not yet reached for those with a normal lactate dehydrogenase. There were no grade 3 or 4 toxicities. Measurement of specific CD8+ T-cell responses by direct ex vivo ELISpot revealed a significant increase in interferon gamma–producing T cells against Melan-A (P = .015) after vaccination, but not against an Epstein-Barr virus control peptide (P = .86). There was a correlation between the magnitude of the increase in Melan-A–specific cells and clinical response (P = .046). Conclusion: This immunization approach may be more straightforward than dendritic cell strategies and seems to have clinical activity that can be correlated to a biologic end point.
MOST MELANOMA tumors express antigens that can be recognized by CD8+ T cells.1,2 Nonetheless, tumors frequently escape immune destruction, either from a failure to generate an optimal tumor antigen-specific T-cell response or from development of resistance to the T-cell response induced. One strategy to overcome the former hurdle is through active immunization, the opportunity for which has been facilitated by the molecular definition of melanoma antigens.3 Specific CD8+ T cells that are properly activated can home to tumor sites and kill tumor cells, to the extent to which they can overcome negative immunoregulatory pathways and tumor resistance.4
The optimal immunization strategy for inducing tumor antigen-specific CD8+ effector T cells in humans remains undefined. However, antigen-presenting cell-based strategies have shown promise. Both monocyte-derived5,6 and bone marrow–derived7 dendritic cells (DCs) have been loaded with melanoma tumor antigens and administered in the advanced-disease setting, with evidence for immunization and tumor regression in subsets of patients. However, DCs are cumbersome to generate and alternative approaches that are more straightforward yet equally as effective would be useful. One cofactor produced by DCs that contributes to their efficacy is interleukin-12 (IL-12), which facilitates the induction of interferon gamma (IFN- In this article, we describe results of a phase II clinical study of immunization with Melan-A/MART-13 peptide-pulsed autologous PBMCs + rhIL-12 in HLA-A2–positive patients with advanced melanoma. Immune responses were analyzed using a direct ex vivo ELISpot assay. We show that this vaccine approach had clinical activity and that the magnitude of increased T-cell response correlated with clinical outcome.
Patient Enrollment and Eligibility This was an open-label, nonrandomized, single-institution study of Melan-A peptide-pulsed autologous PBMCs + rhIL-12.4 The protocol was approved by the University of Chicago Institutional Review Board and all patients signed written informed consent. Patients who were both HLA-A2–positive and showed Melan-A tumor expression by reverse transcriptase polymerase chain reaction (RT-PCR) were considered for inclusion. Additional inclusion criteria were life expectancy more than 12 weeks, Karnofsky performance status ≥70, and adequate hematopoietic, renal, and hepatic function. Delayed-type hypersensitivity (DTH) skin testing was performed against mumps, Candida, and Trichophytin, not for eligibility but to correlate subsequently with clinical outcome and immunization potential. Patients were excluded if they had severe cardiovascular disease or arrhythmia, were pregnant or nursing, had biologic therapy received within 4 weeks, tested positive for hepatitis B surface antigen or human immunodeficiency virus (HIV), had clinically significant autoimmune disease or any illness requiring immunosuppressive therapy, had a psychiatric illness that would interfere with patient compliance and informed consent, had active gastrointestinal bleeding or uncontrolled peptic ulcer disease, or had uncontrolled brain metastases. Patients with treated brain metastases who were clinically and radiographically stable and did not require corticosteroids were allowed to enter onto the trial.
Patient Characteristics
RT-PCR Analysis RNA was isolated from fresh tumor cells using guanidine and cesium chloride. cDNA was synthesized and PCR was performed for Melan-A and beta-actin using the primer pairs and reaction conditions described previously.21 Control reactions without reverse transcriptase were performed to rule out a contribution of genomic DNA. PCR products were visualized using a 1.5% ethidium bromide–stained agarose gel. No formal quantitation was performed.
Vaccine Preparation rhIL-12 was provided by Genetics Institute (Cambridge, MA) as a lyophilized powder of 10 µg under vacuum. Each vial was intended for single use only and was stored as a powder in our research pharmacy at 2 to 8°C until reconstituted with sterile water for injection. Once reconstituted, rhIL-12 was loaded into 3-mL syringes and used within 4 hours. rhIL-12 (4 µg) was administered sc with a 25-gauge needle just after pulsed PBMC inoculation and immediately adjacent to one of the two immunization sites on days 1, 3, and 5. The same approximate location was used for each injection of peptide-pulsed PBMCs and rhIL-12 for each cycle.
Toxicity Assessment and Criteria for Clinical Response
CD8+ T-Cell Preparation
ELISpot Assays
Statistical Analysis
Immunization Treatment and Toxicities Each 3-week cycle consisted of immunization on day 1 and sc rhIL-12 administration on days 1, 3, and 5, as described in Methods. Three cycles constituted one course of therapy and patients were evaluated for response after each course. Patients were observed as inpatients in our General Clinical Research Center for the first 24 hours of each cycle.
Adverse reactions are listed in Table 2
Clinical Outcome Clinical response outcomes are listed in Table 3  . Two patients had a CR, for an overall response rate of 10%. In addition, four patients (20%) had a mixed response, one patient (5%) had an MR, four patients (20%) had SD, and the remaining nine patients (45%) had PD. The sites of tumor response were diverse. The two patients who experienced a CR both had numerous metastases of 2 cm or less and a normal LDH. One patient was female, had multiple cutaneous lesions, and no prior therapy; the other patient was male, had multiple lung lesions, and had experienced prior treatment failure from chemoimmunotherapy. Neither patient experienced a recurrence with a mean follow-up time of 28 months at the time of data analysis. Of the five other patients who showed a decrease in size of at least one tumor mass, three had responses in skin, one had a response in bone, and one had a response in an adrenal lesion. Three of the four patients with SD had visceral metastases.
Peptide-Specific T-Cell Responses by ELISpot A carefully controlled IFN-  ELISpot assay was used to monitor the immune response to immunization. Cryopreserved CD8+ T cells were thawed in batch fashion and stimulated in triplicate directly ex vivo with T2 cells loaded with peptides derived from either HIV, EBV, or Melan-A. The HIV values were subtracted from those obtained with either Melan-A or EBV as an internal control at each time point. Seventeen of the enrolled patients had adequate cryopreserved material with which to perform immunologic assessments.
As shown in Fig 1
The changes in Melan-A–specific ELISpot frequencies were compared among patients who had a mixed response or better and those who had no clinical response. As shown in Fig 2 , the mean increase in Melan-A–specific T cells for the clinical responders was 112 ± 45 and for nonresponders was 26 ± 16, indicating that a greater absolute increase in Melan-A–specific T cells was associated with tumor regression (P = .046).
Survival and Associations Between Immunologic Parameters and Clinical Outcome The overall median survival was 12.25 months and is shown in Fig 3A . Seven patients remained alive at the time of data analysis, with all patients followed beyond 12 months. Because the presence of elevated levels of serum LDH is a known negative prognostic factor,23 survival was also compared in response to this vaccine on the basis of LDH level (Fig 3B). The median survival for patients with an elevated LDH level was 9.25 months, whereas the median had not yet been reached for those with a normal LDH (P = .005). In addition, the median survival for patients who experienced a significant increase in Melan-A–specific T cells was not yet reached, compared with 8.5 months for patients without a significant increase in Melan-A–specific cells (Fig 3C; P = .120).
Additional immunologic parameters that had been measured were also analyzed for associations with either clinical response or survival and are summarized in Table 4 . Neither a positive recall DTH to standard antigens nor a relatively high number of EBV- or Melan-A–specific CD8+ T cells before immunization correlated with either outcome. The median pretreatment Melan-A–specific T cell frequency was 23 in clinical nonresponders and -26 in responders. To increase the sensitivity of the assay to detect Melan-A–specific T cells, an in vitro expansion was performed on the preimmunization samples and analyzed by ELISpot as described in Methods. Ten patients showed high Melan-A–specific T cell frequencies after in vitro expansion. However, this also failed to correlate with clinical outcome. Finally, although a normal LDH level was associated with survival, it did not correlate with clinical response and also did not correlate with immune response. Collectively, these results reinforce the specificity of the result showing a significant association between an increased number of Melan-A–specific T cells and clinical outcome.
Expression of Melan-A in Resected Tumors After Immunization It was conceivable that some patients developed PD despite immunization because of outgrowth of Melan-A–negative tumor cells. Posttreatment tumor samples were obtained from progressing tumors from three patients and analyzed by RT-PCR. Although the new metastasis that developed in patient 1 was negative for Melan-A expression, those samples from patients 4 and 6 retained detectable expression of Melan-A mRNA (Fig 1  ). These results indicate that, although outgrowth of antigen-negative tumors can occur, other mechanisms of resistance to immune destruction likely explain the lack of clinical response in other patients.
In this study we used Melan-A peptide-pulsed autologous PBMC + rhIL-12 as a vaccine to treat HLA-A2–positive patients with advanced melanoma. We observed a significant increase in Melan-A–specific IFN- –producing CD8+ T cells after immunization, and found a statistical association between clinical response and the magnitude of the specific T-cell increase. Although it is difficult to compare across individual, small phase II studies, these results are similar to those that have been reported using antigen-loaded dendritic cells, but with a strategy that may be more straightforward to execute. Preparation of the peptide-loaded PBMCs typically took 5 hours from phlebotomy to injection, and quality control of the cell product was facilitated by the lack of an extended in vitro culture period and absence of exposure to culture medium or serum proteins that is required for dendritic cell preparations. Conversely, dendritic cell vaccines have been prepared in batches and cryopreserved in individual doses in some studies, which obviates the need to prepare a fresh vaccine at each time point. Cryopreservation of vaccines has not yet been examined with our current approach. A comparative trial between PBMC/rhIL-12 and dendritic cell-based vaccination may, therefore, be of interest as the technologies continue to develop. Our results support the notion developed in preclinical models that IL-12 can contribute to effective antitumor immunity, and are consistent with the results of a recent adjuvant vaccine study using rhIL-12 in melanoma.24
We used a direct ex vivo ELISpot assay to assess antigen-specific T-cell responses in this study. Control experiments testing EBV reactivity from normal donors revealed that ELISpot analysis could be performed accurately on cryopreserved CD8+ T cell samples immediately after thawing (H. Harlin and T. Gajewski, unpublished data). We found that background reactivity against the control HIV peptide varied among patients and to some extent among time points for an individual patient. The magnitude of increase in apparent Melan-A–reactive T cells would have been greater in some patients had the values obtained with the HIV control peptide not been subtracted. We believe that this experimental detail is critical because it normalizes the samples for background differences and provides an internal control for minor variation between individual vials of cryopreserved T cells. We also compared the Melan-A frequencies to those against an EBV control peptide, to determine whether the treatment was altering ELISpot results. We performed our analyses on purified CD8+ T cells to control for variable numbers between patients and across time points. It is possible that we excluded subpopulations of CD8- T cells, CD4+ T cells, and natural killer T cells that could have produced IFN-
High frequencies of Melan-A–specific, IFN- The median overall survival in our study was 12.25 months from treatment initiation, which is greater than the expected 6 to 9 months for this patient population. Although it was a relatively small study and subject to selection bias, most patients were pretreated and had visceral disease, one half of the patients had elevated serum LDH levels, and four patients had treated brain metastases. As has been seen in melanoma patients treated with standard therapies, we found that an elevated serum LDH level was a negative prognostic factor for survival. Whether this is reflective of tumor burden or the metabolic state of the tumor cells that have adapted to an anaerobic environment is unclear. Some patients developed increases in Melan-A–specific T cells and developed progressive tumor growth despite retained expression of the antigen on posttreatment biopsies. This observation is similar to that seen in murine studies28 and indicates mechanisms of tumor resistance downstream from initial T-cell priming, presumably within the tumor microenvironment. Potential explanations include poor T-cell trafficking to tumor sites, presence of negative regulatory cells, T-cell anergy or death, expression of inhibitory molecules by tumor cells, or downregulation of class I major histocompatibility complex or antigen-processing molecules.29,30 Future studies should investigate definable mechanisms of tumor escape that allow tumor cells to resist elimination by antigen-specific T cells in vivo.
We thank Genetics Institute/Wyeth for rhIL-12, and T. Karrison, M. Sherman, S. Swiger, and M. Posner for important contributions.
Supported by the Burroughs Wellcome Fund, Research Triangle Park, NC, and the Cancer Research Institute, New York, NY. A.P. and H.H. contributed equally to this work.
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ABSTRACT
INTRODUCTION
