Dendritic cells (DCs) show promise as adjuvants in anticancer immunotherapeutic strategies. Flt3 ligand (FL) is a hematopoietic growth factor that increases the number of immature DCs in the blood and other tissues. We treated 27 patients with metastatic or high-risk resected melanoma with s.c. FL daily for 14 d in three 28 d cycles. Eighteen of these patients also received vaccination with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides. To induce local DC maturation, 8 of the vaccinated patients had imiquimod, a Toll-like receptor-7 ligand (TLR7L), applied topically to their vaccine sites. Patients were monitored for clinical and hematological effects. Immune responses were assessed by cutaneous reactivity to vaccination and by the induction of peptide-specific CD8+ T-cells. Eight patients did not complete the protocol due to adverse events related to their cancer. The treatment was generally safe and well tolerated, although some patients developed clinically significant toxicities related to FL. FL induced increases in immature CD11c+ and CD123+ peripheral blood (PB) DCs. Other hematological effects included monocytosis, granulocytosis, and thrombocytosis, which were marked in some patients. Cutaneous reactions to peptide vaccination and circulating peptide-specific CD8+ T-cells were more frequent in imiquimod-treated patients. FL treatment of melanoma patients has pleiotropic clinical and hematological effects. In vivo maturation of FL-generated DCs using imiquimod may increase immune responses to tumor antigens.
This article was published in Cancer Immunity, a Cancer Research Institute journal that ceased publication in 2013 and is now provided online in association with Cancer Immunology Research.
DCs facilitate the presentation of tumor antigens to the immune system and enable specific antitumor effector responses (1, 2). Efforts to optimize tumor antigen presentation by DCs in patients with cancer have utilized different approaches. DCs derived directly from PB or generated from CD34+ hematopoietic progenitors or CD14+ monocytes can be activated in vitro and administered safely to patients (3, 4, 5, 6, 7, 8). Alternatively, cytokines such as FL can be used in vivo to increase the number of DCs in the blood and other tissues. This may enhance immune responses against either endogenous or exogenous tumor antigens (9, 10, 11, 12).
FL binds to Flt3, a tyrosine kinase growth-factor receptor that is expressed on progenitors of multiple hematopoietic lineages, including DCs (13, 14). In animal models, FL caused tumor regressions that were associated with the development of antigen-specific, cell-mediated antitumor immunity (15, 16). Clinical studies in normal human volunteers have shown that FL is safe and can increase the number of circulating DCs and DC precursors (10). In patients with metastatic colon or non-small cell lung cancer, mobilization of DCs into PB by FL has enabled these cells to be harvested for ex vivo DC vaccine preparation (5). Another study showed increased numbers of DCs at tumor sites when FL was administered to patients with carcinoma of the colon (11). In addition, adjuvant administration of FL with HER-2/neu intracellular domain protein vaccination was reported to increase the precursor frequency of IFN-gamma-secreting T-cells specific for intracellular domain peptides (12). Although DCs mobilized by FL are immature, they are capable of maturing in vitro when provided with appropriate stimuli such as proinflammatory, T-cell-derived or pathogen-derived signals (17). In patients treated with FL, stimuli to induce maturation of DCs are thus likely to be required in order to optimize antigen presentation and consequent cell-mediated immune responses against cancer antigens.
Topical cutaneous application of the TLR7L imiquimod can induce the maturation and migration of cutaneous DCs (18, 19). This occurs in association with an induction of monocyte-derived cytokines, including IFN-alpha (20, 21), TNF-alpha, IL-1alpha, IL-6, and IL-8 (22, 23, 24, 25, 26). Imiquimod is used for the treatment of anogenital warts (27) and is efficacious against cutaneous tumors, including basal cell carcinoma (28) and melanoma metastases (29). These studies provide a rationale for using imiquimod as a DC maturation stimulus at vaccination sites.
Our study sought to examine the effects of immunotherapy with FL, peptide vaccination, and imiquimod in patients with melanoma. Melanoma is an attractive target for DC-based treatment approaches because its natural history may be modified by host immune responses (30, 31). In addition, the discovery of tumor antigens that are associated with melanoma means that antigen-specific vaccination treatment approaches can be evaluated (32). We therefore undertook to determine the safety and tolerability of FL, administered with or without melanoma-specific peptide vaccination and imiquimod, in patients with melanoma, and to document the immunological and clinical effects of these treatments.
Patient characteristics and treatment received
Twenty-seven patients were treated as part of the study. Their clinical characteristics are shown in Table 1. Eight patients were removed from the study before completing all protocol requirements: six because of disease progression that necessitated alternative treatment, and two because of disease-related adverse events that were evaluated as grade 3 on the National Cancer Institute Common Toxicity Criteria (CTC) scale and delayed treatment for over 1 mo. Three of these patients were replaced because the minimal period for evaluation had not been completed.
Details regarding prior therapy for melanoma are listed in Table 1. All patients had undergone previous surgical resection of melanoma. Different types of immunotherapy received included IL-12 (33) (two patients), IFN-alpha (three patients), GM-CSF (one patient), KM871 monoclonal antibody (34) (one patient), and/or vaccination with vaccinia oncolysate (35) (four patients), Mel peptide (33) (two patients), or NY-ESO-1 ISCOMATRIX® (36) (two patients).
Systemic inflammatory symptoms in patients treated with FL
All 27 patients received at least one dose of FL and were evaluable for toxicity. The most frequent toxicities are shown in Table 2. FL, with or without peptide vaccination and imiquimod, was generally well tolerated. Systemic inflammatory symptoms such as lethargy, fever, myalgia, and sweats were common, although the severity varied among patients. Cutaneous reactions to FL administration were of two types: (i) local injection-site reactions characterized by induration, erythema, and pruritus; and (ii) distant, low-grade inflammatory phenomena such as tender nodules of the fingers, toes, and ears (one patient) and erythema in old scars (three patients). All cutaneous reactions resolved within 2 wk of cessation of treatment with FL. Often, coryzal symptoms were also temporally related to FL treatment.
Five patients, all with metastatic disease that included lymph node involvement, experienced significant cyclical, systemic inflammatory symptoms associated with deterioration in their Karnofsky performance status (KPS) (Table 3). Figure 1 illustrates this phenomenon in one patient. In general, such symptoms developed toward the end of a 14-d FL treatment period, continued for several days, and resolved within 2 wk. Table 3 shows that some of these patients had marked leukocytic responses associated with this clinical syndrome.
A protocol amendment was thus obtained to take extra blood for analysis of the cytokines IL-6, IL-10, IL-1beta, and TNF-alpha. Six patients had sufficient samples taken during the first cycle of treatment. Three of these patients had no metastatic disease and no elevation of serum cytokines. One patient with liver and lung metastases had elevated cytokine levels at baseline, which did not alter significantly. The remaining two patients, both of whom exhibited the inflammatory syndrome described previously, had elevated cytokine levels during the first FL treatment period that decreased after stopping FL (Table 3). Figure 2 details the course of cytokine elevation in one of these patients.
Hematological effects of FL in patients with melanoma
Twenty-six patients received at least one cycle of FL and were evaluable for hematological effects. Hematological parameters are shown in Figures 3, 4, and 5. Although the median leukocyte count for the study population remained stable throughout, some patients developed a significant leukocytosis (Figure 3B). For example, four patients had day 15 leukocyte counts of 44.4, 36.4, 31.5, and 29.9 x 109/L, respectively, after the first FL treatment period. Median platelet counts increased with FL administration, although the peak number was generally seen 2 wk after cessation of FL treatment (Figure 3C). Thrombocytosis was marked in four patients, with day 29 counts ranging from 712 to 1093 x 109/L. No patient experienced adverse effects relating to this. Median neutrophil counts for the study population were stable (Figure 4A), although several patients, including two with the inflammatory syndrome described previously, had a greater than two-fold increase in neutrophils at the end of the first FL treatment period. Flow cytometric analysis of the CD4+, CD8+, and CD56+ lymphocyte populations revealed no consistent effect of FL on the numbers or proportions of these cells (data not shown), although one patient had markedly increased CD56+ cell numbers (7.19 x 109/L) after the first cycle of FL.
All patients developed a two-fold or greater increase in PB monocytes during FL treatment, determined by a SYSMEX SE-9000 Automated Hematology Analyzer. In some patients, the monocytosis was marked (Figure 4C). Monocyte numbers demonstrated a cyclical pattern associated with FL dosing, peaking at the end of each FL treatment period (Figure 4C). Since PB DCs may be present in the machine-detected monocyte population, we performed flow cytometric analysis to determine the numbers of CD14+ (monocytes), CD11c+ (so-called myeloid DCs), and CD123+ (so-called plasmacytoid DCs) cells in PB. Cyclical increases in these cellular subsets were seen in association with FL treatment; the peak values noted at the end of the FL administration period (Figure 5) declined after 7 d. Some patients had significant increases in PB DCs, the highest absolute numbers being 6.17, 3.06, 2.97, and 2.08 x 109/L for CD11c+ DCs, and 0.81, 0.56, 0.34, and 0.28 x 109/L for CD123+ DCs.
Imiquimod enhances immunity against vaccinated peptides
Peptide-specific immunity was assessed only in vaccinated patients from cohorts 2 and 3. Two methods of assessment were used: (i) measurement of cutaneous reactions to vaccinated peptides, and (ii) quantitation of peptide-specific CD8+ T-cells in PB.
Cutaneous reactivity data for each vaccination cohort are presented in Figure 6, panels A and B. Cutaneous reactions were induced over the course of vaccination in a significant proportion of patients, although they varied according to the peptide administered. For example, although responses to Tyr were minimal, the majority of patients responded to Flu and ESO1a peptide vaccination. An analysis of individual patients on the basis of defined response criteria (see Materials and Methods) indicated that imiquimod increased the frequency of induced cutaneous reaction responses to the Flu, Mel, Tyr, NY-ESO1b, and NY-ESO1c peptides (Figure 6C).
Peptide-specific CD8+ T-cell responses were evaluated in all patients using both intracellular cytokine staining (ICS) and tetramer assays, and also varied according to the peptide involved. For instance, assays for Flu and Mel were frequently positive at baseline (Table 4), indicating prior reactivity to these antigens. In light of these preexisting responses, it was not possible to assess induced T-cell responses to these peptides. For the Tyr and ESO1c peptides, no preexisting or induced response was seen in any patient. Assessment of the impact of imiquimod on specific CD8+ T-cell responses therefore focused on the ESO1a and ESO1b peptides. The results of assays relating to these peptides are presented in detail for all vaccinated patients in Tables 5 and 6. On the basis of defined response criteria (see Materials and Methods), only 2/8 patients who did not receive imiquimod developed a specific CD8+ T-cell response to NY-ESO1 peptide vaccination. In contrast, 5/8 patients who received imiquimod developed such a response. The ESO1a- and ESO1b-specific CD8+ T-cell response patterns of representative patients from each cohort are shown in Figure 7.
Twelve patients were evaluable for tumor response. One had a partial response of hepatic and mediastinal lymph node metastases. A second patient had unchanged small-volume lung, adrenal, and pre-sacral disease. The remainder of the patients had progressive disease during the course of the study.
DCs have potent immune stimulatory and regulatory effects, which make them attractive for clinical immunotherapy. The quality of these activities depends on DC phenotype and function, which in turn are dependent on several factors, such as ontogeny, context, maturity, and activation. A variety of clinical strategies are being pursued to evaluate the best ways to apply DCs to cancer treatment. In this study, we tested FL, a growth factor that expands the number of DCs, in patients with metastatic or high-risk primary melanoma. FL was evaluated alone, in conjunction with peptide antigens, and combined with a DC-activating TLR7L, imiquimod.
The main findings of this trial were that daily s.c. dosing with FL for 14 d was safe, and that it increased the number of PB white blood cells, including the number of immature DCs. We observed hematological and clinical effects of FL that had not been reported in earlier studies, in which FL was administered to normal volunteers or to patients with cancer (10, 11, 12). In particular, we noted systemic inflammatory phenomena such as lethargy and fevers that were often temporally related to FL dosing. In association with these symptoms, patients exhibited cyclic increases in PB monocytes, platelets, and occasionally, neutrophils. In some cases, serum levels of monocyte-derived cytokines increased in parallel. Despite dramatic increases in DC numbers, the quality of immune responses to vaccinated melanoma antigens was unexceptional, an observation that likely reflects the immature state of most circulating DCs. These responses, however, could be improved by preconditioning the vaccine sites with imiquimod, a TLR7L that was applied to the skin in order to induce local DC maturation.
The laboratory and clinical effects of FL in this study were similar to those reported previously (10, 11) and chiefly involved increases in circulating CD11c+ and CD123+ PB DCs. Our findings differ from these previous reports in two main regards. First, in some patients, the quality of the hematologic responses differed, and a variety of non-DC-lineage cell types were expanded. Second, we report for the first time an association with a systemic flu-like syndrome in some patients.
Advanced melanoma is an aggressive cancer, and such systemic symptoms could be disease-related. Nonetheless, we formed the impression that these clinical features likely resulted from an interaction between FL and the disease. This is because FL acts on early hematopoietic progenitor cells, and its effects are therefore susceptible to downstream modulation by cytokines that act on more mature cells. Indeed, working with primitive murine hematopoietic progenitors, Jacobsen et al. (37) found that the combination of FL with IL-3 or stem cell factor produced mainly mature myeloid cells, whereas the combination of FL with granulocyte-colony stimulating factor, IL-11, or IL-12 produced mainly immature cell types.
Melanoma frequently induces stromal (38) and inflammatory (39) responses, a process that presumably involves the generation of cytokines either by melanoma cells or by host cells responding to the tumor. Thus, whereas normal volunteers demonstrate a predictable response to FL, mainly comprising increased DC numbers and only mild inflammatory phenomena (10), the presence of melanoma in our patients may have created an in vivo cytokine environment that skewed hematopoiesis and increased production of monocytes, granulocytes, and platelets, and also exaggerated systemic inflammatory symptoms in some patients.
The other important findings from this study relate to the impact of FL on vaccine responses since, despite the very substantial increases in DC numbers induced by FL, the immune responses to vaccinated melanoma antigens were unsatisfactory without the addition of a signal to induce DC maturation. We previously reported that the PB DCs mobilized by FL in this study were immature (17, 40), and such cells are unlikely to induce effective immunity in the absence of appropriate activation signals (41). Indeed, antigen presentation by immature DCs may lead to the induction of antigenic tolerance (42, 43, 44). Consequently, contemporary DC vaccine approaches often utilize a maturation step in which cells are cultured with cytokines in vitro prior to administration (45). Recently, an alternative approach was adopted, wherein DCs were activated in vivo by injecting immature cells into a cutaneous site that had been treated with the topical TLR7L, imiquimod (19). This approach enhanced DC migration, indicating that in vivo maturation may offer a superior method for generating immunostimulatory DCs. In consideration of this, we utilized imiquimod to precondition the vaccine sites of a cohort of patients receiving FL and peptides. We found that the application of imiquimod increased the proportion of patients who developed both cutaneous and peptide-specific CD8+ T-cell responses to peptide vaccination. As this observation was made during an uncontrolled phase 1 study, no definitive conclusions regarding the effectiveness of imiquimod as a vaccine adjuvant can be made. Nonetheless, we have shown that in vivo activation of FL-generated DCs has the potential to enhance immune responses in patients receiving a cancer vaccine.
Our study demonstrates that while most patients with melanoma will be minimally affected by FL administration, a subgroup may react adversely to the treatment. We have also revealed previously unreported hematological effects of FL that may be related to altered cellular effects of FL in the presence of melanoma. Modulation of the biological effects of FL by tumor-bearing patients may thus affect the clinical utility of this molecule. Finally, we have shown that the in vivo use of a DC-activating agent such as the TLR7L imiquimod may enhance immune responses to vaccination in the context of FL treatment. This should be considered in the design of future clinical trials that seek to optimize DC-based immunotherapy.
Materials and methods
The study was an open-label protocol in which all participants received FL, with or without peptide vaccination, with or without imiquimod. The Human Research and Ethics Committee of Austin Health, Heidelberg, Australia approved the protocol. Written consent was obtained from all patients prior to any study-specific procedures. Treatment cohorts were: (1) FL alone, (2) FL plus peptides, and (3) FL plus peptides and imiquimod. Participants were randomly assigned to cohorts 1 and 2 initially. Cohort 3 patients were enrolled subsequently, when approval for the use of imiquimod was obtained. Protocol adherence and clinical data were independently monitored by a contract research organization (Kendle, Australia).
Eligible patients were HLA-A2+ and had a history of stage II, III, or IV malignant melanoma that had protein or mRNA expression of any of the tumor antigens Tyr, Mel, or NY-ESO-1. All had an expected survival of at least 4 mo, had fully recovered from surgery, and were aged 18 to 75 years. All had a Karnofsky performance status (KPS) of at least 70% and adequate organ function as defined by a serum creatinine level of <0.2 mmol/L, a bilirubin level of <25 µmol/L, AST/ALT less than two times the upper limit of normal (unless due to the presence of hepatic metastases), >3.0 x 109/L total leukocytes, >2.5 x 109/L granulocytes, >0.5 x 109/L lymphocytes, and >100 x 109/L platelets.
Patients were ineligible if they were pregnant, had a New York Heart Association (NYHA) class III or IV heart failure, a bleeding disorder, active infection, autoimmune disease (except for vitiligo or type I diabetes mellitus), coexistent malignancy (apart from in situ carcinoma of the cervix or basal cell carcinoma of the skin), untreated or progressive brain metastases, or had received anticancer treatment or immunotherapy within 4 wk of the study's commencement (6 wk for nitrosourea treatment). Patients with hyposplenism or a positive antibody titer to the Human Immunodeficiency Virus (HIV) and those receiving immunosuppressive medication were also excluded.
Patients who were removed from the study prior to the completion of one cycle of treatment were replaced, unless the removal was due to toxicity attributable to the study agents.
Study agents and treatment schedule
The treatment schedule is summarized in Figure 8. FL (Immunex/Amgen Corporation, Seattle, WA, USA) was administered daily for 14 d, s.c. at a dose of 20 µg per kilogram per day (up to a maximum of 1500 µg per day), followed by 14 d without FL. Peptides were administered by intradermal injection on days 8 and 15 of each cycle. Peptides used in the study were the NY-ESO-1 peptides NY-ESO-1157-167 (ESO1a) (SLLMWITQCFL), NY-ESO-1157-165 (ESO1b) (SLLMWITQC), and NY-ESO-1155-163 (ESO1c) (QLSLLMWIT), as well as the influenza (Flu) matrix58-66 peptide (GILGFVFTL), the Tyr368-376 internal sequence peptide (YMDGTMSQV), and the Mel27-35 peptide (A27L substitution, ELAGIGILTV) (all purchased from Multiple Peptide Systems, San Diego, CA, USA). NY-ESO-1 peptides were dissolved in dimethyl sulfoxide prior to dilution in PBS, such that 33 µg was delivered per dose. The other peptides were dissolved in PBS and administered at 100 µg per dose. Imiquimod (Aldara™ 5% cream, 3M Pharmaceuticals, St. Paul, MN, USA) was applied topically to vaccine sites 24 h and 2 h prior to each peptide vaccination. Grade 3 and 4 toxicities attributable to study medication necessitated interruption of all treatment. Treatment could be recommenced if the toxicity improved to grade 1 or 2 within 30 d and did not affect major organ function.
Patients were assessed clinically at least every 2 wk. Adverse events were reported according to the Common Toxicity Criteria (CTC) of the National Cancer Institute (version 2.0, April, 1999). Tumor responses were determined using established criteria (46). Full blood counts were performed on a SYSMEX SE-9000 Automated Hematology Analyzer (TOA Medical Electronics Co. Ltd., Kobe, Japan). Biochemical analyses were performed in the Department of Laboratory Medicine at Austin Health (Heidelberg, Australia).
Hematological and immunological evaluation
Leukapheresis was performed before and after the study to obtain cells for immunological evaluation. PB was taken weekly for analysis of DCs and other cell populations. Flow cytometric analysis of whole blood samples was performed with antibodies to the following antigens: CD14, CD11c, CD123, CD4, CD8, CD56, CD45RA, and CD45RO (Pharmingen, San Diego, CA, USA). OptEIA™ enzyme-linked immunosorbent assays for IL-1beta, IL-6, IL-10, and TNF-alpha were performed according to the manufacturer's instructions (Pharmingen, San Diego, CA, USA).
Antigen-specific T-cells in PB were detected by a modified ICS (47, 48) assay and HLA-peptide tetramers. For the ICS assay, 5 x 106 PBMCs were pulsed with either 0.3-1.0 µg/mL of NY-ESO-1157-165 peptide in the presence of 500 µM Tris (2-carboxyethyl)-phosphine hydrochloride (Pierce Endogen, Rockford, IL, USA) or with another peptide in cysteine-free medium, at room temperature for 30 min. Peptide-pulsed cells were washed and cultured in RP-10 containing 10 U/mL human recombinant IL-2 (Roche Diagnostics, Indianapolis, IN, USA). The medium was changed every 2-3 days, and the cells split according to cell density. The resultant T-cells were harvested on day 7 and assayed for intracellular IFN-gamma production in the presence of Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) against T2 cells pulsed with individual peptides. The peptides used are listed in the Study Agents and Treatment Schedule section, except that for detection of NY-ESO-1a-specific responses, the 159-167 (LMWITQCFL) 9-mer peptide was used. Controls assays were performed using PBLs from a single known responder. Negative and positive control peptides were MAGE-A3271-279 (FLWGPRALV) and EBV.BMLF1280-288 (GLATLVAML), respectively. An aliquot of cells from each culture on the same day was used for tetramer assessment. HLA-A2 tetramers complexed with Flu matrix58-66, Mel27-35, Tyr368-376, and ESO-1b157-165 peptides were used (kindly supplied by Dr. Immanuel Luescher, Ludwig Institute for Cancer Research, Lausanne, Switzerland). Flow cytometric analysis was utilized for both ICS and tetramer assays. A positive assay was defined as the presence of a discrete, peptide-specific population comprising at least 0.1% of all CD8+ events. An induced cellular response to vaccination was recorded if: (i) the prestudy assay was negative, and (ii) a positive assay was present on at least two study time points. For the NY-ESO-1 peptides, a positive result required confirmation in at least one of the other assays (ICS for ESO1a, 1b, and 1c, and tetramer for ESO1b) from the same time point.
Cutaneous reactivity to intradermal peptide injections was assessed 48 h after administration by measuring the maximum diameter of induration at the injection site. An induced cutaneous response to vaccination was recorded if at least two measurements were at least 4 mm greater than the baseline reading.
This work was supported by the Ludwig Institute for Cancer Research and by the Sylvia and Charles Viertel Foundation. Michael Jefford was supported by the Stewardson Family Trust.
We are most grateful to the patients who participated in this study, as well as to their families. We thank the Ludwig Institute in New York, particularly Drs. L. J. Old and E. W. Hoffman for their support and encouragement, the staff of the Cancer Clinical Trials Center at Austin Health, Heidelberg, Australia, and Immunex/Amgen Corporation for providing the Flt3 ligand for this clinical trial. This work was presented in part at the 2003 Annual Meeting of the American Society of Clinical Oncology.
- Received July 6, 2004.
- Accepted July 29, 2004.
- Copyright © 2004 by Jonathan Cebon