1. Top of page
  2. Abstract
  6. Acknowledgements

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34+ peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-α. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1+ or HLA-A2+ patients received at least 4 i.v. infusions of 5 × 106 to 5 × 107 DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I–II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2+ patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects. Int. J. Cancer 86:385–392, 2000. © 2000 Wiley-Liss, Inc.

The cloning and characterization of tumor-associated antigens and tumor-associated antigen-derived peptides recognized by human cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion has opened new possibilities for immunotherapeutic approaches to the treatment of human cancers, particularly malignant melanoma (van den Eynde and van der Bruggen, 1997). Encouraging results have been obtained in vivo with different vaccination strategies using antigenic peptides, plasmid DNA or recombinant viruses encoding tumor-associated antigens (Rosenberg et al.,1998a,b). The dominant role of professional antigen-presenting cells (APCs) in the induction of peptide-specific CTLs has been demonstrated in most of these approaches (Iwasaki et al.,1997). In addition, Toes et al. (1998) have shown that the in vivo CTL-tolerizing potential of some peptides can be converted to specific immunostimulation depending on the nature of the APCs.

Dendritic cells (DCs) are potent APCs and thus specifically involved in the initiation of antigen–specific immune responses (Banchereau and Steinman, 1998). Due to their potent co-stimulatory activity, they are well suited to activate T cells toward various antigens, such as tumor antigens.

The availability of large numbers of DCs, generated from hematopoietic progenitor cells or monocytes in vitro, has profoundly changed pre-clinical research as well as the clinical evaluation of these cells (Herbst et al.,1996, 1997). DCs are attractive for the in vivo induction and activation of antigens and peptide-specific T cells. Accordingly, appropriately pulsed or transfected DCs may be used for vaccination in the field of infectious diseases or tumor immunotherapy, to induce specific CTLs.

Previous studies on the vaccination of melanoma patients with peptide-pulsed DCs provided evidence of clinical and immunological anti-tumor responses (Nestle et al.,1998; Hu et al.,1996).

Here, we report on the vaccination of melanoma patients with peptide-pulsed DCs generated in vitro from CD34+ peripheral blood progenitor cells (PBPCs). We have demonstrated that the sequential use of early-acting hematopoietic growth factors, stem cell factor (SCF), IL-3 and IL-6 in vitro followed on day 8 by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) allows the generation of large numbers of immature DCs from CD34+ PBPCs (Herbst et al.,1996). Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-α. Autologous DCs were pulsed with a cocktail of melanoma-associated peptides depending on the HLA haplotype of the patient. Since effective immunization with antigen-pulsed DCs injected i.v. has been demonstrated in studies on both humans and other species (Mayordomo et al.,1995; Hsu et al.,1996; Höltl et al.,1998), we injected the DCs i.v. after pulsing them with peptides. We report here on the results of 14 melanoma patients treated with this type of active immunotherapy.


  1. Top of page
  2. Abstract
  6. Acknowledgements


Patients with advanced malignant melanoma refractory to standard therapeutic regimens were eligible for treatment if they were shown to be HLA-A1+ or HLA-A2+. Patients were required to be between 18 and 70 years of age, to have a life expectancy of at least 3 months and to have a Karnofsky performance status of 60% or higher. Exclusion criteria were severe concomitant diseases, pregnancy or lactation, any treatment or immunosuppressive therapy including steroids in the prior month and clinically evident brain metastasis. The trial was designed and conducted in accordance with the Declaration of Helsinki. The therapeutic protocol was approved by the institutional ethics committee and registered with the regulatory state authority as well as the federal Paul-Ehrlich-Institute. All patients gave written informed consent before enrolling in the study. HLA typing of peripheral blood lymphocytes was carried out at the Freiburg University HLA Typing Laboratory.


Each of the peptides utilized in this study was prepared under GMP-like conditions by Bachem (Bubendorf, Switzerland): Melan-A 26-35 (HLA-A2), EAAGIGILTV; gp100 280–288 (HLA-A2), YLEPGPVTA; tyrosinase 369–377 (HLA-A2), YMDGTMSQV; MAGE-1 161–169 (HLA-A1), EADPTGHSY; MAGE-3 168–176 (HLA-A1), EVDPIGHLY. The identity of each peptide was confirmed by mass spectral analysis. Peptides were >98% pure as assessed by HPLC. The endotoxin level was <0.1 endotoxin units/ml.

Media and reagents

Cells were cultured in RPMI-1640 supplemented with 200 mM L-glutamine, 50 μM β-mercaptoethanol, 100 mM sodium pyruvate, 50 μg/ml streptomycin, 50 units/ml penicillin, MEM vitamins and 10% FCS (treated at 56°C for 30 sec; PAN Systems, Aidenbach, Germany) (standard culture medium, SCM). The following recombinant human cytokines were employed: IL-3, IL-4, IL-6, TNF-α, SCF (all from CellGenix, Freiburg, Germany) and GM-CSF (Essex Pharma, Novartis Pharma, Basel, Switzerland). Human recombinant cytokines were used at a concentration of 10 ng/ml (IL-3, SCF), 20 ng/ml (TNF-α), 50 ng/ml (IL-4) or 100 ng/ml (IL-6, GM-CSF).

Mobilization of PBPCs and CD34+ selection

To mobilize PBPCs, patients received daily s.c. injections of G-CSF (10 μg/kg body weight) (Neupogen; Amgen, Munich, Germany) for 4 days. PBPCs were harvested by leukapheresis and collected from the apheresis product by centrifugation on Ficoll-Hypaque (Pharmacia, Freiburg, Germany) to isolate mononuclear cells (MNCs). CD34+ progenitor cells were positively selected using the Ceprate avidin–biotin immuno-adsorption column (CellPro, Bothell, WA). Aliquots of purified CD34+ PBPCs were frozen in liquid nitrogen and DC cultures initiated for each vaccination.

Generation of DCs from CD34+ PBPCs

DCs were propagated as described previously with minor modifications (Herbst et al.,1996). In brief, CD34+ PBPCs were thawed and cultured for 7 days in SCM supplemented with IL-3, IL-6 and SCF. Subsequently, cells were transferred to SCM supplemented with IL-4/GM-CSF and cultured for the next 3 weeks while medium was replenished weekly. Cells were seeded at a concentration of 5 × 104 cells/ml at the beginning of culture and at a concentration of 5 × 105 cells/ml on days 7, 14 and 21. Maturation to interdigitating DCs was induced during 24 hr by adding TNF-α to the culture medium including IL-4/GM-CSF.

Flow-cytometric analysis

Surface marker analysis of DCs was performed using a FACScan (Becton Dickinson, Mountain View, CA) and the Lysis II software. FITC- or PE-conjugated monoclonal antibodies (MAbs) used for phenotyping included anti-CD1a (T6, IgG1; Coulter, Hialeah, FL), HLA-DR (L243, IgG2a; Becton Dickinson), anti-CD80 (BB-1, IgM) and anti-CD14 (WM15, IgG2a; PharmMingen, San Diego, CA).

Pulsing of in vitro generated DCs

Twenty-four hours after adding TNF-α, 5 × 106 to 5 × 107 CD1a+ DCs were pulsed with either a pool of HLA-A1-restricted peptides (MAGE-1 and MAGE-3) or a pool of HLA-A2-restricted peptides (Melan-A, gp100 and tyrosinase), selected on the basis of the patient HLA haplotype. For patient 3 (HLA-A1+/A2+), a pool of HLA-A1- plus -A2-restricted peptides was used. DCs were pulsed at 10 μg/ml final peptide concentrations for 2 hr at 37°C in PBS.

Treatment schedule

Eligible HLA-A1+ or HLA-A2+ patients received at least 4 vaccinations of 5 × 106 to 5 × 107 peptide-pulsed DCs suspended in 20 ml of PBS. The vaccine was administered i.v. Treatment was repeated at 2-week intervals. Patients were monitored before each vaccination and 4 weeks after final vaccination by physical examination, measurement of performance status and laboratory analysis. Tumor assessment (2-dimensional radiography in the case of solid tumors) was performed immediately prior to the first vaccination and 4 weeks after the 4th vaccination. Tumor progression led to termination of therapy. In the case of stable disease or tumor regression, further vaccinations were given after an interval of 4 weeks until progression was documented. Immune monitoring was assessed before the 1st and 4th vaccinations and monthly thereafter by collecting PBMCs for functional analysis of T cells.

Specific delayed-type hypersensitivity (DTH) reaction

DTH skin tests were performed with peptide-pulsed and non-pulsed DCs before the 1st and at the 4th vaccinations; 5 × 105 unpulsed DCs, 5 × 105 DCs pulsed with each peptide or peptides alone were injected intradermally (i.d.) into the forearm. A positive skin reaction was defined as >5 mm diameter erythema and induration 48 hr after i.d. injection. For immuno-histochemical analysis of vaccination-infiltrating lymphocytes (VILs), 6 mm punch biopsies of i.d. vaccination sites were performed.


Frozen sections (5 μm) of skin biopsies were incubated with MAbs to CD4 and CD8 (DAKO, Hamburg, Germany), and bound antibody was detected by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. Paraffin sections (5 μm) were stained with MAbs to CD1a and CD3 (DAKO). 3-Amino-9-ethylcarbazole served as chromogen in a 1-step peroxidase staining technique (EPOS-System, DAKO). Sections were counterstained with Mayer's hemalum. For the detection of melanoma-associated antigens in formalin-fixed tumor tissues embedded in paraffin, the following antibodies were used: anti-Melan-A (A103; Novocastra, Newcastle, UK), anti-tyrosinase (T311, Novocastra) and anti-gp100 (HMB-45, DAKO). Bound antibody was detected using the avidin–biotin complex (ABC) method.

Expression of melanoma antigens by RT-PCR

Poly-A mRNA was isolated from melanoma biopsies using the Quick Prep Micro mRNA purification kit (Pharmacia, Dubendorf, Switzerland) as described by Noppen et al. (1996). First-strand cDNA was synthesized from poly-A mRNA by taking advantage of a commercial RT-PCR kit (Perkin-Elmer Cetus, Norwalk, CT). The following primers were used: β-actin sense 5′-CACCCACACTGTGCCCATC and β-actin anti-sense 5′-CTAGAAGCATTTGCGGTGGAC, amplifying a 650 bp gene fragment; gp100 sense 5′-CTGTGCCAGCCTGTGCTAC and gp100 anti-sense 5′-CACCAATGGGACAAGAGCAG, amplifying a 334 bp fragment; Melan-A/MART-1 sense 5′-AGATGCCAAGAGAAGATGCTC and Melan-A/MART-1 anti-sense 5′-GCTCTTAAGGTGAATAAGGTGG, amplifying a 364 bp gene fragment; tyrosinase sense 5′-TTGGCAGATTGTCTGTAGCC and tyrosinase anti-sense 5′-AGGCATTGTGCATGCTGCTT, amplifying a 284 bp gene fragment; MAGE-1 sense 5′-CGGCCGAAGGAACCTGACCCAG and MAGE-1 anti-sense 5′-GCTGGAACCCTCACTGGGTTGCC, amplifying a 421 bp fragment; MAGE-3 sense 5′-TGGAGGACCAGAGGCCCCC and MAGE-3 anti-sense 5′-GGACGATTATCAGGAGGCCTGC, amplifying a 725 bp fragment.

In the case of β-actin, tyrosinase, gp100 and Melan-A/MART-1 PCR was performed using the following profile: 20 sec of denaturation at 94°C, 20 sec of annealing at 58°C and 40 sec of extension at 72°C using the “hot-start” technique. For detection of β-actin, Melan-A/MART-1, gp100 and tyrosinase, the PCR was cycled 30 and 35 times. For detection of MAGE-1 and MAGE-3, a different PCR profile was used: 1 min of denaturation at 94°C, followed by 2 min annealing at 72°C and 2 min extension at 72°C. PCR products were run on 1.5% agarose gels containing ethidium bromide and photographed under transillumination.

HLA–peptide tetrameric complexes and flow cytometry

HLA–peptide tetrameric complexes were synthesized as previously described (Dunbar et al.,1998). In brief, purified HLA heavy chain and β2-microglobulin were synthesized using a prokaryotic expression system (pET; Novagen, Milwaukee, WI). The heavy chain was modified by deletion of the transmembrane/cytosolic tail and COOH-terminal addition of a sequence containing the BirA enzymatic biotinylation site. Heavy chain, β2-microglobulin and peptide were refolded by dilution. A*0201-binding peptides were tyrosinase 369–377 YMDGTMSQV, MelanA 26–35 ELAGIGILTV and gp100 280–288 YLEPGPVTA. The 45 kDa refolded products were isolated using fast protein liquid chromatography (FPLC), biotinylated by BirA (Avidity, Denver, CO) in the presence of biotin (Sigma, St. Louis, MO), ATP (Sigma) and Mg2+ (Sigma). Biotinylated products were separated from free biotin by gel filtration and ion exchange using FPLC. Streptavidin–PE conjugate (Sigma) was added in a 1:4 molar ratio and the tetrameric product concentrated to 1 mg/ml. Cells were analyzed for expression of cell-surface markers using FACScan and CellQuest software (both from Becton Dickinson). Frozen PBMCs were thawed and incubated for 24 hr in RPMI-1640 supplemented with 5% human serum to allow recovery of cell viability. For direct tetramer staining, 106 PBMCs were centrifuged at 300 g for 5 min and resuspended in 50 μl cold PBS. Tetramer and anti-CD8-tricolor (Caltag, Burlingame, CA) were added and incubated for another 30 min. Samples were washed 2 more times with PBS before formaldehyde fixation. Double-color analysis was performed with tetramer–PE and anti-CD8-tricolor. Controls for the tetramers included staining A*0201-negative individuals and the use of an irrelevant A*0201 tetramer (SLYNTVATL p17Gag 77–85). For in vitro sensitization, PBMCs were cultured in RPMI-1640 supplemented with 2.5% human serum and 200 units/ml of IL-2; 10 μM of the relevant peptide were added to 4 × 106 cells/well in a final volume of 2 ml. Tetramer staining was performed after 2 weeks in culture.


The technique used for the IFN-γ ELISPOT assay has been described before (Herr et al.,1997). Briefly, wells of MultiScreen-HA plates (Millipore, Bedford, MA) were coated with 50 μl of murine MAb anti-human IFN-γ at a concentration of 10 μg/ml (clone 1-D1K; Mabtech, Nacka, Sweden). After incubation overnight at 4°C, unbound MAb was removed and coated cells were blocked with RPMI-1640 supplemented with 10% human AB serum. After 1 hr at 37°C, the blocking medium was discarded and 1 × 105 CD8+ T cells/well, purified from patient PBMCs by positive selection with CD8 MicroBeads (Miltenyi, Bergisch-Gladbach, Germany), were added. T2 cells (7.5 × 104/well), pulsed with the relevant peptides in the presence of β2-microglobulin, were added. After a culture period of 20 hr at 37°C, cells were removed and biotinylated anti-IFN-γ MAb (clone 7-B6-1, Mabtech) was added for 2 hr at 37°C. A 100 μl volume of ABC (ABC Vectastain-Elite kit; Vector, Burlingame, CA) was added at a dilution of 1/100 and incubated for 1 hr at room temperature. After unbound complex was removed, peroxidase staining was performed using the substrate 3-amino-9-ethylcarbazole (Sigma). Spots appeared within 4 to 5 min. The color reaction was stopped, and numbers and areas of resulting spots were determined with the use of computer-assisted video image analysis (Herr et al.,1997).

Melanoma-inhibiting activity (MIA) assay

Bosserhoff et al. (1997) have demonstrated that MIA can be a useful marker of tumor progression during follow-up of melanoma patients and in monitoring therapy of advanced disease. Human MIA was measured by a 1-step ELISA (Roche, Mannheim, Germany), following the instructions of the manufacturer. The assay is sensitive up to 0.1 ng MIA/ml.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Fourteen patients with refractory malignant melanoma completed at least 4 vaccinations and were evaluable (Table I). All patients had extensive metastatic disease; the majority had undergone multiple previous therapies including surgery, chemotherapy, chemo-immunotherapy or immunotherapy. Six patients were HLA-A1+ and 9 HLA-A2+ (Table II). Autologous peptide-pulsed DCs, generated in vitro, were injected i.v. through a peripheral venous catheter. Patients received at least 4 to 12 vaccinations at 2-week intervals with escalating cell doses ranging fom 5 × 106 to 5 × 107 DCs. Dose escalation was applied interindividually with the exception of 4 patients receiving 2 dose levels (Table II). Four patients were vaccinated with 5 × 106 DCs, 10 patients with 1 × 107 DCs and 4 patients with 5 × 107 DCs (Table II).

Table I. Patient Characteristics
  • 1


  • 2

    Dacarbazine + vinblastine + cisplatin + IL-2 + IFN-α2a.

  • 3


Age (years) 
Performance status (Karnovksy) 
Prior therapy 
Metastatic site 
   Lymph node6
   Soft tissue2
Table II. Clinical Results
Patient numberHLA-AMetastatic siteNumber of vaccinationsNumber of DCs injectedClinical course (months)
  • 1

    NC, no change.

  • 2

    NED, no evidence of disease.

  • 3

    MR, minor response [complete regression of s.c. metastasis (see Fig. 2), stable disease of another cutaneous metastasis].

1HLA-A1LN, skin85 × 106, 1 × 107NC1 (3)
2HLA-A2LN125 × 106, 1 × 107NED2 (19)
3HLA-A1,A2Lung45 × 106PD
4HLA-A2LN, soft tissue75 × 106, 1 × 107NC (4)
5HLA-A2Lung, liver81 × 107NC (8)
6HLA-A1Lung, liver41 × 107PD
7HLA-A2Soft tissue41 × 107PD
8HLA-A1Skin81 × 107, 5 × 107MR3 (6)
9HLA-A2Lung, liver, bone41 × 107NC (3)
10HLA-A2Lung81 × 107NC (6)
11HLA-A1Lung, liver, LN41 × 107PD
12HLA-A2Lung, LN45 × 107PD
13HLA-A1Liver, LN45 × 107PD
14HLA-A2Lung45 × 107NC (6+)

Expression of melanoma-associated antigens in tumor samples

The expression pattern of melanoma-associated antigens in melanoma patients was analyzed either by RT-PCR, when fresh tumor biopsies were available, or by immuno-histochemistry using formalin-fixed paraffin sections. Total cellular RNA was extracted from melanoma biopsies and reverse-transcribed. Expression of gp100, tyrosinase, Melan-A, MAGE-1 and MAGE-3 genes was analyzed by RT-PCR in 7 melanoma patients. The data are shown in Table III. Three patients were HLA-A1+, 3 patients HLA-A2+ and 1 patient HLA-A1+/A2+. Four of 4 HLA-A2+ patients were positive for Melan-A, gp100 and tyrosinase; 3 of 4 HLA-A2+ patients expressed MAGE-1; and only 2 of 4 were positive for MAGE-3.

Table III. Expression of Melanoma-Associated Antigens in Tumor Biopsies
Patient numberMelan-Agp100TyrosinaseMAGE-1MAGE-3
  • 1

    Expression of genes encoding tumor-associated antigens in tumor specimens: freshly excised metastatic melanoma specimens were mechanically minced, and poly-A mRNA was extracted and reverse transcribed. cDNA was then assayed in PCR tests using specific primer pairs for Melan-A, gp100, tyrosinase, MAGE-1 and MAGE-3. For other details, see text.

  • 2

    For detection of melanoma-associated antigens in formalin-fixed tumor tissues embedded in paraffin, the following antibodies were used: anti-Melan-A (A103), anti-tyrosinase (T311) and anti-gp100 (HMB-45). For details, see Material and Methods. n.d., not determined.


Immuno-histochemistry of formalin-fixed paraffin sections was performed for 6 patients using anti-Melan-A, anti-gp100 and anti-tyrosinase MAbs. Results revealed expression of gp100 in 6 of 6 patients; 5 of 6 patients were positive for Melan-A and tyrosinase (Table III).

Ex vivo generation of DCs

Harvested G-CSF-recruited PBPCs contained an average of 46% (range 10%–75%) positively selected CD34+ cells after immuno-adsorption (Table IV). The mean total number of CD34+ cells was 1.4 × 108 (range 0.35 × 108 to 3.7 × 108). After ex vivo culture of CD34+ cells for 28 days, immunophenotyping demonstrated a mean percentage of 34.4% CD1a+ DCs (range 12%–87%) (Table IV). CD1a+ DCs co-expressed HLA-DR and low amounts of CD80, while they were negative for CD14. After addition of TNF-α, expression of CD1a was down-regulated and that of CD80 and HLA-DR increased (data not shown).

Table IV. Characteristics of CD34+ Peripheral Blood Progenitor Cells and CD1a+ DENDRITIC CELLS
Patient numberPurity of CD34+day 0 (%)Total CD34+ (×107)Purity of CD1a+day 28 (%)

Adverse events

A total of 83 vaccinations were performed. Clinical side effects were usually mild and consisted of fever WHO grade I–II (<39.5°C) in 4 of 14 patients, peaking at about 12 to 14 hr after the 2nd vaccination. One patient (patient 2) developed a local inflammatory response with erythema and induration at the tumor site 1 to 2 days following vaccination. With the exception of 1 patient, no physical signs of auto-immune disease were observed in any of the patients. Interestingly, in this HLA-A2+ patient (patient 12), spontaneous appearance of generalized vitiligo occurred 1 week after the 2nd vaccination (Fig. 1a).

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Figure 1. (a) Development of generalized vitiligo in patient 12 (HLA-A2+) 1 week following the 2nd vaccination with peptide-pulsed DCs. (b) Serum levels of melanoma-inhibiting activity (MIA) and lactate dehydrogenase (LDH), indicators of disease activity. Serum samples of patient 12 were collected at various time points (x axis) and measured for MIA (ng/ml) and LDH (units/ml) activity. Time points of 4 different vaccinations with peptide-pulsed DCs are indicated by the syringes.

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Clinical response

All patients had progressive disease prior to inclusion in the vaccination protocol. The only patient with a minor tumor load (patient 8) responded to vaccination with complete regression of s.c. metastasis (Fig. 2), while another small skin metastasis remained unchanged. Another patient (patient 2), suffering from refractory cervical lymph node metastasis, surgically resected 3 times within 12 weeks, showed no evidence of disease when entering the study. He was vaccinated out of study and remained without radiographic evidence of disease over a period of 19 months. Six patients (patients 1, 4, 5, 9, 10 and 14) showed stable disease during therapy for 3 to 8 months (Table II). Furthermore, 1 patient (patient 12) with extensive metastatic disease, who had a marked increase in serum MIA and lactate dehydrogenase levels before vaccination, revealed a marked decrease of both serum levels in parallel with the occurrence of generalized vitiligo (Fig. 1b). At the 4th vaccination, she was diagnosed with brain metastasis and died 4 weeks later.

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Figure 2. Cancer regression in a patient receiving vaccination with peptide-pulsed DCs. Regression of s.c. metastasis at the right leg after 8 cycles of vaccination with peptide-pulsed DCs (patient 8).

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Immunological responses

We used the classical DTH skin reaction to analyze whether peptide-specific immune responses were induced locally after vaccination with peptide-pulsed DCs. Peptide-pulsed and non-pulsed DCs were injected i.d. before the 1st and at the 4th vaccinations. Before the 1st vaccination, no DTH reactivity to DCs alone or to peptide-pulsed DCs was observed in any of the patients tested. Significant DTH reactivity (induration >5 mm in diameter) against DCs pulsed with different melanoma-associated peptides was observed in 4 HLA-A2+ patients (patients 2, 4, 10 and 12) (Fig. 3a,b). All of them showed a positive DTH reaction against Melan-A-pulsed DCs, while control injection of Melan-A peptide in 3 patients was negative (Fig. 3b). Patient 2 had a positive DTH response against gp100-pulsed DCs as well, while patient 10 had a strong reaction against gp100 and tyrosinase. There was no DTH reactivity detectable against DCs alone.

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Figure 3. Peptide-specific DTH reactivity after vaccination with peptide-pulsed DCs. (a) DTH reactivity in patient 10 observed 48 hr after i.d. injection of peptide-pulsed or non-pulsed DCs before the 1st and at the 4th vaccinations. (b) Changes in local inflammatory responses (diameter of induration) of 4 patients 48 hr after i.d. injection of peptide-pulsed or non-pulsed DCs before the 1st and at the 4th vaccinations.

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Punch biopsies of i.d. injection sites, obtained from 4 patients with positive DTH reactions, demonstrated a dense mononuclear infiltrate composed predominantly of CD3+ cells (Fig. 4). Evaluation of T-cell subpopulations usually revealed a predominance of CD4+ cells, but various degrees of CD8+ T-cell infiltration were also observed.

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Figure 4. Immuno-histochemical staining of punch biopsies of the i.d. vaccination site of patient 10 performed 48 hr after the 4th vaccination, showing infiltration mainly of T cells. Anti-CD3 MAb was used for detection of T cells, and staining was performed using the APAAP complex method. Scale bar = 500 μm.

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Frequency of circulating peptide-specific T cells after vaccination

We next analyzed the frequency of circulating CD8+ T cells specifc for the HLA-A*0201-binding melanoma-associated peptides tyrosinase YMDGTMSQV, Melan-A ELAGIGILTV and gp100 YLEPGPVTA using MHC–peptide tetrameric complexes. PBLs from 4 patients before and after vaccination with peptide-pulsed DCs were stained with the PE-conjugated tetrameric complexes and with an anti-CD8 antibody conjugated to tricolor. Tetramer-reactive T cells could not be detected in the pre-vaccination samples. In 3 of the 4 patients, no change in the frequency before and after vaccination was observed. However, patient 2 revealed significant expansion of Melan-A- and gp100-specific CD8+ CTLs after vaccination. Circulating Melan-A-ELAGIGILTV-reactive CTLs, which were undetectable before vaccination, could be detected with a low frequency of 0.04% in 2 post-vaccination samples in the absence of any stimulation in vitro (data not shown). After 2 weeks of in vitro culture in the presence of the relevant peptide, the frequency of Melan-A ELAGIGILTV-reactive T cells from patient 2 increased significantly from 0.09% (2 months after the last vaccination) to 0.6% (4 months after the last vaccination), while T cells obtained at the last vaccination remained negative (Fig. 5a–c). The number of gp100 YLEPGPVTA-specific CD8+ T cells increased from 0.01% (at the last vaccination) to 0.3% (2 months after the last vaccination) upon in vitro sensitization with the peptide, demonstrating a subsequent decrease to 0.01% in the 4 months post-vaccination sample (data not shown).

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Figure 5. Frequency of tetramer-reactive CD8+ T cells in PBMCs after 2 weeks of peptide restimulation in vitro. PBMCs from patient 2 were stained with tricolor-conjugated anti-CD8 and PE-conjugated Melan-A–ELAGIGILTV tetrameric complex. Percentages of CD8+/tetramer+ T cells, obtained from PBMCs at the last vaccination (a) and 2 months (b) and 4 months (c) after the last vaccination, are shown in the upper right quadrant of the dot blot.

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CD8+ T cells isolated from PBLs of 5 other HLA-A1+ or HLA-A2+ melanoma patients before the 1st and after the 4th vaccinations were tested for reactivity against HLA-A1-binding peptides from MAGE-1 and MAGE-3 or HLA-A2-binding peptides from Melan-A, gp100 and tyrosinase in IFN-γ ELISPOT assays. No significant increase in peptide-specific CD8+ T cells was observed after vaccination (data not shown).


  1. Top of page
  2. Abstract
  6. Acknowledgements

We report a clinical phase I vaccination study in melanoma patients performed with peptide-pulsed DCs generated in vitro from CD34+ hematopoietic progenitor cells. The following conclusions emerge from this study, which included 83 vaccinations in 14 melanoma patients: (i) sufficient numbers of DCs can be generated from CD34+ PBPCs for a minimum of 4 vaccinations with 5 × 106 to 5 × 107 CD1a+ DCs; (ii) vaccination with peptide-pulsed DCs is possible without major side effects or signs of auto-immune disease; (iii) immunological responses consisted of peptide-specific DTH responses in 4 patients, a significant increase in circulating Melan-A- and gp100-reactive CD8+ CTLs after vaccination in 1 patient showing signs of clinical anti-tumor response and development of generalized vitiligo in 1 HLA-A2+ patient during vaccination; (iv) clinical anti-tumor responses were observed in 2 patients, including regression of s.c. metastasis.

Our study is a clinical trial with DCs derived from CD34+ hematopoietic progenitor cells. Previous studies were conducted with monocyte-derived DCs (Nestle et al.,1998; Höltl et al.,1998). We have demonstrated that the sequential use of early-acting hematopoietic growth factors, SCF, IL-3 and IL-6 followed on day 8 by differentiation with IL-4 and GM-CSF allows generation of large numbers of immature DCs that mature to interdigitating DCs within 24 hr of addition of TNF-α (Herbst et al.,1996). Comparative analysis of CD34+ PBPCs and monocyte-derived DCs revealed similar phenotypic and functional properties (Herbst et al.,1997). However, Mortarini et al. (1997) demonstrated that DCs derived from CD34+ progenitors are more efficient APCs for the activation of low-frequency, peptide-specific CTLs than DCs derived from monocytes.

In the present study, patients received peptide-pulsed DCs in 3 different cell-dose levels, ranging from 5 × 106 to 5 × 107 CD1a+ DCs. Clinical and immunological responses were observed independently of the dose level, thus supporting the view that relatively small numbers of DCs are effective at stimulating specific immune responses (Nestle et al.,1998; Hsu et al.,1996).

So far, 2 studies on vaccination with peptide-pulsed DCs have been published. Nestle et al. (1998) showed a specific DTH response in 11 of 15 patients but an objective response in 5 of 16 evaluable patients after vaccination with peptide- or tumor lysate–pulsed DCs. However, the tumor load in these selected patients was reported to be low, suggesting efficiency of this approach in melanoma patients with minimal residual disease, as achieved by cytoreductive therapy. Mukherji et al. (1995) vaccinated patients with MAGE-1 peptide-pulsed APCs and demonstrated accumulation of MAGE-1-specific CTLs at the vaccination site, though it required 3 in vitro restimulations to detect this response (Hu et al.,1996).

In the present study, 4 of 14 patients revealed a peptide-specific DTH response after vaccination. Interestingly, in 2 of these patients, positive DTH response correlated well with specific immune reponses. Patient 12, showing a positive DTH response against Melan-A-pulsed DCs after vaccination, developed generalized vitiligo after the 2nd vaccination. An association between high frequencies of Melan-A-specific CTLs and development of auto-immune vitiligo has been reported (Ogg et al.,1998). Vitiligo is a common progressive depigmentary skin disorder believed to be due to the auto-immune-mediated destruction of epidermal melanocytes. Patient 2 revealed a positive DTH response against Melan-A and gp100. In parallel, we detected a significant increase of circulating Melan-A- and gp100-reactive CTLs in PBLs after vaccination. Melan-A-specific CTLs are present ex vivo in melanoma-infiltrated lymph nodes (Romero et al.,1998) and circulating CD8+ T cells specific for Melan-A and tyrosinase in unvaccinated melanoma patients (Lee et al.,1999). However, antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth (Lee et al.,1999).

The optimal mode of DC administration with respect to practical aspects and induction of specific immune responses remains unclear. We and others have shown that i.v. administration of in vitro generated human DCs delivers these APCs preferentially to the spleen and liver (Mackensen et al.,1999; Morse et al.,1999). Clinical anti-tumor responses after DC vaccination have been reported after i.v. injection (Hsu et al.,1996; Höltl et al.,1998) and after direct injection into lymph nodes (Nestle et al.,1998). Comparative analysis of clinical and immunological responses after different routes of DC vaccination are thus warranted.

In conclusion, our pilot study demonstrates that it is possible to significantly increase the number of circulating peptide-specific CTLs by vaccination with peptide-pulsed autologous DCs derived from hematopoietic progenitors and to mediate tumor regression in some patients with metastatic melanoma.


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  2. Abstract
  6. Acknowledgements

We thank Dr. R. Mertelsmann for laboratory space and helpful disccussions. The excellent technical assistance of Ms. K. Schweda is gratefully acknowledged.


  1. Top of page
  2. Abstract
  6. Acknowledgements
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