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. Author manuscript; available in PMC: 2007 Nov 15.
Published in final edited form as: Hum Gene Ther. 2003 May 20;14(8):709–714. doi: 10.1089/104303403765255110

Inability to Immunize Patients with Metastatic Melanoma Using Plasmid DNA Encoding the gp100 Melanoma-Melanocyte Antigen

Steven A Rosenberg 1, James C Yang 1, Richard M Sherry 1, Patrick Hwu 1, Suzanne L Topalian 1, Douglas J Schwartzentruber 1, Nicholas P Restifo 1, Leah R Haworth 1, Claudia A Seipp 1, Linda J Freezer 1, Kathleen E Morton 1, Sharon A Mavroukakis 1, Donald E White 1
PMCID: PMC2078240  NIHMSID: NIHMS30918  PMID: 12804135

Abstract

Immunization with plasmid DNA represents a theoretically attractive method for increasing T cell responses against cancer antigens. We administered plasmid DNA encoding the gp100 melanoma-melanocyte differentiation antigen to 22 patients with metastatic melanoma and evaluated immunologic and clinical responses. Patients were randomized to receive plasmid DNA either intradermally (n = 10) or intramuscularly (n = 12). One patient (4.5%) exhibited a partial response of several subcentimeter cutaneous nodules. All other patients had progressive disease. Of 13 patients with cells available before and after immunization, no patient exhibited evidence of the development of anti-gp100 cell responses using in vitro boost assays. The same assays were capable of demonstrating immunologic precursors after immunization with fowl poxvirus encoding gp100 or with gp100 peptides. We were thus unable to demonstrate significant clinical or immunologic responses to plasmid DNA encoding the “self” nonmutated gp100 tumor antigen.

OVERVIEW SUMMARY

Multiple approaches to immunizing patients against cancer antigens are being explored. Immunization with DNA encoding cancer antigens has the advantage of immunizing only against the transgene and not against other vector components. In this work we have explored the ability to immunize patients with metastatic melanoma utilizing DNA encoding the melanoma-melanocyte differentiation antigen gp100. We were unable to demonstrate any consistent immunization against gp100 by this approach, using sensitive assays for the detection of immune T cell precursors.

INTRODUCTION

Active immunization strategies for the immunotherapy of patients with cancer are based on the ability to raise T lymphocytes capable of reacting against tumor antigens. Many of the genes encoding cancer antigens have been cloned and extensive studies are being performed to determine the most effective means for immunizing patients against these autologous tumor antigens (Rosenberg, 2001). Immunization with recombinant viruses such as adenovirus, fowl poxvirus, and vaccinia virus engineered to encode full-length tumor antigens can generate modest levels of immune precursors in some patients; however, the ability to perform repeated immunizations with these viruses is severely impeded by the neutralizing immune reactions that occur against the viral envelope proteins (Rosenberg et al., 1998b). Thus alternative approaches have been sought that enable repeated boosting immunizations against the same antigen. Attempts to immunize with full-length recombinant proteins have been limited by the difficulties and expense of producing sufficient quantities under GMP (Good Manufacturing Practice) conditions suitable for human use. The relative ease of producing large amounts of plasmid DNA has thus led to exploration of the use of DNA encoding full-length antigens as an immunizing vector in animals and in humans.

DNA-based immunization strategies have been shown to elicit cellular and humoral immunity to a broad range of agents including bacteria, viruses, and cancer in many nonhuman species (reviewed in White and Conry, 2000). Preliminary studies in humans, using bacterial or viral antigens, have suggested that cellular immune responses can be generated in humans as well. These studies have prompted us to explore the ability of DNA immunization to generate cellular immune responses against the gp100 human cancer antigen and to explore the therapeutic efficacy of this approach in patients with metastatic melanoma.

gp100 is a nonmutated, differentiation antigen expressed on melanocytes and overexpressed on melanomas (Kawakami et al., 1994, 1995). The immunogenicity of gp100 can be significantly increased by introducing amino acid modifications in the protein at positions 210 (methionine for threonine) and 288 (valine for alanine) that produce peptides with increased binding affinity for HLA-A*0201 MHC class I molecules (Parkhurst et al., 1996; Rosenberg et al., 1998a). In previous studies we have shown that immunization with these modified gp100 peptides or with fowl poxvirus encoding full-length gp100 containing these modifications can generate specific anti-gp100 T cells in patients with metastatic melanoma (Rosenberg et al., 1998a, 2003).

In the present study we have administered plasmid DNA encoding the modified gp100 molecule either intradermally or intramuscularly to patients with metastatic melanoma and evaluated the impact on tumor growth and the generation of anti-gp100 T cells.

MATERIALS AND METHODS

Patient protocol

All patients were treated in the Surgery Branch, National Cancer Institute (NCI, Bethesda, MD) and signed an informed consent approved by the NCI Institutional Review Board before entering this clinical trial. All patients were HLA-A*0201 positive, had measurable metastatic melanoma, and were expected to survive more than 3 months. All patients had progressive disease and had not undergone any other form of therapy in the 3 weeks before entering this protocol. Patients requiring steroid therapy or who had any form of primary or secondary immunodeficiency disease were not included. All patients were evaluated by complete physical and radiologic examination before and at 2-month intervals during the protocol.

Patients were prospectively randomized to receive 1 mg of DNA either intradermally or intramuscularly every 4 weeks for up to four doses. Patient clinical status was evaluated after two doses and if disease progressed, patients were removed from the protocol or switched to other treatments. Patients randomized to receive DNA by the intramuscular route received 0.5 mg intramuscularly into each of two proximal extremities. Patients randomized to receive DNA by intradermal injection received 0.1 mg of DNA at five sites within 10 cm² of skin on each of two proximal extremities.

A partial response was defined as a 50% reduction in the sum of the products of perpendicular diameters of all lesions, lasting at least 1 month with no growth of any lesions or appearance of any new lesions. Any patient not achieving at least a partial response was considered a nonresponder.

DNA encoding the gp100 melanoma-melanocyte antigen

Plasmid DNA encoding the full-length gp100 molecule containing methionine and valine amino acid substitutions at positions 210 and 288, respectively, was provided by Vical (San Diego, CA) under a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute. The DNA was produced in Escherichia coli, using the VR4951 plasmid designed to optimize expression of the transgene. The plasmid contained the gp100 gene promoted by the human cytomegalovirus immediate-early (IE) promoter, intron A derived from human cytomegalovirus, the rabbit β-globin/Proudfoot terminator sequence, and the kanamycin resistance gene. The purified DNA was resuspended in phosphate-buffered saline, filtered through a 0.1-µm pore size filter, and stored in vials containing 1.2 ml at 1 mg/ml. The DNA was stored at −20°C and injected immediately after thawing at room temperature.

The plasmid underwent extensive testing for functionality in mice. A single DNA immunization with the plasmid was found to induce vigorous gp100-specific antibody responses. A single immunization with the DNA plasmid used also induced gp100-specific T cell responses in HLA-A*0201-transgenic mice. Finally, when coated onto gold beads and administered by Helios gene gun (Bio-Rad, Hercules, CA), the plasmid was shown to be effective at inducing protection from challenge with the gp100-expressing B16 mouse melanoma.

In vitro assessment of gp100-specific immune precursors

Immunologic reactivity against the immunizing gp100 antigen was tested with a 12-day in vitro boost assay as previously described (Rosenberg et al., 1998a). In brief, cryopreserved peripheral blood mononuclear cells (PBMCs) obtained before and 4 weeks after immunization were thawed and washed in complete culture medium containing 10% human AB serum. Peptides from the gp100 molecule (gp100:209–217 and gp100:280–288) were added at 1 µM concentration and the cells were cultured in interleukin 2 (IL-2) at 300 IU/ml (Chiron, Emeryville, CA) for approximately 12 days. Cells were harvested and tested for specific cytokine release after coculture for 18 hr with T2 cells pulsed with peptides or with HLA-A*0201-positive and -negative tumor cells. As in previous studies a positive response was defined as secretion of interferon γ at greater than 100 pg/ml in response to the specific peptide and at least two times that released in response to a control peptide (Rosenberg et al., 1998a).

RESULTS

Eleven patients were randomized to receive DNA by the intramuscular route and 12 patients were randomized to receive DNA by the intradermal route. One patient randomized to receive intramuscular DNA was subsequently found not to have measurable melanoma and was excluded from the analysis. The details of the 22 evaluable patients are shown in Table 1. Patients ranged from 26 to 70 years of age and all had received systemic treatment for metastatic melanoma before entering the protocol. Sixteen patients had received prior chemotherapy, 20 had received some prior form of immunotherapy, and 8 had received prior radiation therapy. Most patients had multiple sites of metastatic melanoma. Most patients received at least two cycles of DNA treatment although four patients were taken off protocol at 1 month after one cycle of treatment because of substantial disease progression. Five patients received more than two cycles of immunization.

TABLE 1.

PATIENT CHARACTERISTICS

Patient Age/sex Prior treatmenta Site(s) of disease DNA, cycles (number) Responseb
1 70/F S, I Cutaneous 8 PR
2 44/F S, C, I Lung 2 NR
3 26/F S, C, I Subcutaneous 2 NR
4 39/M S, C, I, R Liver, lymph nodes, spleen 4 NR
5 49/M S, I Liver, lung, adrenal 2 NR
6 42/F S, C, I Lung, lymph nodes 1 NR
7 68/M C, I, R Liver, lung 2 NR
8 62/F S, C, I Subcutaneous 2 NR
9 69/M S, C, I Lung 4 NR
10 60/M S, C, I, R Liver, lung, subcutaneous 2 NR
      Intradermal    
1 51/M S, C, I Lymph nodes, intraperitoneal 2 NR
2 46/F S, C, I Lung 2 NR
3 67/M S, I Lung, intestine 8 NR
4 42/M S, C, I Liver, lung 2 NR
5 42/M S, C, R Lymph nodes 2 NR
6 67/F S, C, I Cutaneous 4 NR
7 51/M S, I Subcutaneous, lymph nodes 1 NR
8 59/M S, C, I, R Lung, adrenal 1 NR
9 75/M S, C, I, R Lung, cutaneous, subcutaneous 2 NR
10 58/F S, I, R Lung, subcutaneous 1 NR
11 49/F S, C, I, R Lung, subcutaneous 2 NR
12 36/F S, C Pancreas 2 NR
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a

S, Surgery; I, immunotherapy; C, chemotherapy; R, radiotherapy.

b

PR, Partial response; NR, no response.

Mild but transient pain at the injection sites in some patients was the only toxicity seen. One of the 22 patients (4.5%) experienced a partial response of three subcentimeter metastatic deposits on the ankle that were the only sites of disease. All other patients had progressive disease.

Of the 22 evaluable patients, 6 had previously received immunization to different forms of gp100, either as peptides or recombinant viruses, and thus were not evaluable for immunologic assessment of precursor generation. Three patients had no postimmunization cells available for assay. Thus 13 patients (5 had received intramuscular injections and 8 had received intradermal injections) were tested for the ability of the DNA immunization to raise immunologic precursors directed against the two gp100 immunodominant peptides: gp100:209–217 and gp100:280–288. An example of an assay performed is shown in Table 2. In vitro boosting was performed with the native gp100:280–288 peptide or with the native gp100:209–217 peptide, and the coculture was tested against the native peptides as well. To assess the immunologic competence of the cells, an HLA-A*0201-restricted flu peptide was often included in the assays because most patients had previous exposure to influenza virus. As a positive control in the assay, patients who had been successfully immunized with the immunodominant gp100:209–217 peptide in other protocols were included as well (patient 4 in Table 2) (Rosenberg et al., 1998a). As shown in Table 2, there was no generation of reactivity against the immunodominant gp100 peptides tested, although lymphocytes were immunologically competent, as evidenced by reactivity to flu peptide. PBMCs from the patient who had received prior peptide immunization (included as a control in this assay) were strongly positive against the gp100:209–217 peptide.

TABLE 2.

In vitro ASSAY OF PBMCs

      Targeta: T2 cells pulsed with:
Patient Vaccine In vitro peptide boost T2 gp100:280–288 gp10:209–217 Flu peptide
1 DNA, intramuscular gp100:280–288 81 136 38 82
    gp100:209–217 164 142 119 588
    Flu 160 140 145 292,100
2 DNA, intramuscular gp10:280–288 446 96 110 121
    gp100:209–217 172 79 164 101
    Flu 101 130 106 2,175
3 DNA, intradermal gp100:280–288 91 94 53 76
    gp100:209–217 158 117 120 560
    Flu 85 49 118 18,810
4b Peptide gp10:280–288 38 42 592 24
    gp100:209–217 34 28 11,860 0
    Flu 0 51 2,023 26,210
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a

Targets were T2 cells unpulsed or pulsed with gp100:280–288, gp100:209–217, or flu peptide. Results expressed as picograms of IFN-γ per milliliter.

b

PBMCs from a patient who received the gp100:209–217 (210M) peptide in Freund’s incomplete adjuvant.

None of the 13 patients tested showed any evidence of immunization against the gp100:209–217 or gp100:280–288 peptide when tested 4 weeks after two cycles of immunizations. In addition, three patients were assessed after four cycles of immunization (one intramuscular and two intradermal) and neither had any in vitro evidence of immunization, nor did the patient who received eight sequential cycles of intradermal injections. No attempt was made to measure antibody responses to gp100.

Because we could identify no evidence of immunogenicity against these peptides after a single in vitro boost assay, PBMCs obtained after two administrations of plasmid DNA were tested after three weekly in vitro stimulations with the immunizing peptide. We had previously shown that repeated in vitro stimulations (rather than the single 12-day in vitro sensitization reported above) were more sensitive at eliciting low levels of immunization (Salgaller et al., 1996; Cormier et al., 1997). PBMCs from 10 patients were subjected to three in vitro stimulations (4 intramuscular and 6 intradermal patients) and again, no reactivity could be elicited against the two immunodominant peptides in postimmunization samples.

Results obtained from PBMCs of patients in this study were compared with similar patients with metastatic melanoma treated during approximately the same time period with fowl poxvirus encoding the same modified DNA and utilizing the same 12-day in vitro sensitization assay (Table 3). Four of 14 patients and 7 of 14 patients receiving this recombinant fowl poxvirus were successfully immunized against the gp100:209–217 peptide and the gp100:280–288 peptide, respectively. In another clinical trial, 9 of 10 patients were successfully immunized after receiving the gp100:209–217(210M) peptide administered in Freund’s incomplete adjuvant, also assessed using this same 12-day in vitro sensitization assay (our unpublished data).

TABLE 3.

IMMUNIZATION OF MELANOMA PATIENTS WITH THE gp100 ANTIGEN

  Reactivity againsta:
Vaccine gp100:209–217 gp100:280–288 gp100:254–162
gp100 DNA, intramuscular 0/5 0/5 0/3
gp100 DNA, intradermal 0/8 0/8 0/6
Fowl poxvirus gp100 4/14 7/14 1/14
gp100:209–217 (210M) 9/10 – –
  peptide in Freund’s      
  incomplete adjuvant      
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a

No. of patients positive/total.

DISCUSSION

DNA-mediated immunization is a theoretically attractive approach to immunization against full-length cancer antigens. DNA is relatively easy to prepare, purify, and sequence in large amounts compared with full-length proteins or recombinant viruses. Plasmid DNA does not result in the expression of non-relevant proteins and, thus, no immune reactions are generated to neutralize the immunizing vector, as is the case for recombinant viruses, thus enabling the repetitive administration of DNA. Unlike many recombinant viruses, DNA vaccines do not express proteins that can downregulate MHC class I gene expression in transfected cells. In addition, DNA preparations are stable and, because they do not integrate into the genome, represent little risk of insertional mutagenesis or recombinational events that can lead to pathogenic viruses (Nicholas et al., 1995).

Since early reports by Wolff et al. demonstrated that skeletal muscle cells could internalize plasmid DNA and result in the episomal expression of the encoded gene (Wolff et al., 1990, 1992), many attempts have been made to utilize DNA-based immunization to raise humoral and cellular immunity to a broad range of agents including bacteria, viruses, and cancer in a wide variety of species (Tang et al., 1992; Cox et al., 1993; Davis et al., 1993; Fynan et al., 1993; Ulmer et al., 1993; Wang et al., 1993; Hoffman et al., 1994; Conry et al., 1995; Xu and Liew, 1995; Irvine et al., 1996). Multiple studies have shown that direct injection of DNA encoding “foreign” model tumor antigens could protect mice from the growth of tumors, including those expressing simian virus 40 (SV40) large T antigen, human carcinoembryonic antigen, mutated p53 antigen, β-galactosidase, as well as a variety of human immunodeficiency virus (HIV)-related proteins (Conry et al., 1995; Wang et al., 1995; Bright et al., 1996; Irvine et al., 1996). The best route to immunize with DNA is unknown, and although the intramuscular route is most commonly used, studies of mouse and nonhuman primates have suggested that intradermal injection or ballistic introduction of DNA-coated gold particles, utilizing a “gene gun,” can be more effective in raising immune responses (Tang et al., 1992; Fynan et al., 1993; Ulmer et al., 1993; Wang et al., 1993).

These preclinical studies led to attempts to use DNA to immunize humans against bacterial or viral proteins. Multiple trials in which HIV-infected individuals were immunized with DNA encoding envelope proteins or the Nef, Rev, and Tat regulatory proteins from HIV have been difficult to interpret because of preexisting immunity in these patients, although several have suggested an increase in cellular and humoral immune reactions (Goepfert et al., 1998; MacGregor et al., 1998; Boyer et al., 1999). DNA vaccine trials against HIV proteins in normal volunteers have resulted in modest immunization in a minority of patients (Goepfert et al., 1998; Gold and Avrett, 1998). DNA vaccination resulted in humoral and cellular responses in humans against malarial, hepatitis virus, and papillomavirus proteins in patients receiving repeated injections of up to 2.5 mg of DNA either by the intramuscular or intradermal route or encapsulated in biodegradable polymer particles (Wang et al., 1998; White and Conry, 2000; Conry et al., 2002; Klencke et al., 2002).

Few data are available concerning the ability of DNA-mediated immunization to raise immune responses against non-mutated but overexpressed “self” cancer antigens in humans. A dual expression plasmid encoding carcinoembryonic antigen, as well as hepatitis B surface antigen, injected intramuscularly into 17 patients with metastatic colorectal cancer demonstrated effective generation of antibodies against the hepatitis protein but little or no humoral or cellular reactivity against carcinoembryonic antigen (Conry et al., 2002). DNA vaccines encoding a chimeric immunoglobulin molecule containing autologous variable heavy and light chain immunoglobulin sequences derived from each patient’s B cell lymphoma linked to mouse immunoglobulin constant region sequences resulted in cellular immune reactivity against the human idiotype in 1 of 12 patients (Timmerman et al., 2002). No antitumor clinical responses were seen in these trials. DNA encoding the HLA-B7 MHC class I protein was injected into HLA-B7-negative patients, using DNA-liposome complexes designed to enhance the immunogenicity of tumor cells by activating immune responses against these foreign MHC antigens. Some regression of injected nodules was reported in these studies (Nabel et al., 1993).

On the basis of these preclinical and clinical efforts we have conducted a study attempting to immunize HLA-A*0201-positive melanoma patients with DNA encoding the gp100 melanoma antigen containing two amino acid substitutions designed to increase the immunogenicity of the immunodominant epitopes of this antigen (Parkhurst et al., 1996). To determine the most effective method for immunizing patients, they were randomized to receive repeated injections of 1 mg of DNA either intramuscularly or intradermally. The vector we used in these clinical trials (produced and supplied by Vical) had several features designed to optimize expression of the encoded gene, including a eukaryotic cytomegalovirus intron A that has been reported to augment protein production (Chapman et al., 1991). Only one partial response of subcentimeter cutaneous nodules was seen in the 22 patients we treated. All other patients had progressive disease. We were unable to demonstrate any evidence of cellular immune reactions against the two immunodominant epitopes of the gp100 molecule, using assays that were successful in demonstrating immune reactions against these antigens after injection of recombinant fowl poxvirus encoding gp100 or by the direct injection of gp100 peptides in Freund’s incomplete adjuvant (Table 2 and Table 3). The in vitro sensitization boost assay we used is highly sensitive and can detect immune reactions in patients in whom enzyme-linked immunospot and tetramer assays are negative. Despite the high level of sensitivity of this assay, including efforts at repeated in vitro stimulations, we were unable to demonstrate any evidence of cellular immune reactivity in these patients.

We thus conclude that neither intramuscular nor intradermal injection of DNA encoding the gp100 nonmutated melanoma-melanocyte antigen was capable of raising cellular immune reactivity or a significant incidence of antitumor effects in patients with metastatic melanoma. Studies performed in experimental animals have suggested that addition of DNA encoding cytokines (Kim et al., 1997; Xin et al., 1998), additional CpG sequences (Sato et al., 1996), or replicon-based vectors (Leitner et al., 2003) may be more potent approaches to DNA immunization in future. We conclude, however, that although DNA immunization in humans can raise cellular and immune reactivity to “foreign” proteins, little evidence exists today that such reactivity can be generated against nonmutated cancer antigens in humans.

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