1 N.N. Petrov Research Institute of Oncology, St Petersburg; 2 Institute of Gene Biology, Moscow, Russian Federation
* Correspondence to: Dr N. Tyukavina, N.N.Petrov Research Institute of Oncology, pos. Pesochny-2, 68 Leningradskaia str., 197758 St Petersburg, Russian Federation. Tel: +7-812-596-65-44; Fax: +7-812-596-65-23; Email: tyukavina{at}yandex.ru
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Abstract |
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Patients and methods: Twenty-one patients (17 with disseminated malignant melanoma and four with metastatic renal cell carcinoma) were enrolled in this study. Cytoreduction was performed in all cases prior to therapy. Autologous tumor cells were transfected with the tag7/PGRP-S gene, irradiated and injected intradermally every 3 weeks.
Results: Vaccinations were well tolerated by all patients, without clinically significant signs of toxicity. Delayed-type hypersensitivity was observed in 48% of cases. Antitumor immune response was observed in 95% of patients. There were no complete or partial responses; however, a minor response was achieved in one patient with renal cell carcinoma. The stabilization of neoplastic disease was observed in eight patients (seven with malignant melanoma and one with renal cell carcinoma). Median time to tumor progression was 3 months.
Conclusions: The approach suggested here appears to be well tolerated and produces a number of durable clinical effects. Further studies are required to determine whether promising effects on immune activation will result in an actual clinical benefit for patients with malignant melanoma and renal cell carcinoma.
Key words: antitumor vaccination, gene therapy, melanoma, phase I/II trial, renal cell carcinoma, tag7 gene
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Introduction |
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Dendritic cells primed with tumor antigens in vitro are also considered to be a promising vaccine approach. At the same time, limited knowledge about tumor rejection antigens dictates the development of certain approaches based on the whole tumor cells as a source of antigens.
To activate specific immune responses against these antigens, tumor cells are genetically modified to secrete cytokines, enhancing immune response. Genes encoding granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin (IL)-2 and IL-12 have been used most commonly, both in preclinical studies and clinical trials [610
]. These cytokines are well known to participate in systemic immune response. Several studies have shown that the professional antigen-presenting cells (APCs) of the host, rather than the vaccinating tumor cells themselves, are responsible for priming CD4+ and CD8+ T cells, both of which are required to generate systemic antitumor immunity [11
, 12
]. Recent findings indicate that the adaptive arm of immunity is governed by the innate immunity mechanisms that control co-stimulatory signaling of APCs [13
16
]. Recently, we identified a novel gene, tag7, also know as PGRP-S [17
]. The insect ortholog of the tag7/PGRP-S was shown to be involved in the innate immune response in Drosophila [18
, 19
]. In preclinical studies we have demonstrated that tag7-modified mouse tumor cells induced long-lasting T-cell-dependent immune response in mice [20
]. The effectiveness of antitumor vaccination was demonstrated on different models of mouse tumor, particularly for melanoma cells (M3, B16, F10). Preclinical studies allowed us to develop a protocol for a phase I/II clinical trial of the autologous tag7-modified tumor cell vaccination of patients with disseminated solid tumors.
Clinically important results of vaccinotherapy were achieved in patients with melanoma and renal carcinoma in a number of studies. The results with this treatment are comparable to chemotherapy and immunotherapy [21]. We assume that to make anticancer vaccines more effective we have first to activate the innate, followed by the activation of the adaptive, component of immunity. Thus, to evaluate the feasibility and toxicity of treatment with autologous tumor cells modified with tag7 gene, which has been shown to be involved in innate immunity mechanisms, a phase I/II clinical trial has been performed.
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Patients and methods |
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Tumor cells lines establishment
Tumor specimens (no less than 1 cm3) were obtained during surgery. Single-cell suspensions of tumors were prepared by mechanical disaggregation using Medimachine System (Dako, Glostrup, Denmark). Cells were then washed, resuspended in Dulbecco's modified Eagle's medium/F12 medium containing 20% fetal bovine serum, 100 µ/ml penicillin and 100 µg/ml streptomycin (all from Invitrogen Grand Island, NY, USA). Suspension was plated on 100-mm cell culture Petri dishes (Costar, Actor, MA, USA) at a density of 104 cells/cm2, and cultured in the presence of 5% CO2 in humidified atmosphere. After 36 weeks of culture, the origin of the cells was confirmed using immunocytochemical staining with S-100 and HMB-45 for melanoma, and renal cell carcinoma antigen antibodies for renal cell carcinoma (NCL-RCC; Novocastra, Newcastle, UK). Transient transfections of primary tumor cell lines were performed with Effectene reagent (Qiagen). Optimal transfection conditions were determinate during preliminary experiments with reporter plasmid pEGFP-C2 (BD Biosciences Clontech, San Diego, CA, USA) according to optimization protocol provided by Qiagen. Reporter gene expression assays were performed with fluorescent microscopy (Figure 1).
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Vaccine preparation
Tumor cells were transiently transfected in vitro by Effectene reagent with plasmid containing human tag7 cDNA in the same vector as described previously [20]. Plasmid DNA was purified by EndoFree Plasmid Kit (Qiagen, Valencia, CA, USA) and was tested for endotoxin level using the LAL-test (Sigma). Each batch of vaccine was analyzed for Tag7 secretion by immunobloting. Routinely, quantities of the secreted protein varied between 5 and 80 ng/ml of culture medium/24 h/106 cells. After expression assay cells were irradiated with 2000 cGy [23
] and cryopreserved in liquid nitrogen. Frozen cells (107) were thawed and washed in Hanks' balanced salt solution before injection [24
].
Treatment scheme
Patients were vaccinated intradermally at three spots (3 cm from each other) in the paravertebral area 46 weeks after surgery. Non-transfected irradiated cells were injected intradermally at the time of vaccination. A complete treatment course consists of six injections at 21-day intervals.
Assessment of immunological response
To determine immunological response after vaccine administration, immunity status analysis was performed. This included absolute and relative content of subpopulations of T lymphocytes CD3, CD4 and CD8 (Sorbent, Perm, Russia), B lymphocytes CD20 (Sorbent), natural killer cells CD16 (Sorbent), activated T and B lymphocytes CD25, CD38, CD71, CD95 (Sorbent) and CD30 (Dako, Denmark), determined by indirect immunofluorescence [25]. The level of serum immunoglobulins G, A and M was determined by radial immunodiffusion [26
]. Phagocytic activity of neutrophils and monocytes was determined with latex particles [27
]. Metabolical activity of neutrophils and monocytes was determined by the spontaneous activity of neutrophils test according to the manufacturer's recommendations. Functional activity of T lymphocytes was determined by their reaction on concovalin A and phytohemagglutinin mitogens [28
]. Delayed-type hypersensitivity (DTH) was measured as bidimensional induration at 2448 h at the site of vaccine administration.
Assessment of toxicities
Toxicities were graded using National Cancer Institute Common Toxicity Criteria (NCI-CTC) (version 2.0, 1998).
Clinical response
Evaluation of antitumor response included physical examination, computed tomography scans or other tests appropriate for the specific tumor location. The sum of the maximum perpendicular measurements for all lesions was used to determine response (WHO, 1981) [29]. A complete response (CR) was defined as complete disappearance of all metastatic lesions, a partial response (PR) as a >50% reduction in the size of metastatic lesions, a minor response (MR) as a 25% to 50% decrease of lesions, and stable disease (SD) as a <25% decrease in the size of metastatic lesions. Survival was measured from the time of the first vaccination. The probability of survival was determined using the KaplanMeier method and GraphPad Prism 6.0 statistics software.
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Results |
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Toxicity
There was no systemic toxicity associated with the vaccine. The injections were well tolerated with minimal local pain and no ulceration.
DTH skin testing
Ten of 17 patients (48%) (three patients with renal carcinoma and seven with melanoma) developed DTH reactivity after intradermal injection of modified with the tag7 gene autologous tumor cells, which was clinically characterized by extensive erythema and induration, with a peak at 48 h, which then gradually resolved. It is important to mention that residual effects of DTH reactivity in patients with renal carcinoma remained for 20 days after the injection.
Clinical responses
No major clinical response (CR or PR) was observed in any patient. Eight patients (38%) showed SD (seven patients with melanoma and one with renal carcinoma) (Table 2). One patient with renal cancer had MR of lung metastases lasting 10 months.
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Immunological responses
The analyses of immunological monitoring showed that vaccinotherapy induced immune response in 95% (19 of 20 patients) of primary immunized patients. However, only eight patients received a complete course of vaccination. Immunological analysis of these patients showed a reliable increase of the relative amount of functional CD3+ lymphocytes from 65.6 ± 1.58% up to 72.7 ± 1.25% (P=0.013 according to Wilcoxon criteria) over 1 month from the first vaccination (Figure 2).
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Discussion |
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It is clear that treatment with autologous vaccines will be more efficient in patients with small tumors and high risk of relapse after surgical treatment. Taking into consideration the observed immunological effect, this group of patients should be the target for immunotherapy with autologous vaccine, in particular with the tag7-modified tumor cells.
It was reported previously that patients with renal carcinoma and melanoma were treated with the vaccines based on autologous tumor cells modified with the genes encoding such cytokines as GM-CSF, and IL-2, -7 and -12 [3134
]. These patients had a DTH reaction and activation of cell and humoral antitumor immunity, but experienced no clinical effects. In particular, the use of GM-CSF-based vaccine gave a stabilization period of 2036 months in three of 33 patients with skin melanoma [14
], and one of 16 patients with renal carcinoma had regression of multiple lung metastases [32
] (see also Table 3).
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Autologous vaccination may be considered as a promising method of treatment; however, it is not widely used because of the difficulties of vaccine preparation and thus the high price of these vaccines. Undoubtedly this method will have an important place in the treatment of human malignant tumors in future.
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Acknowledgements |
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Received for publication February 21, 2004. Revision received April 18, 2004. Accepted for publication September 7, 2004.
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