Gene-modified Tumor Vaccines Research Articles

A Novel Therapeutic Vaccine of Mouse GM-CSF Surface Modified MB49 Cells Against Metastatic Bladder Cancer

Immunotherapy is considered effective for muscle invasive bladder cancer mini metastasis. We developed what is to our knowledge a novel technology by which streptavidin tagged mouse GM-CSF was displayed on the surface of biotinylated bladder cancer cells to induce antitumor immunity. Mouse subcutaneous and lung metastasis bladder cancer models were established. Mice were injected subcutaneously with 1 × 10(6) mouse GM-CSF surface modified MB49 bladder cancer cells and monitored for tumor growth and survival. Immunohistochemical and flow cytometric assay were done to assess the proportion of T lymphocytes. The T-lymphocyte cytotoxicity assay was performed to assess MB49 specific cytotoxicity. On day 60 after MB49 implantation the vaccine cured mice were injected subcutaneously with MB49 or RM-1 cells in the left or right hind leg, respectively. They were monitored for survival and T-lymphocyte cytotoxicity. Mouse GM-CSF surface modified vaccine significantly inhibited tumor growth in the subcutaneous model and extended survival in the lung model. More CD4 and CD8 T cells appeared at tumor sites and in peripheral blood in the vaccine treated group than in other control groups. Splenocytes from the vaccine treated group showed the most potent cytotoxicity on MB49 cells. Cured mice in the vaccine treated group resisted the second injection of MB49 bladder cancer cells but not the RM-1 prostate cancer cell challenge. Mouse GM-CSF surface modified MB49 bladder cancer cell vaccine induced specific antitumor immunity and was efficient for metastatic bladder cancer.

Gene-modified tumor vaccine secreting a designer cytokine Hyper-Interleukin-6 is an effective therapy in mice bearing orthotopic renal cell cancer

Although renal cell cancer (RCC) is known to be immunogenic, clinical efficacy of various immunotherapeutic approaches remains unsatisfactory. Novel targeted therapies showing cytostatic rather than cytotoxic activity are unable to cure RCC patients. In our studies, we evaluated the therapeutic efficacy of whole-cell vaccine based on irradiated murine RENCA cells genetically modified to secrete designer cytokine--Hyper-IL6 (H6)--comprising IL-6 and soluble IL-6 receptor. An orthotopic RCC model based on a subcapsular implantation of RENCA cells into kidneys of Balb/C mice was employed. The efficacy of RENCA-H6 vaccine was compared with control vaccine (RENCA-wt) in relation to naive (non-immunized) animals. Three sets of vaccination experiments were carried out in a (i) protective, (ii) palliative and (iii) adjuvant (following nephrectomy) setting. The influence of vaccination on survival of RCC-bearing animals was analyzed. Specificity of vaccine-induced immune response was studied using model antigen-GFP. RCC-bearing animals immunized with RENCA-H6 vaccine showed prolonged survival compared with other groups. In palliative and adjuvant settings the survival RENCA-H6-immunized animals exceeded 75%. Administration of RENCA-H6 inhibited formation and recruitment of Treg cells (CD4+CD25+Foxp3+) and increased maturation of DCs. RENCA tumors in RENCA-H6- vaccinated animals contained large populations of NK cells and activated CD4+, CD8+ T cells. In addition, in mice vaccinated with RENCA-H6 cells large population of CD4+ and CD8+ memory cells (CD62Llow) were detected. In the orthotopic RCC model, RENCA-H6 vaccine showed high therapeutic potential, which resulted from modulation of numerous immunological mechanisms.

Immune Responses Are Induced Against Side-Population B-CLL “Stem Cells ” by Patient Vaccination with hCD40L/IL2 Gene Modified Tumor Cells.

An apparent stem cell population can be identified in many normal and malignant tissues by their side-population (SP) phenotype, characterized by a high level of Hoechst dye efflux. Malignant SP cells have increased resistance to many cytotoxic drugs and may contribute to relapse and disease progression. We have looked for SP cells in patients with B cell chronic lymphocytic leukemia (B-CLL) and determined whether these cells could be targeted by an immune response after immunization with hCD40L/IL2 gene modified tumor vaccines. We also assessed the impact of any anti-SP immune response on overall disease. For SP analysis, PBMC containing >90% CD5+CD19+ cells were obtained pre-treatment. We incubated the cells with 5mg/ml Hoechst 33342 dye for 2 hours at 37°C. Cells were then labeled with CD5, CD19 and propidium iodide and sorted by flow cytometry into SP and non-SP populations. 11 of 12 patient tumor samples contained a distinct CD5+CD19+ SP population (0.04%–2%), whereas SP were undetectable in PBMC from healthy donors. SP cells had increased gene expression compared to non-SP for ABCG2 (4±2-fold) and the “stemness” genes p21cip1/waf1 (8±12-fold) and Bmi-1 (12±21-fold) supporting their function as a precursor population for the bulk of tumor cells. In 5 of the patients, SP analysis was done on samples taken before, during and after receiving tumor vaccines. We observed a significant decrease (p< 0.05) in B-CLL SP cells in all 5, although in 4/5, the decrease was transient, returning to pre-treatment levels by 4 months post-immunization. To determine if the observed reduction in tumor SP was associated with an immune response to these cells, B-CLL-specific cytotoxic T lymphocytes (CTL) were generated from T cells during immunization (2–4 weeks). These CTLs were specific for autologous bulk B-CLL cells (week 2 CTL, 40±4 IFN-gamma spot forming units (SFU) versus unstimulated controls 20±12 SFU, rising to 334±14 SFU versus control 20±5 SFU by week 4, P<0.01). Moreover, CTL lines obtained at 4 weeks after immunization were highly reactive against SP B-CLL cells. To determine the effects of the induced anti-B-CLL SP reactivity on the patients' disease, we followed the 5 patients for >1 year. In the 4 individuals in whom the anti-SP cell response was transient, no sustained effect on disease burden was evident. In one patient, however, the tumor SP cells remained at undetectable levels after immunization. This individual subsequently had a delayed (from 6 months onwards) but progressive disease response, and by 15 months had resolution of lymphadenopathy, a reduction in splenomegaly and a >90% decrease in the circulating B-CLL count. Hence, B-CLL cells contain a primitive side-population, which can be targeted by an immune response after immunization with autologous CD40L and IL2 expressing tumor cells. A sustained anti-B-CLL SP response was associated with a delayed but progressive response in the bulk tumor population. This effect is consistent with “stem cell” activity in the SP of B-CLL, and suggests that tumor SP cells may be an important target for therapy.

Small RNAs and Non-Small Cell Lung Cancer

Patients with non-small cell lung cancer (NSCLC) are commonly diagnosed with advanced disease and have limited therapeutic options. Experimental treatment approaches including small molecule targeted therapeutics, gene modified tumor vaccines, and viral-based gene therapy have induced tumor regression in a small proportion of patients, suggesting that advanced NSCLC is susceptible to molecular perturbations. RNA interference (RNAi) has generated considerable excitement as a potential cancer therapeutic application. RNAi is the process by which small, double stranded RNA molecules (small interfering RNA, or siRNA) can initiate sequence-specific, post-transcriptional gene silencing (PTGS). Cancer growth inhibition was attained through siRNA-knockdown of unique or overexpressed cancer oncogenetic messages that are relevant to NSCLC pathophysiology. As with other loss-of-function cancer gene therapy approaches, clinical efficacy of siRNA depends largely on the extent of cell target coverage at the locoregional and/or systemic level. Cationic liposomes as well as viral vectors have been used successfully for siRNA delivery. However, viral delivery may have more immediate relevance due to its wider clinical acceptance in the cancer gene therapy arena. We advocate the use of conditional replicative, oncolytic adenovirus for siRNA delivery, which offers potential benefits of restricted and renewable siRNA expression within the tumor microenvironment, and an additive anti-tumor outcome through viral oncolysis and siRNA-mediated oncogene-silencing, which we have demonstrated with the A549 NSCLC cell line. Several oncolytic adenoviral constructs are potentially applicable clinical platforms with proven infectivity and safety, which are feasible also for the delivery of microRNAs (miRNA), a recently discovered group of endogenous, small RNA with PTGS activity that is downregulated in lung cancer.

631. Phase 1/2 Trial of Autologous Tumor Mixed with an Allogeneic GVAX® Immunotherapy in Advanced Stage Non-Small Cell Lung Cancer

We have previously demonstrated activity with immunotherapy composed of autologous tumor cells genetically modified to secrete GM-CSF (GVAX®) in advanced stage NSCLC1. In an effort to remove the requirement and variability associated with genetic transduction of individual tumors we developed a “bystander” GVAX® platform composed of autologous tumor cells mixed with an allogeneic GM- CSF secreting cell line. We conducted a phase I/II trial of this immunotherapy (3 to 12 biweekly injections) in advanced stage NSCLC. Tumors were harvested from 86 patients, tumor cell processing was successful in 76, and 49 received study treatment. The most common toxicity was local injection site reactions. Serum GM-CSF pharmacokinetics were consistent with secretion of GM- CSF from the injected cells for up to 4 days with associated transient leukocytosis confirming GM-CSF bioactivity. Evidence of treatment-induced immune activation was demonstrated; however, objective tumor responses were not seen. Compared with autologous GVAX® immunotherapy for lung cancer prepared by transduction of individual tumors with an adenoviral GM-CSF vector, GM-CSF secretion was approximately 25-fold higher with the bystander GVAX® product used in this trial. However, the frequency of injection site reactions, tumor response, time to disease progression, and survival were all less favorable in the current study in comparison to prior results. Follow-up results will be presented. 1. Nemunaitis J, Sterman D, Jablons D, et al. Granulocyte- macrophage colony-stimulating factor gene-modified autologous tumor vaccines in non-small-cell lung cancer. J Natl Cancer Inst 2004;96(4):326–31.

CpG immunostimulatory oligodeoxynucleotide 1826 enhances antitumor effect of interleukin 12 gene-modified tumor vaccine in a melanoma model in mice.

The effectiveness of interleukin (IL)-12-secreting tumor vaccines in the treatment of mouse tumors could be enhanced by concurrent application of cytokines and costimulatory molecules. We investigated the therapeutic potential of IL-12 gene-transduced melanoma vaccine in combination with CpG immunostimulatory oligodeoxynucleotide (ODN) 1826, an adjuvant known to favor development of Th1-biased immune response, in a B78-H1 (B78) melanoma model in mice. Mice injected with B78 melanoma cells were treated with irradiated IL-12 gene-transduced B78 cells [B78/IL-12(X)] and/or ODN 1826. Mechanisms responsible for the antitumor effects of the treatment were investigated using fluorescence-activated cell sorter analysis, a standard (51)Cr releasing assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ELISA. Single injection of B78/IL-12(X) cells had no effect on tumor growth, whereas seven consecutive daily injections of ODN 1826 markedly inhibited tumor progression with occasional curative effects. When used in combination, B78/IL-12(X) cells and ODN 1826 caused additional tumor growth reduction and eradication of tumors in 62% of treated mice. The combined treatment activated local inflammatory response against tumor but also induced systemic antitumor immunity. In vitro studies have shown that when used together, B78/IL-12(X) cells and ODN 1826 induced a potent Th1 response and suggested the role of IFN-gamma in activation of the host immune response. The antitumor effects in double-treated mice were accompanied by the development of cytotoxic effectors in the spleen and activation of macrophages. The results provided the evidence that the combination of IL-12 gene-modified melanoma vaccine and ODN 1826 induces synergistically systemic and local antitumor immunity.

Superantigen-SEA gene modified tumor vaccine for hepatocellular carcinoma: an in vitro study.

To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to express staphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC. SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80. Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine, positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFgamma-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells. Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by (51)Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control. Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P<0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P<0.05). Tumor cell membrane expressed superantigen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.

GVAX (GMCSF gene modified tumor vaccine) in advanced stage non small cell lung cancer

Treatment for advanced stage non-small cell lung cancer is limited. In an attempt to determine whether immune stimulating activity can be induced with a GMCSF gene transduced autologous tumor vaccine (GVAX), 83 patients were entered into trial (20 with stage 1B/IIB disease and 63 with stage IIIB/IV disease). Patients fulfilling eligibility criteria underwent surgical harvest of autologous lung tumor tissue. The tissue was then processed, transfected with an E1 deleted, E3 deleted adenoviral GMCSF gene vector irradiated prior to injection. Patients received a series of six vaccinations (1 every two weeks). Forty-three of the 83 patients were eligible for treatment (10 in cohort A and 33 in cohort B). These results have previously been reported. GVAX was well tolerated. Three of 33 patients with advanced disease achieved complete response. Evidence of immunologic activity following vaccination in a subset of patients was suggested, based on antibody response to either autologous SV40 transformed tumor cells or allogeneic non-small cell lung cancer tumor cells. These results supported further clinical investigation with GVAX in non-small cell lung cancer.

368. Therapy with IL-6 and Soulble IL-6R Gene Modified Tumor Vaccine (GMTV) Extends Survival of Advanced Melanoma Patients

368. Therapy with IL-6 and Soulble IL-6R Gene Modified Tumor Vaccine (GMTV) Extends Survival of Advanced Melanoma Patients

Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DC IMAT) or relative mature DCs (DC RMAT). DC RMAT displayed an up-regulated expression of immune molecules (Ia d, CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DC IMAT. These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DC RMAT/J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1×10 6 cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DC IMAT/J558 hybrids. Furthermore, immunization of mice with engineered DC RMAT/J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DC RMAT/J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.

Melanoma vaccines.

Several different approaches to fighting melanoma are currently being actively explored utilizing the host's immune system. These include augmentation of the immunogenicity of tumor cells by gene modification with cytokine genes and the use of T cell-defined melanoma-associated peptides, either directly for immunization (peptide vaccines) or after loading onto professional antigen-presenting cells such as dendritic cells (dendritic cell-based vaccines). Preclinical studies provided the regulatory, mechanistic and functional principles for cytokine gene-modified tumor vaccines and led to a number of phase I/II studies, including trials with IL-7-, IL-12-, GM-CSF- or interferon gamma-secreting tumor cell vaccines or IL-2 gene-transfected cell-based vaccines. Results from other clinical studies suggest that peptide immunization may represent a potentially effective means of treatment and that vaccination with autologous dendritic cells generated from peripheral blood is a safe and promising approach. The above strategies for the treatment of melanoma have shown encouraging results, although the number of patients treated in each clinical trial was small.

Induction of antitumor immunity with modified autologous cells expressing membrane-bound murine cytokines.

Development of cytokine gene-modified autologous tumor vaccines must take into account the strictly paracrine physiology of cytokines whose expression at the tumor microenvironment is important for the successful induction of tumor-specific immunity. In this study, we investigated the efficacy of a tumor vaccine composed of inactivated autologous cells transfected with two plasmid vectors encoding a mutant membrane-bound murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and murine interferon-gamma (MuIFN-gamma). Expression of both cytokines as cell surface ligands on the highly metastatic D122 clone of Lewis lung carcinoma led to abrogation of their tumorigenicity and metastatic phenotype. More importantly, vaccination with irradiated tumor cells expressing the membrane-bound GM-CSF and IFN-gamma induced a cytotoxic T lymphocyte (CTL) response that protected syngeneic mice against a subsequent challenge with D122 cells as a primary tumor in preimmunized mice as well as against lung metastasis developing after surgical removal of the primary tumor in naive mice. Autologous cells expressing the membrane-bound GM-CSF and IFN-gamma exhibited comparable efficacy as an antimetastatic vaccine to a vaccine composed of transfectants expressing wild-type secreted cytokine molecules. These results indicate that membrane-bound cytokines can cause enhanced immunogenicity when transfected into tumor cells for the induction of antitumor immunity.

Efficient activation of antitumor immunity by IL-6 gene-modified leukemia vaccine in combination with low dose cyclophosphamide and low dose IL-2

Objective: To investigate the antitumor effect of the IL-6 gene-modified erythroleukemia cells combined with low dose cyclophosphamide (Cy) and low dose IL-2. Methods: Mice inoculated with FBL-3-IL-6 in combination with low dose IL-2 and low dose cyclophosphamide (Cy). Results: Mice received combined therapy of FBL-3-IL-6, IL-2 and Cy developed tumors most slowly and survived much longer when compared with mice in control groups, with 5 out of 8 leukemia-bearing mice being tumor free 100 days after the combined treatment. To further explain the mechanism of the antitumor effects by the combined therapy. It was found that combined therapy with low dose Cy, low dose IL-2 and FBL-3-IL-6 achieved maximal cytotoxic effects of nature killer cells and specific cytotoxic T lymphocytes, increased production IL-2, TNF and GM-CSF from spleen lymphocytes in tumor-bearing mice. Vaccination with the FBL3-IL-6 also enhanced the cytotoxic activity of the peritoneal macrophages. The results demonstrated that administration of low dose Cy and low dose IL-2 in combination with IL-6 genemodified leukemia vaccine could elicit potent antileukemia effects, and the mechanisms involved in the antitumor process may include the induction of specific and nonspecific antitumor immunity, reversal of T suppressor cells that mediated local immuno-suppression in tumor bearing mice. Conclusion: The combined therapy with cytokine gene-modified tumor vaccine, low dose of Cy and IL-2 might be a promising approach for the treatment of leukemia.

Gene-Modified Tumor Vaccine with Therapeutic Potential Shifts Tumor-Specific T Cell Response from a Type 2 to a Type 1 Cytokine Profile

Vaccination with a poorly immunogenic/nonimmunogenic tumor fails to protect the host from a subsequent challenge with the same tumor. The mechanisms underlying the failure of these tumors to sensitize therapeutic T cells are not clearly understood, but the inability of host T cells to recognize tumor has been implicated. In this study, vaccination with the poorly immunogenic B16BL6-D5 (D5 H-2b) tumor fails to generate therapeutic T cells from the tumor vaccine-draining lymph nodes (TVDLN) in our adoptive immunotherapy model. However, if vaccination is performed with an allogeneic MHC class I gene (H-2 Kd)-modified tumor, the T cells obtained from the TVDLN are therapeutic after activation with anti-CD3 and IL-2. Lymph nodes (LN) draining both D5 and D5-Kd tumor vaccines contained increased numbers of cells with reduced expression of L-selectin (L-selectin(low/-)) compared with naive LN. This implies that vaccination led to sensitization of T cells even in LN draining the unmodified D5 tumor. L-selectin(low/-) cells from D5-Kd, but not D5, TVDLN were therapeutic in our animal model. No antitumor activity was seen in the high level L-selectin T cells. L-selectin(low/-) T cells exhibited tumor-specific cytokine release that was type 2 (IL-4, IL-10) following vaccination with native D5 and type 1 (IFN-gamma) following vaccination with gene-modified D5-Kd. Our data suggest that the failure of unmodified D5 to generate therapeutic T cells is not due to an inability to recognize tumor Ags, but, rather, to the induction of an immune response that is ineffective in mediating tumor regression, i.e., immune deviation.

Gene and cell transfer for specific immunotherapy.

The realisation that human tumor cells may express and process tumor specific and tumor associated antigens has increased interest in immunotherapeutic approaches to cancer treatment. This interest has been coupled with a burgeoning ability to genetically modify tumor cells and components of the immune system, in an effort to maximize the anti-neoplastic response. In a number of settings, gene modified tumor vaccines, cytotoxic T cells and dendritic cells are producing both immunomodulation and clinically evident benefits. Continued exploration of this approach seems well justified.

Is gene therapy of advanced prostatic carcinoma on the verge of clinical application?

Gene transfer strategies for prostate cancer may yield promising future perspectives. In this regard, gene modified tumor vaccines, suicide gene therapy and gene replacement of nonfunctioning tumor suppressor genes are of special interest. Clinical phase I and early phase II studies with gene modified vaccines have to demonstrate the clinical implication of these new therapeutic options. Apart from this approach the development of more efficient delivery systems for in vivo gene transfer is highly desirable.

The humoral antitumor responses induced by IL-4 gene-modified tumor vaccine

It is well known that IL-4 plays important roles in the induction of humoral immunity. In the present study, the humoral antitumor responses induced by IL-4 gene-modified tumor vaccine were investigated. The mice were vaccinated with the IL-4 gene-modified B16 melanoma cells. Then the proliferative capacity of the splenic lymphocytes, the levels of the antibodies in the murine serum against wild-type B16 cells and the cytotoxicity of the serum to wild-type melanoma cells were detected. Our data showed that the LPS-induced proliferation of the splenic lymphocytes from the mice vaccinated with the IL-4 gene-modified tumor vaccine increased more significantly than that from mice vaccinated with wild-type tumor vaccine. The cytotoxicity of the serum to wild-type melanoma cells also increased markedly when detected. It was also observed that the number of pulmonary metastases decreased more obviously when the mice were intravenously injected with the mixture of wild-type B16 cells and the serum from the mice vaccinated with the IL-4 gene-modified B16 cells. Our data demonstrated that humoral immunity might contribute to the antitumor effect of IL-4 gene therapy.

Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors.

Recent studies have shown that the brain is not a barrier to successful active immunotherapy that uses gene-modified autologous tumor cell vaccines. In this study, we compared the efficacy of two types of vaccines for the treatment of tumors within the central nervous system (CNS): dendritic cell (DC)-based vaccines pulsed with either tumor extract or tumor RNA, and cytokine gene-modified tumor vaccines. Using the B16/F10 murine melanoma (B16) as a model for CNS tumor, we show that vaccination with bone marrow-generated DCs, pulsed with either B16 cell extract or B16 total RNA, can induce specific cytotoxic T lymphocytes against B16 tumor cells. Both types of DC vaccines were able to protect animals from tumors located in the CNS. DC-based vaccines also led to prolonged survival in mice with tumors placed before the initiation of vaccine therapy. The DC-based vaccines were at least as effective, if not more so, as vaccines containing B16 tumor cells in which the granulocytic macrophage colony-stimulating factor gene had been modified. These data support the use of DC-based vaccines for the treatment of patients with CNS tumors.

Efficient adenovirus-mediated gene transduction of normal and leukemic hematopoietic cells.

We evaluated the efficiency of adenovirus-mediated gene transfer into normal and malignant human hematopoietic cells. An E-1 and E-3 deleted, replication-defective recombinant Ad.RSV beta gal vector was used and the transduction efficiency was studied at a multiplicity of infection of 13 p.f.u. per cell. Approximately 40-50% of normal monocytes were transduced, whereas purified normal resting T cells and B cells were resistant to infection. We showed that 50-80% of primary chronic myeloid leukemia cells (CML, n = 12) were efficiently transduced in contrast to CML, successful transduction of resting primary chronic B lymphocytic leukemia cells required appropriate preactivation of targeted cells. A novel protocol for the efficient transduction of adenovirus into B-CLL cells was presented. We showed that anti-CD40 mAb or CD40 ligand acts in synergy with rhIL-4 to enable the transduction of approximately 50-75% of B-CLL cells (B-CLL, n = 6). Expression of beta-galactosidase in transduced CML cells and B-CLL cells was detected for at least 15 days after transduction. The present studies underline the utility of adenovirus vectors for the construction of cytokine gene-modified tumor vaccines for the treatment of hematopoietic malignancies such as CML and B-CLL.

Grundlagen der Gentherapie

The rapid development of recombinant DNA technology and our enhanced understanding of the genetic basis of human disease has facilitated the development of new molecular therapeutic modalities, termed gene therapy. Gene therapy involves the transfer of functional genes into somatic cells and their expression in target tissues in order to replace absent genes, correct defective genes, or induce antitumoral activity in the tumor-bearing host. Currently, an increasing number of gene therapy strategies are being investigated in experimental and clinical trials. Despite substantial progress, a number of technical and logistical hurdles must still be overcome before gene therapy can be safety and effectively applied in the human patient. Since gene therapy involves complex cell processing and can be time consuming and costly, simplifications or even alternative approaches will be necessary in order to establish this therapy as suitable for clinical use. This report reviews various gene therapy strategies and gene delivery techniques currently under clinical or experimental investigation. Special emphasis is given to cytokine gene therapy using gene-modified tumor vaccines for cancer treatment.