Anti-Gene IGF-I Vaccines in Cancer Gene Therapy: A Review of a Case of Glioblastoma.

OBJECTIVE: Vaccines for the deadliest brain tumor - glioblastoma (GBM) - are generally based on targeting growth factors or their receptors, often using antibodies. The vaccines described in the review were prepared to suppress the principal cancer growth factor - IGF-I, using anti-gene approaches either of antisense (AS) or of triple helix (TH) type. Our objective was to increase the median survival of patients treated with AS and TH cell vaccines. METHODOLOGY: The cells were transfected in vitro by both constructed IGF-I AS and IGF-I TH expression episomal vectors; part of these cells was co-cultured with plant phytochemicals, modulating IGF-I expression. Both AS and TH approaches completely suppressed IGF-I expression and induced MHC-1 / B7 immunogenicity related to the IGF-I receptor signal. RESULTS: This immunogenicity proved to be stronger in IGF-I TH than in IGF-I AS-prepared cell vaccines, especially in TH / phytochemical cells. The AS and TH vaccines generated an important TCD8+ and TCD8+CD11b- immune response in treated GBM patients and increased the median survival of patients up to 17-18 months, particularly using TH vaccines; in some cases, 2- and 3-year survival was reported. These clinical results were compared with those obtained in therapies targeting other growth factors. CONCLUSION: The anti-gene IGF-I vaccines continue to be applied in current GBM personalized medicine. Technical improvements in the preparation of AS and TH vaccines to increase MHC-1 and B7 immunogenicity have, in parallel, allowed to increase in the median survival of patients.

Characterization of effector components from the humoral and cellular immune response stimulated by melanoma cells exhibiting modified IGF-1 expression.

Modified melanoma B16 cells inhibited in their IGF-1 expression (B16MOD), on the contrary to the IGF-1 fully expressed parental wild-type (B16WT) counterpart, were shown to stimulate humoral as well as cellular immune responses. Among humoral components, the neutralizing and complement-fixing antibodies of IgM and essentially IgG2 (a+b) isotypes exhibited in vitro and in vivo effects upon tumour growth, while the IgG1 antibody isotype promoted enhanced tumour proliferation. As for the cellular immunity, it was found that the T CD8(+) lymphocyte subpopulation remained the main potent and long lasting immune active effector regulating tumour growth.

Rescue of MHC-1 antigen processing machinery by down-regulation in expression of IGF-1 in human glioblastoma cells.

Escape from immune recognition has been hypothesized to be a factor in carcinogenesis. It may be mediated for many cancers through down-regulation in the MHC class 1 antigen processing and presentation pathway. TAP-1, TAP-2, tightly linked to LMP-2 and LMP-7 are multiple components of the endogenous, antigen presentation pathway machinery. We addressed the question of alterations in this pathway in human Glioblastoma (HGB) and of its relationship to modulation in expression of IGF-1 that is highly expressed in this cancer. Deficiencies in expression of TAP-1 were demonstrated by RT-PCR and/or by immuno-flow cytometry in the HGB cell line T98G obtained from ATCC, and in 3 of 4 human cell lines established from patients with Glioblastoma Multiforme. Deficiencies in expression of TAP-2 were observed in 3 of 4, deficiencies in expression of LMP-2 in 4 of 4 and deficiencies in LMP-7 in 3 of 4 HGB cell lines examined by RT-PCR and Western blot. Following down-regulation of IGF-1 by transfection with the pAnti IGF-1 vector that expresses IGF-1 RNA in antisense orientation, or by the exogenous addition of IGF-1 receptor monoclonal antibody to cell culture media, the deficiencies in components of the MHC-1 antigen presentation pathway were up-regulated and/or rescued in all HGB cell lines tested. Moreover, this up-regulation in expression was aborted by addition of 100 ng/ml of IGF-1 to the culture media. Unlike in the case of IFN-γ, the restoration of TAP-1 and LMP-2 by down-regulation of IGF-1 in Glioblastoma cells was not correlated to the tyrosine phosphorylation of STAT 1. In summary, the simultaneous reversion in expression of the multiple constituents of MHC-1 antigen processing path and up-regulation in expression of MHC-1 occurring with down-regulation in IGF-1 may have a role in reinforcement of immunity against tumor antigen(s) in some animal cancers and in humans with Glioblastoma Multiforme.

Methodology for Anti-Gene Anti-IGF-I Therapy of Malignant Tumours.

The aim of this study was to establish the criteria for methodology of cellular "anti-IGF-I" therapy of malignant tumours and particularly for glioblastoma multiforme. The treatment of primary glioblastoma patients using surgery, radiotherapy, and chemotherapy was followed by subcutaneous injection of autologous cancer cells transfected by IGF-I antisense/triple helix expression vectors. The prepared cell "vaccines" should it be in the case of glioblastomas or other tumours, have shown a change of phenotype, the absence of IGF-I protein, and expression of MHC-I and B7. The peripheral blood lymphocytes, PBL cells, removed after each of two successive vaccinations, have demonstrated for all the types of tumour tested an increasing level of CD8(+) and CD8(+)28(+) molecules and a switch from CD8(+)11b(+) to CD8(+)11. All cancer patients were supervised for up to 19 months, the period corresponding to minimum survival of glioblastoma patients. The obtained results have permitted to specify the common criteria for "anti-IGF-I" strategy: characteristics sine qua non of injected "vaccines" (cloned cells IGF-I(-) and MHC-I(+)) and of PBL cells (CD8(+) increased level).

Stimulation of anti-melanoma immune effectors via modified tumour cells exhibiting inhibited IGF-I and low CD9.

Modified melanoma cells (B16-F0.MOD) characterized by inhibited IGF-I, CD9 low but not their wild-type counterparts (B16-F0.WT), IGF-I positive, CD9 high, were shown to be immunogenic for syngeneic hosts. C57BL/6 syngeneic recipients vaccinated with B16-F0.MOD cells developed immune effectors that were observed at the humoral as well as cellular levels. These immune effectors were shown to be capable of controlling in vitro tumour growth and in vivo tumour progression.

A new putative target for antisense gene therapy of glioma: glycogen synthase.

The treatment of malignant brain gliomas remains a challenge, despite the availability of the classical triad of surgery, radiotherapy, and chemotherapy. There is thus the need for investigations into other forms of treatment strategies, such as gene therapy. Using antisense technology we have targeted glycogen metabolism, since malignant astrocytes present a high content of glycogen. In vitro rat C6­glioma cells, transfected with antisense glycogen synthase (C6­AS cells) exhibited a decreased expression of glycogen synthase and reduced activity of glycogen synthesis, along with attenuated invasiveness. In vivo tumors induced by C6­AS cells in nude mice exhibited a significant reduction in tumor growth compared with controls. This reduction could be mediated by the induction of MCH­I expression. The inhibition of glycogen synthesis by antisense glycogen synthase validates a putative target and a new approach for further study to advance the much­needed efficacy of intervention strategies for malignant gliomas.

[Usefulness of the antisense and triplex anti-IGF-1 techniques for postoperative cellular gene therapy of malignant gliomas expressing IGF-1]. / Pooperacyjna terapia genokomórkowa z ekspresja IGF-1 glejaka wielopostaciowego przy zastosowaniu techniki antysensu i tripleksu anty-IGF-1.

BACKGROUND AND PURPOSE: The aim of the study was to estimate the usefulness of the antineoplastic vaccination in treatment of malignant brain tumors. According to medical knowledge there is no cure for this kind of tumors. MATERIAL AND METHODS: Between 2001 and 2005, ten patients suffering from malignant glial tumors were treated. There were 5 male and 5 female individuals, aged from 17 to 76 (mean age: 40.8 years). The histopathological examination showed 4 cases of glioblastoma and 6 cases of anaplastic astrocytoma. Initially, patients were operated on with dissection of 1 cm(3) of the most representative part of tumor. The neoplasm cells were cultured, transfected with episomal pMT EP vector (expressing alternatively oligonucleotide sequence forming triple helix with IGF-I gene or antisense against IGF-1 mRNA), re-cultured, irradiated and resuspended in medium to prepare antineoplastic vaccine. The patients were vaccinated subcutaneously. We examined peripheral blood lymphocyte subsets to assess the immunological response of the patients. RESULTS: We observed prolongation of the survival time to 21.7 months compared to 9-11 months observed in literature. The patients were additionally treated oncologically with radiotherapy and chemotherapy (temozolomide) according to the reasonable indications. Therefore, the comprehensive assessment of the genotherapy as the supplemental monotherapy was impossible. CONCLUSIONS: The method of treatment used in this study prolongs the survival time of patients with high-grade gliomas of the central nervous system. This gene therapy needs further investigations as a method of oncological monotherapy of brain malignant gliomas.

[Studies on insulin-like growth factor-I expression in human lower airways]. / Badania nad ekspresja insulinopodobnego czynnika wzrostu I w dolnych drogach oddechowych u ludzi.

UNLABELLED: Antisense techniques that inhibit intracellular expression of insulin-like growth factor-I (IGF-I) were efficient in gene therapy of some tumor diseases. IGF-I in the airways is considered to induce lung fibrosis in interstitial lung diseases, inhibit apoptosis of epithelial cells and participate in local carcinogenesis. AIM OF THE STUDY: The aim of the study was--by examining the IGF-I expression in the lower airways--to evaluate preliminary the efficacy of anti-IGF-I antisense technicques in the treatment of airways diseases. MATERIAL AND METHODS: The IGF-I expression was examined in the reverse transcriptase--polimerase chain reaction (RT-PCR) applied to A549 human cell line, that is representative for lower airway epithelium and exemplifies the model of bronchioloalveolar adenocarcinoma. IGF-I expression in non-neoplastic lower airways cells was assessed by immunocytochemical staining (anti-IGF-I) in cytological materials originating from bronchoalveolar lavage (BAL) of sarcoidosis and asbestosis patients and in individuals free of lung pathology. RESULTS: The IGF-I expression was detected in A549 cells with use of RT-PCR method (3 independent probes). In BAL cytological specimens the appearance of IGF-I was found, mainly in alveolar macrophages (62 +/- 6, 5 in sarcoidosis vs. 36 +/- 6 in controls, p=0,09), as well as in BAL lymphocytes. CONCLUSIONS: Summing up, the antisense technicques, blocking the intracellular IGF-I expression may be potentially useful in treatment of selected lower airways, both tumor (non-small cell lung carcinoma) and non-tumor (ILD complicated with lung fibrosis) conditions.

[Apoptosis of bronchoalveolar lavage lymphocytes (L-BAL) in pneumoconiosis]. / Apoptoza limfocytów pochodzacych z plukania oskrzelowo-pecherzykowego (L-BAL) w pylicach pluc.

BACKGROUND: The appearance and extension of apoptosis phenomenon in lymphocytes originated from the lower airways of patients with pneumoconioses, including silicosis and asbestosis, as well as its relation to the clinical data, remains unclear. METHODS: Bronchoalveolar lavage (BAL) was carried out in 11 patients with silicosis, 8 with asbestosis and in 7 control subjects. L-BAL were a) studied for CD95 and CD95 Ligand expression, b) permeabilized and stained with PI (flow cytometry, ModFit software) for apoptosis/cell cycle analyses and c) stained with Annexin V FITC/PI. RESULTS: The low number of L-BAL enter apoptosis. No significant changes between studied groups were found in PI determined apoptosis (silicosis: 1.8 +/- 0.7%, asbestosis: 3.1 +/- 0.9%, controls: 1 +/- 0.7%, median +/- SEM). Similar results were obtained, if tested with Annexin V FITC. However, the asbestosis group was characterized by higher CD4/CD8 ratio and increased percent of L-BAL CD95 Ligand expression (21.0 +/- 4.5 vs 13.7 +/- 3.3 in controls). In silicosis L-BAL apoptosis was inversely correlated with FEV1/VC values (r=-0.26, p<0.05). Surprisingly the majority of BAL lymphocytes expressed CD95, the marker of cell susceptibility to apoptotic stimuli; no difference between studied groups was found. CONCLUSIONS: Lower airways lymphocytes seem to be prevented from excessive apoptosis. Tendency to slightly increased percent of apoptotic and/or Fas Ligand cells reflects likely the local immunity alterations in asbestosis patients.

Analysis of coding and promoter sequences of the IGF-I gene in children with growth disorders presenting with normal level of growth hormone.

The insulin-like growth factor-I (IGF-I) gene was analyzed in a population of children with growth disorders presenting normal GH and low IGF-I. We thus tried to detect any mutation in the IGF-I gene that could be responsible for short stature in children, using PCR, single-strand conformation polymorphism (SSCP) analysis, followed by DNA cloning and sequencing. We demonstrated in all examined children significant changes in the promoter region of the IGF-I gene (P1 IGF-I). Nucleotide sequence changes, such as CC-->GT and A-->G, and their localization are described. The results obtained excluded mutations in the coding sequence of the IGF-I gene. We conclude that testing the IGF-I P1 region, using PCR/SSCP analysis, could be useful in the diagnosis of growth disorders.

IGF-I: from diagnostic to triple-helix gene therapy of solid tumors.

Alterations in the expression of growth factors and their receptors are associated with the growth and development of human tumors. One such growth factor is IGF-I (insulin-like growth factor I ), a 70-amino-acid polypeptide expressed in many tissues, including brain. IGF-I is also expressed at high levels in some nervous system-derived tumors, especially in glioblastoma. When using IGF-I as a diagnostic marker, 17 different tumors are considered as expressing the IGF-I gene. Malignant glioma, the most common human brain cancer, is usually fatal. Average survival is less than one year. Our strategy of gene therapy for the treatment of gliomas and other solid tumors is based on: 1) diagnostic using IGF-I gene expression as a differential marker, and 2) application of "triple-helix anti-IGF-I" therapy. In the latter approach, tumor cells are transfected with a vector, which encodes an oligoribonucleotide--an RNA strand containing oligopurine sequence which might be capable of forming a triple helix with an oligopurine and/or oligopyrimidine sequence of the promotor of IGF-I gene (RNA-IGF-I DNA triple helix). Human tumor cells transfected in vitro become down-regulated in the production of IGF-I and present immunogenic (MHC-I and B7 expression) and apoptotic characteristics. Similar results were obtained when IGF-I antisense strategy was applied. In both strategies the transfected cells reimplanted in vivo lose tumorigenicity and elicit tumor specific immunity which leads to elimination of established tumors.