Technical hurdles in a pilot clinical trial of combined B7-2 and GM-CSF immunogene therapy for glioblastomas and melanomas

Summary Objective Malignant glioblastomas and melanomas continue to have a dismal prognosis despite advances in conventional therapy. This has led to investigations of novel treatment strategies including immunogene therapy. We report a pilot clinical trial of combined B7-2 and GM-CSF immunogene therapy for gliomas and melanomas and discuss technical hurdles encountered. Methods Patients with recurrent malignant gliomas or medically refractory melanomas were vaccinated with irradiated autologous tumor cells transduced with B7-2 and GM-CSF genes using a retroviral vector. Patients were monitored for toxicity, inflammatory/immune reactions, and clinical status. Results Vaccine preparation was attempted from 116 malignant glioma and 32 melanoma specimens. Adequate vaccines could only be prepared for five glioblastoma and three melanoma patients. Six patients (three recurrent glioblastomas and three melanomas) were actually vaccinated. Minor toxicities included flu-like symptoms (3/6), injection site erythema (4/6), and asymptomatic elevations in liver enzymes (3/6). Most patients showed evidence of an inflammatory response but specific anti-tumor immunity was not demonstrated. All six patients have died, although three patients with minimal residual disease at treatment had prolonged recurrence-free intervals after vaccination. Conclusions Combined B7-2 and GM-CSF immunogene therapy for glioblastomas and melanomas using autologous tumor cells has many technical pitfalls hindering large scale application and evaluation. As a result, this pilot study was too limited to draw meaningful conclusions regarding safety or anti-tumor immunity. While immunotherapy has been promising in pre-clinical studies, alternate strategies will be required to bring these benefits to patients.     

Improved Technique for Establishing Short Term Human Brain Tumor Cultures

Culturing human central nervous system tumors has been difficult compared to other neoplasms. We report improved success rates for establishing short term human brain tumor cultures using a modified tissue processing technique. Eighty-seven brain tumor specimens (56 glioblastomas, 8 mid grade astrocytomas, 8 oligodendrogliomas, 15 other) were obtained from June 1988 to March 1997. The first twenty-three samples were processed by dissection, partial enzyme dissociation, and filtration through a tissue culture sieve. Subsequent samples were processed identically except tumor cells were centrifuged on a density gradient prior to plating. Successful cultures were defined as those surviving greater than three passages in tissue culture and growing to sufficient numbers (>10⁶ cells) to allow freezing. Success rate was 42% (10/23) using standard processing methods and 86% (55/64) with the addition of density gradient centrifugation. Glial fibrillary acidic protein (GFAP) and vimentin staining, karyotypes, and growth curves were obtained for representative glioma cultures. All cultures tested were positive for vimentin (29/29) while 62% (18/29) were positive for GFAP. Of four cultures karyotyped (two glioblastomas, two oligodendrogliomas), all but one oligodendroglioma culture exhibited clonal cytogenetic abnormalities. These immunohistochemical and karyotypic results are consistent with the malignant glial origin of these cells. Of note, low passage human glioma cultures grew slower and exhibited more contact inhibition than immortalized human glioblastoma cell lines. Nevertheless, this simple method for establishing short term human brain tumor cultures should aid in further developing human brain tumor pre-clinical models as well as enhancing clinical applications dependent on in vitro human brain tumor cell growth adjust.     

Gene Gun Transfection of Human Glioma and Melanoma Cell Lines with Genes Encoding Human IL-12 and GM-CSF

We used particle-mediated gene transfer by a custom-built gene gun to transfect two well-established human glioma (D54MG and U251) and melanoma (SK mel 28 and Ed 141) cell lines, as well as two glioma lines locally established from primary patient tumors (Ed 147 and Ed 149). Using -galactosidase as a reporter gene, D54MG, U251, Ed 141 and SK mel 28 showed an average transfection efficiency of 15–40%, whereas Ed 147 and Ed 149 had mean transfection efficiencies of 3% and 5% respectively. Twenty-four hours after transfection with the gene encoding human interleukin-12 (IL-12), ELISA was performed on cell supernatants (mean of n=12 for each cell line). IL-12 expression was extremely variable between the different cell lines, ranging from 52 to 1151pg/10⁶ cells/24h. Results were very similar when cells were exposed to 20,000rads of gamma irradiation 2h after transfection. When the cell lines were transfected with human granulocyte-macrophage colony-stimulating factor, 24h levels were: 13.0 (Ed 147), 17.8 (Ed 149), 18.6 (Ed 141), 27.4 (D54MG) and 27.7ng/10⁶ cells/24h (U251). SK mel 28 produced 88.1ng/10⁶ cells/24h. We conclude that the gene gun can efficiently transfect a variety of immortalized, well-established and locally-established glioma and melanoma cell lines. High dose gamma irradiation does not adversely affect the expression of the foreign gene (IL-12) at 24h. Significantly, transfected cell lines show different levels of expression depending on the particular gene/plasmid introduced. Therefore, each cell line has to be assessed individually for the level of expression of each introduced gene.