Habib Fakhrai

Phase II study of belagenpumatucel-L, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non-small-cell lung cancer.

PURPOSE Belagenpumatucel-L is a nonviral gene-based allogeneic tumor cell vaccine that demonstrates enhancement of tumor antigen recognition as a result of transforming growth factor beta-2 inhibition. PATIENTS AND METHODS We performed a randomized, dose-variable, phase II trial involving stages II, IIIA, IIIB, and IV non-small-cell lung cancer patients. Each patient received one of three doses (1.25, 2.5, or 5.0 x 10(7) cells/injection) of belagenpumatucel-L on a monthly or every other month schedule to a maximum of 16 injections. Immune function, safety, and anticancer activity were monitored. RESULTS Seventy-five patients (two stage II, 12 stage IIIA, 15 stage IIIB, and 46 stage IV patients) received a total of 550 vaccinations. No significant adverse events were observed. A dose-related survival difference was demonstrated in patients who received > or = 2.5 x 10(7) cells/injection (P = .0069). Focusing on the 61 late-stage (IIIB and IV) assessable patients, a 15% partial response rate was achieved. The estimated probabilities of surviving 1 and 2 years were 68% and 52%, respectively for the higher dose groups combined and 39% and 20%, respectively, for the low-dose group. Immune function was explored in the 61 advanced-stage (IIIB and IV) patients. Increased cytokine production (at week 12 compared with patients with progressive disease) was observed among clinical responders (interferon gamma, P = .006; interleukin [IL] -6, P = .004; IL-4, P = .007), who also displayed an elevated antibody-mediated response to vaccine HLAs (P = .014). Furthermore, positive enzyme-linked immunospot reactions to belagenpumatucel-L showed a correlation trend (P = .086) with clinical responsiveness in patients achieving stable disease or better. CONCLUSION Belagenpumatucel-L is well tolerated, and the survival advantage justifies further phase III evaluation.

Antigenic Profiling of Glioma Cells to Generate Allogeneic Vaccines or Dendritic Cell–Based Therapeutics

Purpose: Allogeneic glioma cell lines that are partially matched to the patient at class I human leukocyte antigen (HLA) loci and that display tumor-associated antigens (TAA) or antigenic precursors [tumor antigen precursor proteins (TAPP)] could be used for generating whole tumor cell vaccines or, alternatively, for extraction of TAA peptides to make autologous dendritic cell vaccines. Experimental Design: Twenty human glioma cell lines were characterized by molecular phenotyping and by flow cytometry for HLA class I antigen expression. Twelve of the 20 cell lines, as well as analyses of freshly resected glioma tissues, were further characterized for protein and/or mRNA expression of 16 tumor antigen precursor proteins or TAA. Results: These 20 human glioma cell lines potentially cover 77%, 85%, and 78% of the U.S. Caucasian population at HLA-A, HLA-B, and HLA-C alleles, respectively. All cells exhibited multiple TAA expressions. Most glioma cells expressed antigen isolated from immunoselected melanoma-2 (Aim-2), B-cyclin, EphA2, GP100, β1,6-N-acetylglucosaminyltransferase V (GnT-V), IL13Rα2, Her2/neu, hTert, Mage, Mart-1, Sart-1, and survivin. Real-time PCR technology showed that glioblastoma specimens expressed most of the TAA as well. Tumor-infiltrating lymphocytes and CD8+ CTL killed T2 cells when loaded with specific HLA-A2+ restricted TAA, or gliomas that were both HLA-A2+ and also positive for specific TAA (Mart-1, GP100, Her2/neu, and tyrosinase) but not those cells negative for HLA-A2 and/or lacking the specific epitope. Conclusions: These data provide proof-in-principle for the use of allogeneic, partially HLA patient–matched glioma cells for vaccine generation or for peptide pulsing with allogeneic glioma cell extracts of autologous patient dendritic cells to induce endogenous CTL in brain tumor patients.

Lymphokine gene therapy of cancer

A novel method of tumor immunotherapy is described comprising the genetic modification of cells resulting in the secretion of cytokine gene products to stimulate a patients immune response to tumor antigens. In one embodiment, autologous fibroblasts genetically modified to secrete at least one cytokine gene product are utilized to immunize the patient in a formulation with tumor antigens at a site other than an active tumor site. In another embodiment, cells genetically modified to express at least one tumor antigen gene product and to secrete at least one cytokine gene product are utilized in a formulation to immunize the patient at a site other than an active tumor site.

Comparison of gene therapy with interleukin-2 gene modified fibroblasts and tumor cells in the murine CT-26 model of colorectal carcinoma.

We compared the efficacy of gene therapy mediated by interleukin-2 (IL-2) gene-modified tumor cells to gene therapy mediated by IL-2 transduced fibroblasts in the CT-26 model of murine colorectal carcinoma. We transduced CT-26 tumor cells and BALB/c 3T3 fibroblasts with three different retroviral vectors using three different promoters for the human IL-2 gene: DC/TKIL-2 (thymidine kinase promoter), LXSN-iIL2 (long terminal repeat promoter), and LNCX-iIL2 (cytomegalovirus promoter). These transductions resulted in CT-26 and 3T3 subclones that secreted different amounts of IL-2. Immunization of animals with either CT-26/IL-2 cells or with fibroblast/IL-2 cells mixed with CT-26 induced similar levels of immunity that protected 62-82% of animals against a subsequent tumor challenge with parental CT-26. However, mice developed tumors at the site of inoculation in 46% of the animals immunized with CT-26/IL-2 cells. In a separate experiment, CT-26/IL-2 cells were exposed to 6,000 cGy of gamma irradiation to prevent tumor growth at the site of inoculation. Although the CT-26/IL-2 cells continued to secrete IL-2 after irradiation, they were no longer effective at inducing antitumor immunity. In contrast, both irradiated and nonirradiated fibroblast/IL-2 cells, mixed with irradiated CT-26, were equally effective at inducing antitumor immunity. These data suggest that in the CT-26 model, fibroblast-mediated IL-2 gene therapy has advantages for the induction of antitumor immunity and abrogation of tumorigenic potential at the site of inoculation compared with tumor cell-mediated IL-2 gene therapy.

CD24+/CD38- as new prognostic marker for non-small cell lung cancer

BackgroundLung cancer is the leading cause of death among cancers in the world. The annual death toll due to this disease exceeds the combined deaths caused by colon, breast, prostate, and pancreatic cancers. As a result, there has been a tremendous effort to identify new biomarkers for early detection and diagnosis of lung cancer.MethodsIn this study we report the results of screening a panel of eight non-small cell lung cancer (NSCLC) cell lines originating from different subtypes of lung cancer in an attempt to identify potential biomarkers unique to this disease. We used real-time polymerase chain reaction and flow cytometry techniques to analyze the expression of ALDHA1, EpCAM, CD133, CD24, and CD38 in this panel.ResultsWe demonstrate for the first time that the majority of NSCLC cells do not express levels of CD38 that would qualify it as a new biomarker for the disease. In contrast, we found that CD24 is over-expressed in 6 out of 8 of the cell lines. The combined CD24+/CD38-/low phenotype was detected in 50% of the cell lines that are also positive for CD133 and EpCAM.ConclusionsWe report that CD24+/CD38-/low signature could potentially be used as a new biomarker for the early detection of NSCLC.

The stability of breast cancer progenitor cells during cryopreservation: Maintenance of proliferation, self-renewal, and senescence characteristics.

Cancer stem cells are believed to be the driving force behind tumor progression and development. Despite extensive studies on the effects of cryopreservation on embryonic and hematopoietic stem cells there is only limited data that directly deals with in the cryopreservation of cancer stem cells. In this study, we looked at the effect of cryopreservation on breast cancer progenitor cells known as mammospheres, which are derived from the MCF7 breast carcinoma cell line. We focused on the effect of cryopreservation on the cell biology and function of tumor-initiating cells using a standard method of cryopreservation with 15% dimethyl sulfoxide (Me(2)SO). Cell proliferation and survival was analyzed by alamarBlue solution on cryopreserved cells stored for 1-12 weeks and also by the expression of Ki-67. To assess self-renewal, single cells were harvested by limiting dilution procedure and wells were scored once a week. In order to investigate senescence, the activity of beta-galactosidase was detected by histochemical staining. Our results indicate that cryopreservation of breast cancer initiating cells will not reduce the ability of the cells to proliferate following cryopreservation storage for up to 12 months. Similarly, self-renewal, a unique property of stem cells, was shown to be maintained during cryopreservation. In contrast, cryopreservation of the mammospheres significantly increases the rate of senescence-mediated pathways. These data suggest that although cryopreservation of tumor-initiating cells is feasible but further studies are necessary to achieve a trustable repository of tumor-initiating cells and the design of new therapeutic measures to specifically target these cells.

Construction and characterization of retroviral vectors for interleukin-2 gene therapy.

Summary Several investigators have employed interleukin-2 (IL-2) gene transfer to enhance the immunogenicity of tumor cell vaccines. We describe in this report the construction and characterization of retroviral vectors for IL-2 gene therapy. Human IL-2 cDNA with a chimeric rat preproinsulin/IL-2 DNA leader sequence was sub-cloned into the pLXSN (long terminal repeat promoter) and pLNCX (cytomegalovirus [CMV] promoter) vectors to generate the plasmids pLXSN-iIL2 and pLNCX-iIL2, respectively. Human IL-2 cDNA with a chimeric human tissue factor/IL-2 DNA leader sequence was utilized to construct the vector pLXSN-tIL2. The levels of IL-2 secreted by transduced tumor cells and fibroblasts were evaluated by enzyme-linked immunosorbent assay (ELISA) of culture supernatants and compared with those of normal peripheral blood mononuclear cells (PBMC) activated in vitro with calcium ionophore and phorbol 12-myristate 13-acetate. The highest levels of IL-2 secreted by transduced tumor cells (760 units/106 cells/24 h), adult fibroblasts (625 units/106 cells/24 h), and embryonic fibroblasts (3,975 units/106 cells/24 h) were 150− to 1,000-fold higher than that secreted by the activated PBMC (4 units/106 cells/24 h). Similar levels of IL-2 were expressed by human fibroblasts transduced with pLXSN vectors employing the preproinsulin (pLXSN-iIL2) or tissue factor (pLXSN-tIL2) leader sequences (range in IL-2 units/106 cells/24 h pLXSN-iIL2 = 375–625 vs. pLXSN-tIL2 = 90–440). Because IL-2-transduced cells for clinical applications are generally irradiated to prevent cellular proliferation, we evaluated the effects of radiation on IL-2 production. Radiation doses between 1,500 and 10,000 cGy resulted in gradual decreases in IL-2 secretion by transduced cells. The range of the decrease in IL-2 secretion was 7–11% by day 7, 0–29% by day 14, and 25–50% by day 35. For clinical applications, stable production of the vector in high concentrations is an important consideration. The retroviral vector pLXSN-tIL2 produced the highest viral titer and was chosen for further characterization. Southern blot analysis of SacI-digested genomic DNA from the LXSN-tIL2 producer cell line and SacI-digested pLXSN-tIL2 plasmid DNA revealed the expected 3.2-kbp fragment, suggesting the absence of transgene rearrangement and the suitability of this vector as a candidate for clinical applications.

GENE THERAPY APPROACHES TO ENHANCE ANTITUMOR IMMUNITY

Publisher Summary This chapter summarizes the advances in antitumor immunity enhancement and concentrates on several types of genetic manipulations that have been explored to enhance the efficacy of cancer immunotherapies. A number of cytokines, produced in purified form by recombinant DNA methodology, have been evaluated for their antitumor effects. In several clinical trials, cytokines and related immunomodulators have produced objective tumor responses in some patients afflicted with a variety of neoplasms. The development of antitumor immune responses as a consequence of cytokine gene transfer in the treatment of cancer has been demonstrated in several animal tumor models. A number of approaches have been developed to circumvent the need to establish cell lines or cultures ex vivo for immunogene therapy. Direct tumor injection of cytokine or allogeneic MHC gene vectors has been successful in animal models and this strategy is being evaluated in phase I clinical trials. Additional targets for immunogene therapy approaches include mutated oncogenes, tumor suppressor genes, and viral antigens. These potential tumor-associated antigens (TAAs) have been suggested as appealing targets for immunotherapy as they are not expressed by normal tissues. The chapter also provides a summary of immunogene therapy clinical protocols submitted to regulatory agencies worldwide. The results of these trials should provide insights regarding that of the immunogene therapy approaches are most appropriate for further clinical evaluation.

Characterization of a new human glioblastoma cell line that expresses mutant P53 and lacks activation of the PDGF pathway

SummaryWe have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms. The circumstance of a weak or inactive PDGF-B autocrine mechanism in human glioblastoma paired with a homozygously altered p53 suggests that the loss of suppressor function of p53 may be a major contribution to the transformed phenotype of these cells.

Molecular integrity and global gene expression of breast and lung cancer stem cells under long-term storage and recovery

Cryopreservation is a common procedure widely used in biological and clinical sciences. Similar protocols are also applied in preserving cancer stem cells, a field with high promises and challenges. Specific cell surface membrane proteins are considered to be biomarkers of cancer stem cells and they may play a critical role in differentiating stem cells from non stem cells. We have looked at the possible effect of long-term cryopreservation on the molecular integrity of breast MCF7 and lung, A549 and H460, cancer stem cells and to assess if these cells are more sensitive to long-term storage process. We analyzed the expression of CD24 and CD38 as two potent biomarkers of lung cancer stem cells and EpCAM and ALDH that are used as biomarkers of a wide range of cancer stem cells. We also selected three genes essential for the normal functioning of the cells, Fos, MUC1, and HLA. Our results indicate a pattern of down-regulation in the expression of the genes following freezing, in particular among cell surface marker proteins. Global gene expression of the post-thaw breast and lung cancer stem cells also reveals a significant down-regulation in freeze-thaw cells independent from each other. Analyzing the canonical pathways between two populations reveals a significant alteration in the gene expression of the pathways involved in cell cycle, mitosis, and ataxia telangiectasia mutated pathways. Overall, our results indicate that current protocols for long-term storage of lung and breast cancer stem cells may substantially influence the activity and function of genes.