Cytokine gene therapy of gliomas: effective induction of therapeutic immunity to intracranial tumors by peripheral immunization with interleukin-4 transduced glioma cells.

To provide a means for comparing strategies for cytokine gene therapy against intracranial (i.c.) tumors, we generated rat gliosarcoma 9L cells transfected with interleukin-4 (9L-IL4), interleukin-12 (9L-IL12), granulocyte-macrophage colony-stimulating factor (9L-GMCSF) or interferon-alpha (9L-IFNalpha). To simulate direct and highly efficient cytokine gene delivery, cytokine transfected 9L tumors were implanted i.c. into syngeneic rats. i.c. injection led to tumor-outgrowth in the brain and killed most animals, whereas these cell lines were rejected following intradermal (i.d.) injection. Cytokine-expressing i.c. 9L tumors, however, had a greater degree of infiltration by immune cells compared with control, mock-transfected 9L-neo, but to a lesser degree than i.d. cytokine-expressing tumors. Tumor angiogenesis was suppressed in cytokine-transfected tumors. In a prophylaxis model, i.d. vaccination with 9L-IL4 resulted in long-term survival of 90% of rats challenged i.c. with parental 9L; whereas 40% of 9L-GM-CSF, 40% of 9L-IFNalpha and 0% of 9L-IL12-immunized rats were protected. In a therapy model (day 3 i.c. 9L tumors), only i.d. immunization with 9L-IL4 had long-term therapeutic benefits as 43% of rats survived >100 days. These data indicate that peripheral immunization with 9L-IL4 had the most potent therapeutic benefit among various cytokines and approaches tested against established, i.c. 9L tumors.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

Combination of stereotactic radiosurgery and cytokine gene-transduced tumor cell vaccination: a new strategy against metastatic brain tumors.

OBJECT: To determine if the combination of radiosurgery and tumor cell vaccine would enhance the therapy of metastatic lesions of the central nervous system (CNS), the authors examined the antitumoral effects of radiosurgery and cytokine-transduced tumor cell vaccine. METHODS: Fifty-five rats underwent intracranial implantation of 5 x 10(3) MADB 106 cells. On Day 3 after tumor implantation, 34 rats were inoculated in the flank with nonirradiated MADB 106 cells that had been retrovirally transduced to express granulocyte-macrophage colony-stimulating factor or interleukin-4. Twenty-seven rats (17 animals that had received the vaccine and 10 that had not) underwent radiosurgery performed using a gamma knife at maximum doses of 32 Gy on Day 5. No animals in the untreated group or in the vaccine-alone groups survived longer than 21 days. Animals treated by ra diosurgery alone displayed prolonged survival in comparison with untreated animals (p < 0.0001), but only one of 10 animals survived longer than 55 days. In contrast, 14 of 17 animals that received the combination therapy of radiosurgery and vaccination survived longer than 55 days (p = 0.0003 compared with animals that underwent radiosurgery alone). On Day 55, the long-term survivors were challenged by parental MADB 106 cells, which were implanted in the contralateral hemisphere. All animals from the combination therapy groups survived longer than 50 days after this challenge, but the single survivor from the radiosurgery-alone group died of tumor growth in 27 days. CONCLUSIONS: The combination of radiosurgery and cytokine gene-transduced tumor cell vaccine markedly prolonged animal survival and protected animals from a subsequent challenge by parental tumor cells placed in the CNS. The data provided by this study indicate that this combination therapy represents a strategy that may have clinical applicability for single and/or multiple metastatic brain tumors.

Cytokine gene therapy of gliomas: induction of reactive CD4+ T cells by interleukin-4-transfected 9L gliosarcoma is essential for protective immunity.

Tumor cells genetically modified to secrete cytokines stimulate potent immune responses against peripheral and central nervous system tumors; however, variable results on the efficacy of this strategy for therapeutic intervention against established intracranial neoplasia have been reported. We have found that vaccination with rat 9L gliosarcoma cells expressing interleukin 4 (9LmIL4) induced a specific, protective, immune response against rechallenge with parental 9L tumors. In naive rats, sham-transfected 9L (9Lneo) tumors and 9LmIL4 tumors grew at comparable rates for 12-14 days, and then 9LmIL4 tumors regressed. After regression of 9LmIL4 tumors, rats were resistant to rechallenge with parental 9L cells. To investigate the mechanism(s) responsible for 9LmIL4-induced immunity, the phenotype and function of tumor-infiltrating lymphocytes (TILs) in 9Lneo and 9LmIL4 tumors were compared. In flow cytometric analyses, it was determined that CD4+ T cells were the predominant cell type in both 9Lneo and 9LmIL4 tumors at day 10. However, at the onset of regression (day 14), 9LmIL4 tumors were infiltrated predominantly by CD8+ T cells. To investigate functional aspects of the anti-9L tumor responses, we assessed the capacity of 9LmIL4 TILs to mediate specific lytic function or production of cytokines. In response to parental 9L, TILs isolated from day 14 9LmIL4 tumors were demonstrated to produce substantially greater amounts of IFN-gamma than did TILs from 9Lneo tumors. Although freshly isolated TILs from 9LmIL4 or control tumors did not lyse 9L cells in 51Cr-release cytotoxicity assays, specific cytotoxicity was demonstrable using TILs from day 14 9LmIL4 or splenocytes from 9LmIL4-bearing rats after their restimulation for 5 days with parental 9L tumor cells in vitro. Antibody blocking studies demonstrated that cytokine production and lytic activity by TILs, or splenocytes from 9LmIL4-immunized rats, were mediated in a T-cell receptor-dependent fashion. Because interleukin-4 also promotes humoral responses, quantity and isotype of immunoglobulins in sera from 9Lneo or 9LmIL4-immunized rats were compared. The amount of IgG1 antibodies was significantly increased in sera from 9LmIL4-immunized rats compared to sera from 9Lneo-bearing rats. Experiments using sublethally irradiated, naive rats adoptively transferred with splenocytes and/or sera from 9LmIL4-immunized or naive rats demonstrated that immune cells, with or without immune sera, protected recipients from challenge with parental 9L. Immune sera provided no protection when given with lymphocytes from naive rats, and it did not enhance protection against parental 9L when given in conjunction with lymphocytes for 9LmIL4-immunized rats. In additional adoptive transfer experiments, an essential role for CD4+ T cells in immunity was observed because their depletion from among splenocytes of 9LmIL4-immunized rats eliminated the protective effective against 9L, whereas depletion of CD8+ cells resulted in a more limited effect on protection against 9L. These data suggest that strategies for inducing systemic, long-term tumor-specific reactivity among CD4+ T cells will be critical for the development of immunotherapy of gliomas.

Characterization and transduction of a retroviral vector encoding human interleukin-4 and herpes simplex virus-thymidine kinase for glioma tumor vaccine therapy.

Vaccination with cytokine-transduced tumor cells represents a potentially important approach to the treatment of central nervous system tumors. We have recently demonstrated the therapeutic efficacy of tumor cell vaccines expressing the murine interleukin 4 (IL-4) and the herpes simplex virus-thymidine kinase in a rat brain tumor model in which nonirradiated vaccine cells can be eliminated by the subsequent administration of ganciclovir. In this report, we demonstrate the construction and characterization of a retroviral vector that encodes human IL-4, neomycin phosphotransferase, and herpes simplex virus-thymidine kinase genes for use in human clinical trials. An MFG-based retroviral vector was used to generate the recombinant retrovirus, TFG-hIL4-Neo-Tk, in which a long terminal repeat-driven polycistronic transcript encodes three cDNAs that are linked and coexpressed using two intervening internal ribosome entry site fragments from the encephalomyocarditis virus. The amphotropic retroviral vector TFG-hIL4-Neo-Tk was then used to infect human primary glioma cultures and skin-derived fibroblasts. After infection and G418 selection, cells produced 89-131 ng/10(6) cells/48 hours of human IL-4, which was determined to be biologically active. Transduced glioma cells were highly sensitive to the cytotoxic effect of ganciclovir. These data demonstrate the suitability of the TFG-hIL4-Neo-Tk vector for therapeutic studies of cytokine-transduced autologous tumor vaccination in patients with malignant gliomas.

Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSVtk tumor vaccine.

To explore the potential for molecular immunotherapies in the treatment of malignant gliomas, we evaluated the efficacy of subcutaneous tumor cell vaccines in the treatment of intracranial 9L tumors, using 9L gliosarcoma cell lines stably transduced with the murine interleukin-4 cDNA (9L-IL4), the herpes simplex virus-thymidine kinase cDNA (9L-Tk) or both (9L-IL4-Tk). The expression of multiple genes from a single transcript was achieved by incorporating internal ribosomal entry site (IRES) cassettes in the retroviral constructs. Subcutaneous immunization of rats with nonirradiated 9L-IL4 cells or 9L-IL4-Tk cells followed by treatment with ganciclovir (GCV) completely protected the animals from a subsequent intracranial challenge with wild-type 9L cells. In contrast, only 50% of animals immunized with 9L-Tk cells and 0% of 9L-neo immunized animals rejected the same challenge with wild-type 9L. More importantly, treatment of established (day 3) intracranial 9L tumors with genetically engineered tumor cells resulted in long-term survival (> 100 days) for 25-43% of 9L-IL4-Tk immunized animals and for 27% of nonirradiated 9L-IL4 immunized animals. In striking contrast, no 9L-Tk, 9L-neo or irradiated 9L-IL4 immunized animals survived for more than 33 days. As a marker of a cellular immune response, splenocytes from nonirradiated 9L-IL4, 9L-Tk or 9L-IL4-Tk immunized animals produced interferon-gamma (IFN-gamma) in greater amounts than those from 9L-neo immunized or Hank's balanced salts solution (HBSS) treated animals when stimulated with wild-type 9L in vitro. Our findings support the use of tumor cell vaccines expressing the IL-4 and HSVtk genes for the treatment of malignant gliomas.

Bone marrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effective anti-tumor immunity against intracranial neoplasms.

Although the central nervous system (CNS) is often regarded as an immunologically privileged site, it is well established that specific CNS immunoreactivity can be generated through peripheral vaccination with CNS antigens. Dendritic cells (DC) are potent antigen presenting cells of hematopoietic origin that have emerged as a promising tool for cancer immunotherapy capable of evoking significant anti-tumor immunity when pulsed with tumor-associated peptides. To explore a role for DC-based immunization strategies for the treatment of CNS tumors, we developed a brain tumor model using the C3 sarcoma cell line which expresses the tumor-specific, major histocompatibility complex (MHC) class I-restricted peptide epitope E7(49-57). Syngeneic C57Bl/6 mice receiving intravenous (i.v.) injections of bone marrow-derived DCs pulsed with E7 peptide were effectively protected against a subsequent intracerebral challenge with C3 tumor cells. More importantly, this systemic immunization strategy was effective in a therapy model as 67% of animals (10 of 15) with established (day 7) intracerebral C3 tumors treated with 3 weekly injections of E7 peptide-pulsed DCs achieved a long-term survival (>90 days) while no control animals survived beyond day 41. In vivo depletion of CD8+ cells, but not CD4+ or asialo-GM1+ cells, abrogated the efficacy of E7 peptide-pulsed DC therapy of established tumors, indicating a pivotal role of specific CD8+ T-cell responses in mediating the anti-tumor effect. Our findings support the hypothesis that effective CNS anti-tumor immunoreactivity can be generated with DC-based tumor vaccines.