Therapeutic vaccination against metastatic carcinoma by expression-modulated and immunomodified autologous tumor cells: a first clinical phase I/II trial.

Therapeutic vaccination of tumor patients with cytokine gene-transfected tumor cells leads to tumor regression in animal models but has so far not resulted in significant clinical benefit. We and others demonstrated that tumor cells transfected to mediate overexpression of a cytokine gene activate immunologic effector cells for an improved proliferation rate and significantly higher antitumoral cytotoxic activity. Here, we performed a pilot study of therapeutic vaccination in patients with metastatic disease. Autologous tumor cells were simultaneously transfected with novel minimalistic, immunogenically defined, gene expression constructs (MIDGE) for overexpression of the two cytokines interleukin 7 (IL-7) and GM-CSF and newly designed double stem-loop immunomodulating oligodeoxyribonucleotides (d-SLIM) as a Th1-promoting and NK cell-stimulating adjuvant. Transfection was performed ex vivo by ballistomagnetic gene transfer. Patients received four subcutaneous injections of at least 1 x 10(6) of their expression-modulated and immunomodified autologous tumor cells. Ten patients have been enrolled in the study protocol. In all patients no adverse effects could be detected. IL-7 and interferon gamma levels were elevated in the serum of the patients after treatment. Interestingly, cytotoxicity of patient-derived PBLs increased significantly during treatment. All 10 patients had progressive disease when entering our protocol. One complete, one partial, and one mixed response with progression of abdominal metastases and regression of lung metastases were observed. Two patients showed a stable disease after treatment and five patients remained in progressive disease. Our observations confirm the capability of autologous expression-modified and immunomodulated tumor cell vaccines to stimulate a strong immune response in patients with metastatic cancer even in the presence of a large tumor burden.

Increase in proliferation rate and normalization of TNF-alpha secretion by blockage of gene transfer-induced apoptosis in lymphocytes using low-dose cyclosporine A.

Efficient gene transfer of lymphocytes is extremely difficult. We have shown previously that induction of apoptosis may play a role in the gene transfer resistance of lymphocytes. Anti-CD3 antibody can be used as a surrogate for receptor-mediated gene transfer in T lymphocytes. However, anti-CD3 antibody has been shown to be the causative agent of apoptosis in receptor-mediated gene transfer. In this study, we show that blockage of apoptosis by addition of low-dose cyclosporine A can lead to normalization of elevated TNF-alpha secretion and to a significant increase in the proliferation rate of transfected lymphocytes. In contrast, this had no negative effect on cytotoxic activity of immunologic effector cells called cytokine-induced killer cells. Therefore, blockage of apoptosis should have an impact on the use of lymphocytes transfected with cytokine genes as immunologic effector cells in cancer gene therapy protocols.

Comparison of non-viral transfection methods in melanoma cell primary cultures.

Melanoma primary cultures were transiently transfected via electroporation and lipofection for comparison. Transfection efficiency was superior with electroporation (58+/-9%) as compared to lipofection (23+/-9%) as determined by enhanced green fluorescent plasmid (EGFP) transfection. Secretion of IL-2 persisted for up to 3 weeks after electroporation. The increase in sensitivity against immunologic effector cells by transfection with IL-2 was not significant. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction.

Establishment and characterization of colon carcinoma and renal cell carcinoma primary cultures.

Patients with metastatic renal and colon carcinoma have a very poor prognosis. In many cases, the tumor recurs after surgical excision and chemotherapy. Therefore, it might be beneficial for cancer patients to induce an immune attack against the tumor by inserting a cytokine gene into the tumor cells. Here, two different techniques for isolation of single tumor cells were compared. An enzymatic solution was superior to an EDTA/DTT isolation solution for establishing tumor primary cultures. In total, 18 primary cell cultures could be established from 68 patients with colon and renal cell carcinoma. Cells were further characterized concerning fibroblast contamination, cell proliferation and HLA-typing. These primary tumor cells might be of value for cytokine gene transfer and in vaccination protocols for cancer patients.

Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and lymphoma.

Natural killer-like T lymphocytes termed cytokine-induced killer (CIK) cells have been shown to eradicate established tumours in a severe combined immune deficient (SCID) mouse/human lymphoma model. Recently, we demonstrated that CIK cells transfected with cytokine genes possess an improved proliferation rate and a significantly higher cytotoxic activity as compared to non-transfected cells. Here, in a phase I clinical protocol, autologous CIK cells were generated from peripheral blood obtained by leukapheresis in patients with metastatic renal cell carcinoma, colorectal carcinoma and lymphoma. CIK cells were transfected with a plasmid containing the interleukin-2 (IL-2) gene via electroporation. Transfected cells generated IL-2 in the range of 330-1800 pg 10(-6) cells 24 h(-1) with a mean of 836 pg 10(-6) cells 24 h(-1). Ten patients received 1-5 intravenous infusions of IL-2-transfected CIK cells; five infusions with transfected CIK cells were given. In addition, the same patients received five infusions with untransfected CIK cells for control reasons. In three patients, WHO grade 2 fever was observed. Based on polymerase chain reaction of peripheral blood transfected cells could be detected for up to 2 weeks after infusion. There was a significant increase in serum levels of interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta) during treatment. Interestingly, there was also an increase in CD3+ lymphocytes in the blood of patients during therapy. In accordance, a partial increase in cytotoxic activity in peripheral blood lymphocytes (PBLs) was documented when patient samples before and after therapy were compared. Concerning clinical outcome, six patients remained in progressive disease, three patients showed no change by treatment, and one patient with lymphoma developed a complete response. In conclusion, we were able to demonstrate that CIK cells transfected with the IL-2 gene can be administered without major side-effects and are promising for future therapeutic trials.

TNF-alpha secretion and apoptosis of lymphocytes mediated by gene transfer.

Efficient gene transfer of lymphocytes is extremely difficult. Apoptosis may play a role in this gene transfer resistance of lymphocytes. Here we show that transfection of lymphocytes via non-viral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be mediated via tumor necrosis factor d (TNF-alpha) and the TNF-alpha receptor pathway, we studied the amount of TNF-alpha secreted by lymphocytes transfected without gene insert. TNF-alpha secretion was dependent on the gene transfer method used. High amounts were detected using receptor-mediated gene transfer and lipofection. In contrast, only low amounts of TNF-alpha were detected after electroporation and retroviral gene transfer. In receptor-mediated gene transfer, TNF-alpha secretion was due to the use of anti-CD3 antibody. Transfection of lymphocytes led to selective decrease in CD120b/TNF-alpha receptor II (TNFR-2)-positive cells. Induction of apoptosis and necrosis mediated by TNF-alpha via TNFR-2 (p80) was partially blocked using a neutralizing anti-TNF-alpha antibody. Blockage of apoptosis and necrosis could be further increased by adding anti-Fas-ligand (FasL) antibody, suggesting that induction of apoptosis via FasL and Fas receptor (Apo-1/CD95) may also play a role. This blockage led to a significant increase in the proliferation rate of lymphocytes transfected with cytokine genes. In conclusion, various gene transfer techniques led to TNF-alpha secretion, apoptosis and necrosis of lymphocytes. Apoptosis and necrosis could be partially blocked using a neutralizing anti-TNF-alpha antibody.

Increase of proliferation rate and enhancement of antitumor cytotoxicity of expanded human CD3+ CD56+ immunologic effector cells by receptor-mediated transfection with the interleukin-7 gene.

Cytokine-induced killer (CIK) cells have been shown to eradicate established tumors in a SCID mouse-human lymphoma model. CIK cells depend on exogenous addition of cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7) or interleukin-12 (IL-12) for proliferation. In this study, we used the adenovirus-enhanced CD3 receptor-mediated gene transfer for transfection with the IL-7 gene. An episomally replicating plasmid was used containing cDNA of the human IL-7 gene under the control of a CMV promoter for transfection of CIK cells. Biosynthesis of IL-7 was demonstrated by RT-PCR, an enzyme-linked immunosorbent assay (ELISA) and using a bioassay. Transfected cells produced IL-7 in the range between 200 and 1100 pg/10(6) cells in 24 h. IL-7 was shown to be biologically active, since transfected CIK cells showed an improved proliferation rate as compared with nontransfected cells. Expression of IL-7 altered the secretion of other cytokines by CIK cells, in particular the production of TNF alpha increased after transfection. In contrast, nontransfected CIK cells fed with IL-7 showed no increase in TNF alpha secretion. No significant differences were found in expression of surface antigens linked to the cytotoxic activity of CIK cells. Cytotoxic activity against various tumor cell lines (eg renal cell carcinoma, malignant melanoma and colon carcinoma) was tested. Transfected cells possessed a significantly higher cytotoxic activity as compared with nontransfected cells. Receptor-mediated gene transfer effectively delivers expression plasmids for therapeutic genes into CIK cells and CIK cells transfected with an IL-7 gene expression construct may be valuable for adoptive immunotherapy.

Generation of cytokine-induced killer cells using exogenous interleukin-2, -7 or -12.

Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells.

Increase of cytotoxic sensitivity of primary human melanoma cells transfected with the interleukin-7 gene to autologous and allogeneic immunologic effector cells.

Patients with metastatic melanoma have a very poor prognosis. In many cases, the tumor recurs after surgical excision. Therefore, it might be beneficial for cancer patients to induce an immune attack against the tumor by inserting a cytokine gene into the tumor cells. Here, 14 primary cell cultures could be established from 45 patients with malignant melanoma. Primary cell cultures were transfected via electroporation with the gene encoding for human interleukin-7 (IL-7). Transfection resulted in the production of biologically active IL-7 with an average of 850 pg/mL per 10(6) cells per 24 hours. Irradiation with 10,000 cGy, which inhibited tumor cell growth in vitro, increased the amount of released IL-7 to an average amount of 1050 pg/mL per 10(6) cells per 24 hours. No significant differences in the phenotype were observed in the IL-7-transfected cells compared with nontransfected cells. The expression of HLA class I and II, ICAM-1, and of a melanoma-associated antigen remained unaltered. Transfection with IL-7 had no significant effect on the proliferation of melanoma cells as measured in a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. There was no significant change in the cytokine profile after transfection or irradiation of the cells, but one cell culture expressed a high amount of IL-6 (about 2 ng/mL). IL-6 was expressed in nontransfected cells and was not altered by transfection. Interestingly, transfected cells from primary melanoma cultures possessed a higher sensitivity to immunologic effector cells compared with nontransfected cells. This was true for allogeneic as well as autologous melanoma cells. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction. IL-7-transfected cells might be of value in vaccination protocols for melanoma patients.

Lymphocyte apoptosis: induction by gene transfer techniques.

Efficient gene transfer of lymphocytes has been shown to be extremely difficult. The molecular background for this gene transfer resistance is not completely understood. We reasoned that apoptosis may play a role in this gene transfer resistance of lymphocytes. We show that transfection of lymphocytes via nonviral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be mediated via the TNF alpha and TNF alpha receptor pathway, we studied the amount of TNF secreted by transfected lymphocytes. The percentage of apoptotic lymphocytes correlated well with TNF alpha secretion. TNF secretion was dependent on the gene transfection method used. High amounts of TNF secretion were detected using receptor-mediated gene transfer and lipofection. In contrast, only low amounts of TNF were detected after electroporation and retroviral gene transfer. In receptor-mediated gene transfer, TNF secretion was due to the use of anti-CD3 antibody. Induction of apoptosis and increase in necrosis was blocked using an anti-TNF antibody. This blockage led to a significant increase in the proliferation rate of lymphocytes transfected with the interleukin-2 or interleukin-7 gene. In conclusion, gene transfer techniques led to TNF secretion, apoptosis and necrosis of lymphocytes. This could be blocked using an anti-TNF antibody. Blockage of apoptosis after gene transfer should have an impact on the use of lymphocytes transfected with cytokine genes as immunologic effector cells in cancer gene therapy protocols.