Tomislav Dorbic

Vaccination with IL-12 gene-modified autologous melanoma cells: preclinical results and a first clinical phase I study

Cytokine gene transfer into tumor cells has been shown to mediate tumor regression and antimetastatic effects in several animal models via immunomodulation. Therefore, clinical protocols have been developed to treat cancer patients with cytokine gene-modified tumor cells. We inserted the genes coding for the p35 and p40 chain of interleukin-12 (IL-12) in two independent eukaryotic expression vectors and transduced melanoma cells of 15 different primary tumor cultures with both plasmids by a ballistic gene transfer approach. Secreted IL-12 demonstrated strong bioactivity by inducing interferon-γ release from peripheral blood lymphocytes upon coculture with cell culture supernatants after IL-12 gene transfer which could at least partly be blocked by IL-12-specific antisera. Further enrichment of transduced tumor cells by magnetic separation directly after gene transfer increased cytokine secretion from a mean of 119 pg in the unsorted to 507 pg IL-12 (24 h/106 cells) in the magnetically enriched cell fraction. Irradiation of these cells led to a further elevation of secreted IL-12 (mean 987 pg). Elevated IL-12 levels were detected over 7 days after irradiation in vitro. In a subsequent first clinical phase I study six patients with metastatic melanoma were vaccinated with autologous, interleukin-12 gene-modified tumor cells. Melanoma cells were expanded in vitro from surgically removed metastases, transduced by ballistic gene transfer, irradiated and were then injected subcutaneously (s.c.) at weekly intervals. Clinically, there was no major toxicity except for mild fever. All patients completed more than four s.c. vaccinations over 6 weeks and were eligible for immunological evaluation. Post-vaccination, peripheral mononuclear cells were found to contain an increased number of tumor-reactive proliferative as well as cytolytic cells as determined by a limiting dilution analysis in two patients. Two patients developed DTH reactivity against autologous melanoma cells and one had a minor clinical response. Biopsies taken from that patient’s metastases revealed a heavy infiltration of CD4+ and CD8+ T lymphocytes. In conclusion, vaccination induced immunological changes even in a group of advanced, terminally ill patients. These changes can be interpreted as an increased antitumor immune response.

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.

Increased non-major histocompatibility complex-restricted lytic activity in melanoma patients vaccinated with cytokine gene-transfected autologous tumor cells

Genetically modified antitumoral vaccines focus on eliciting or increasing the T-cell-mediated antitumoral response. Little is known about non-major histocompatibility complex-restricted responses. In two phase I studies, we have immunized advanced melanoma patients with either interleukin-7 (IL-7) gene-transfected or IL-12 gene-transfected, autologous, irradiated melanoma cells. To monitor the immune response, peripheral blood mononuclear cells were collected before the first vaccination and 2 weeks after the third vaccination. Spontaneous lytic activity and lymphokine-activated killer (LAK) activity after a 5-day culture in the presence of 1000 U/mL IL-2 against autologous and against allogeneic melanoma cells were measured. In parallel, a precursor cytotoxic T-cell frequency analysis was performed using a 25-day limiting dilution analysis assay. A total of 10 of 14 immunologically evaluable patients demonstrated a marked increase in LAK activity, and 7 of 14 showed increased spontaneous lytic activities against autologous melanoma cells after three vaccinations. Remarkably, two patients with a good clinical performance status (Karnofsky index of >70; Multitest Merieux of >13.4 mm/3) and the highest cytotoxic T-lymphocyte (CTL) response after vaccination showed the only clear decrease in LAK and spontaneous lytic activity. Otherwise, three patients with no detectable CTL response after vaccination demonstrated an increase in LAK activity and the strongest increase in the autologous spontaneous lytic activity. This group of patients was associated with a poor clinical performance status (Karnofsky index of <70; Multitest Merieux of <4 mm/1) and with no clinical response. In conclusion, in accordance with other studies, a good clinical and immunological performance status appears to be the prerequisite for a successful CTL response. However, even strong non-major histocompatibility complex-restricted responses could be generated in patients with reduced clinical performance in vaccination therapies with gene-transfected autologous tumor cells.

Retinoic acid receptor γ1 expression determines retinoid sensitivity in pancreatic carcinoma cells

Abstract Background & Aims: Retinoids inhibit growth and induce differentiation in a variety of pancreatic carcinoma cells. The goal of this study was to examine the molecular mechanisms responsible for retinoid sensitivity. Methods: Anchorage-independent growth was examined in AR42J, DSL-6A/C1, and Capan-2 cells using a human tumor clonogenic assay. Retinoid receptors were characterized by a reverse-transcription polymerase chain reaction. Retinoic acid receptor γ 1 (RARγ 1 ) was stably transfected into AR42J cells using lipofectamin and into DSL-6A/C1 using ballistomagnetic gene transfer. Receptor expression was verified using Southern and Northern blotting as well as electrophoretic mobility shift assays. Results: Retinoid treatment resulted in a dose-dependent growth inhibition of Capan-2 cells, whereas growth was not affected in AR42J and DSL-6A/C1 cells. A selective loss of RARγ 1 expression was observed in both retinoid-resistant cell lines, whereas all other retinoid receptor subtypes showed an identical expression pattern. Retinoid treatment of three independent RARγ 1 -expressing cell clones of AR42J and DSL-6A/C1 cells resulted in pronounced growth inhibition compared with wild-type control cells. Conclusions: RARγ 1 expression determines sensitivity of pancreatic carcinoma cells to retinoid-mediated growth inhibition and might therefore serve as a valuable predictive marker for retinoid treatment of pancreatic cancer. GASTROENTEROLOGY 1998;115:967-977

IDENTIFICATION OF PROCESSES THAT INFLUENCE NEGATIVE SUPERCOILING IN THE HUMAN C-MYC GENE

DNA elements with sequences suitable for Z-DNA formation are found frequently at various positions in chromatin. Z-DNA formation in these sequences depends largely on the level of local negative supercoiling. We can use binding of a Z-DNA specific antibody at low concentrations in metabolically active permeabilized nuclei to detect naturally occurring Z-DNA formation. Previously we identified three sequence elements in the human c-myc gene that adopt the Z-DNA conformation in the transcribed gene. The three elements are found far upstream (Z1), close to the main transcription start site (Z2) and in the first intron (Z3). Here we measure the persistence of Z-DNA at these three sites under the influence of various metabolic inhibitors. This provides some insight into the varying levels of negative supercoiling. alpha-Amanitin, an inhibitor of transcription, reduced the persistence of Z-DNA in all three elements. Aphidicolin, an inhibitor of replication, increased the persistence of Z-DNA in one element without significantly influencing the other two elements. When camptothecin an inhibitor of topoisomerase I was added in the presence of alpha-amanitin, the persistence of Z-DNA was extended in all three elements. However, in the presence of aphidicolin no effect of camptothecin on Z-DNA formation was observed.

The promoter of the HaCaT keratinocyte differentiation-related gene keratin 4 contains a functional AP-2 binding site

Abstract The nuclear transcription factor AP-2 appears to be a key regulator mediating programmed gene expression during embryonic morphogenesis and adult cell differentiation. AP-2 has also been considered to be involved in epidermal gene regulation, but its precise role is not yet defined. The level of AP-2 transcripts increases during culturing of HaCaT keratinocytes preceding the expression of the differentiation-related gene keratin 4 (K4). The current study was aimed at investigating whether AP-2 transactivates K4 transcription. We cloned and sequenced the promoter region of K4 and found, in addition to canonical sequences, an AP-2 consensus site in the vicinity of the transcriptional start. In order to provide functional evidence for a regulation of K4 transcription by AP-2, we cloned various parts, which did or did not contain the AP-2 site of the K4 upstream sequence, into Cat reporter plasmids. These constructs were ballistically transfected into differentiating HaCaT keratinocytes. The determination of the resulting Cat activity revealed that the AP-2 site in the vicinity of the transcriptional start was functional for K4 transcription. Thus, the role of AP-2 in the process of keratinocyte differentiation appears to be considerable. In addition, further regulatory elements were found to be necessary for full transcription of K4.