Katarzyna Gryska

Genetically modified tumour vaccines (GMTV) in melanoma clinical trials

Since melanoma is a model immunogenic malignancy incurable in the disseminated phase of its natural course different immunotherapeutic approaches are tested in clinical trials. A number of tumour vaccines genetically modified (GMTV), with various immunostimulatory factors, are tested in phase I/II clinical trials. These factors include cytokines, tumour antigens (TA), costimulatory molecules or HLA antigens. We have designed a novel, mixed auto/allogeneic cellular melanoma vaccine modified with the IL-6 and the sIL-6R genes. Preclinical studies in a mouse model demonstrated that the IL-6/sIL-6R based vaccine is able to elicit efficient anti-tumour responses, mediated by CD8+ and NK cells, which resulted in inhibition of the tumour growth, metastases formation and prolonged survival of the animals treated. Irradiation of vaccine cells does not only lead to their sterilisation but also causes increased secretion of exogenous IL-6 and sIL-6R. Since January 1996 we have vaccinated more than one hundred metastatic melanoma patients. Promising clinical results (22% CR+PR, 32% SD) and the evidence of immune responses in the vaccinated patients have prompted us to design a phase III clinical trial which is to be open in 2000.

Antitumor effects of the combination therapy with TNF-α gene–modified tumor cells and interleukin 12 in a melanoma model in mice

In the present study, TNF-α gene–transduced B78 melanoma cells (B78/TNF) were used as a vaccine and combined with interleukin (IL)-12 in the treatment of B78-melanoma-bearing mice. The combined administration of genetically modified melanoma cells and IL-12 induced specific protective antitumor immunity resulting in a decreased rate of the tumor take following a rechallenge with parental B78 cells. When used therapeutically, intratumoral injections of irradiated B78/TNF melanoma cells and IL-12 exerted strong antitumor effects and led to complete regression of established tumors in 50% of mice. Injections of irradiated B78/TNF cells alone did not influence tumor development and IL-12 itself significantly delayed tumor growth but without curative effect. FACS analysis of parental B78 melanoma cells and its B78/TNF genetically modified variant showed that a proportion of cells of both cell lines expressed B7-1 (CD80) costimulatory molecule and that the expression of this molecule was increased during incubation with IFN-γ. Moreover, IFN-γ markedly augmented expression of major histocompatibility class (MHC) class I and II molecules on B78/TNF cells that were primarily MHC class I and II negative with no substantial effect on MHC-negative parental B78 melanoma. IFN-γ also synergized in cytostatic/cytotoxic effects with TNF-α against B78 melanoma in vitro. Lymphocyte depletion studies in vivo showed reduction of the antitumor response in mice treated with anti-NK monoclonal antibodies (mAbs) as well as in mice treated with anti-CD4+anti-CD8 mAbs. The results suggest that, when used therapeutically, IL-12 and a vaccine containing TNF-α gene–transduced tumor cells may reciprocally augment their overall antitumor effectiveness by facilitating development of systemic antitumor immunity and by stimulating local effector mechanisms of the tumor destruction. Cancer Gene Therapy (2000) 7, 1581–1590.

A designer hyper interleukin 11 (H11) is a biologically active cytokine

BackgroundInterleukin 11 (IL-11) is a pleiotropic cytokine with anti-apoptotic, anti-inflammatory and hematopoietic potential. The IL-11 activity is determined by the expression of the IL-11R receptor alpha (IL-11Rα) and the signal transducing subunit β (gp130) on the cell membrane. A recombinant soluble form of the IL-11Rα (sIL-11Rα) in combination with IL-11 acts as an agonist on cells expressing the gp130 molecule. We constructed a designer cytokine Hyper IL-11 (H11), which is exclusively composed of naturally existing components. It contains the full length sIL-11Rα connected with the mature IL-11 protein using their natural sequences only. Such a construct has two major advantages: (i) its components are as close as possible to the natural forms of both proteins and (ii) it lacks an artificial linker what should avoid induction of antibody production.ResultsThe H11 construct was generated, the protein was produced in a baculovirus expression system and was then purified by using ion exchange chromatography. The H11 protein displayed activity in three independent bioassays, (i) it induced acute phase proteins production in HepG2 cells expressing IL-11, IL-11Rα and gp130, (ii) it stimulated the proliferation of B9 cells (cells expressing IL-11Rα and gp130) and (iii) proliferation of Baf/3-gp130 cells (cells not expressing IL-11 and IL-11Rα but gp130). Moreover, the preliminary data indicated that H11 was functionally distinct from Hyper-IL-6, a molecule which utilizes the same homodimer of signal transducing receptor (gp130).ConclusionsThe biologically active H11 may be potentially useful for treatment of thrombocytopenia, infertility, multiple sclerosis, cardiovascular diseases or inflammatory disorders.

Designer cytokine hyper interleukin 11 (H11) is a megakaryopoietic factor.

Interleukin-11 (IL-11) displays megakaryopoietic activity. We constructed super-cytokine Hyper- IL11 (H11) by linking soluble IL-11 receptor α (sIL-11Rα) with IL-11, which directly targets β-receptor (gp130) signal transducing subunit. The effects of H11 on hematopoiesis with a focus on megakaryopoiesis were studied. The expansion, differentiation and type of colony formation of cord blood progenitor Lin-CD34+ cells were analyzed. H11 was more effective than recombinant human IL-11 (rhIL-11) in enhancement of the Lin-CD34+ cells expansion and differentiation into megakaryocytes (Mk). It induced higher expression of CD41a and CD61 antigens, resulting in a substantially larger population of CD34-CD41ahighCD61high cells. H11 treatment led to increased number of small and mainly medium megakaryocyte colony formation (Mk-CFU). Moreover, it induced the formation of a small number of large colonies, which were not observed following rhIL-11 treatment. Significantly higher number of H11 derived Mk colonies released platelets-like particles (PLP). Furthermore, H11 was considerably more potent than rhIL-11 in promoting differentiation of Lin-CD43+ cells toward erythrocytes. Our results indicate that H11 is more effective than rhIL-11 in enhancing expansion of early progenitors and directing them to megakaryocyte and erythroid cells and in inducing maturation of Mk. Thus, H11 may prove beneficial for thrombocytopenia treatment and/or an ex vivo expansion of megakaryocytes.

Humoral responses to melanoma vaccine, genetically modified with interleukin 6 and soluble interleukin 6 receptor.

Production of antibodies directed against the tumour antigens is well recognised in cancer patients. However, role of antibodies in antitumour immune responses and their clinical significance is not clear, especially when specific immune therapies aim boosting of cellular responses to tumour cells1-3. Moreover, antagonisms between cellular and humoral responses were observed mainly in animal models4-5. Recently, so called immune enhancement, in human colon cancer patients was also demonstrated6in which depletion of B lymphocytes correlated with tumour regression. On the other hand, several reports suggested that humoral responses are associated with positive clinical outcomes.

Flow cytometric cytotoxicity assay with GFP gene modified target cells

The cytotoxicity assays have been one of the essential tools in studies of effector functions of the immune system. In order to avoid use of radioactive materials many groups have developed alternative ways to quantitate cell mediated lysis. These methods include release of target cell cytoplasmic enzymes (1), dye inclusion or vital dye uptake reduction (2-4). In most of the assays target cells have to be labeled prior analysis which albeit easy to perform is unnecessarily time consuming Here we describe the measurement by flow cytometry killing activities of human NK cells against K562 cells stably transduced to express enhanced green fluorescent protein (EGFP). After short incubation with effector cells killed EGFP positive K562 cells can be later easily identified by propidium iodide (PI) staining. Since EGFP is expressed in every target cell it requires no further cell labelling, cell purification, or extra washing steps thus, it is more time efficient than current assay methods.

Improving the retroviral vector (RV) systems for immunotherapy of cancer

Most protocols of gene therapy clinical trials are based on immunotherapy strategies with genetically modified tumor vaccines (GMTV). The major issue when designing GMTV is gene delivery system; RV proven to be very useful for ex vivo transduction of therapeutic genes. The advantages of RV include: easy manipulation of small viral genome, stable integration of transduced gene and safety. Most frequently, Gibbon Ape Leukemia Virus (GaLV) and Amphotropic Murine Leukemia Virus (AMuLV) envelope proteins are used to pseudotype RV-based vectors. Such vector pseudotypes infect target cells by their specific receptors: PiT1 and PiT2, respectively. Both receptors belong to the same family’, and despite high degree homology, no cross interference is observed.

Inflammatory Cytokines Controlling Branching of N-Heteroglycans of Acute Phase Protein

Tissue injury, infection, various inflamatory processes or tumor growth cause the activation of a large number of cells responsible for host protection (1). These cells secrete factors which in turn have the ability to stimulate effector cells. One group of these factors is referred to as cytokines. The cytokines involved in stimulation of anti or pro-inflammatory mechanisms are more specifically termed inflammatory cytokines. There is increasing evidence demonstrating that biological effects of these factors do not result from an action of a single cytokine but rather result from coordinated cooperation of a number of cytokines and other factors such as glucocorticoids (2).

Whole cell melanoma vaccine genetically modified to stem cells like phenotype generates specific immune responses to ALDH1A1 and long-term survival in advanced melanoma patients

ABSTRACT Allogeneic whole cell gene modified therapeutic melanoma vaccine (AGI-101H) comprising of two melanoma cell lines transduced with cDNA encoding fusion protein composed of IL-6 linked with the soluble IL-6 receptor (sIL-6R), referred to as H6 was developed. H6 served as a molecular adjuvant, however, it has altered vaccine cells phenotype towards melanoma stem cells (MSC)-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). AGI-101H was applied in advanced melanoma patients with non-resected and resected disease. In the adjuvant setting, it was combined with surgery in case of recurring metastases, which were surgically removed and vaccination continued. A significant fraction of AGI-101H treated melanoma patients is still alive (11–19 years). Out of 106 living patients, 39 were HLA-A2 positive and were the subject of the study. Immunization of melanoma patients resulted in the generation of cytotoxic CD8+ T cells specific for ALDH1A1, which were detected in circulation by HLA-A0201 MHC dextramers loaded with ALDH1A188-96(LLYKLADLI) peptide. Phenotypically they were central memory CD8+ T cells. Re-stimulation with ALDH1A188-96 ex vivo resulted in IFN-γ secretion and cells degranulation. Following each vaccine dose administration, the number of ALDH1A1-CD8+ T cells increased in circulation and returned to the previous level until next dose injection (one month). ALDH1A1-CD8+ T cells were also found, however in the lower number than in vaccinated patients, in the circulation of untreated melanoma with stage IV but were not found in stage II or III and healthy donors. Specific anti-ALDH1 antibodies were present in treated patients. Long-term survival suggests immuno-targeting of MSC in treated patients.

Effects of Designer Hyper-Interleukin 11 (H11) on Hematopoiesis in Myelosuppressed Mice

The incidence of cancer is constantly increasing. Chemo/radiotherapy is one of major methods of treating cancer. Although adverse chemo/radiotherapy events, such as anemia and neutropenia, can be successfully cured, thrombocytopenia is still problematic. We constructed the Hyper-IL11 (H11) cytokine by linking soluble interleukin 11 receptor alpha (sIL-11Ralpha) with IL-11. In vivo H11 activity was examined in myelosuppressed mice. Myelosuppression was induced by either i) sublethal irradiation and carboplatin administration or ii) sublethal irradiation. A dose of 100 μg/kg of H11 or IL-11 was administered subcutaneously for 7 days. IL-11 and H11 accelerated leukocyte, hematocrit and platelet recovery. The effect on the attenuation of thrombocytopenia was significant. Moreover, both cytokines increased the cellularity and numbers of megakaryocyte, erythroid, and granulocyte/macrophage progenitors in the bone morrow and spleen compared with the control. Although H11 was administered at a molar concentration that was three times lower, its effects were comparable with or better than those of IL-11; thus, the activity of H11 was superior to that of IL-11. Because no toxicity was observed after the intravenous administration of H11, this hyper-cytokine may be potentially useful for treatment of thrombocytopenia and other IL-11-dependent disorders.