Therapeutic melanoma vaccine with cancer stem cell phenotype represses exhaustion and maintains antigen-specific T cell stemness by up-regulating BCL6.

We developed a therapeutic, gene-modified, allogeneic melanoma vaccine (AGI-101H), which, upon genetic modification, acquired melanoma stem cell-like phenotype. Since its initial clinical trial in 1997, the vaccine has resulted in the long-term survival of a substantial fraction of immunized patients (up to 20 years). Here, we investigated the potential molecular mechanisms underlying the long-lasting effect of AGI-101H using transcriptome profiling of patients' peripheral T lymphocytes. Magnetically-separated T lymphocytes from AGI-101H-immunized long-term survivors, untreated melanoma patients, and healthy controls were subjected to transcriptome profiling using the microarray analyses. Data were analyzed with a multitude of bioinformatics tools (WebGestalt, DAVID, GSEA) and the results were validated with RT-qPCR. We found substantial differences in the transcriptomes of healthy controls and melanoma patients (both untreated and AGI-101H-vaccinated). AGI-101H immunization induced similar profiles of peripheral T cells as tumor residing in untreated patients. This suggests that whole stem cells immunization mobilizes analogous peripheral T cells to the natural adaptive anti-melanoma response. Moreover, AGI-101H treatment activated the TNF-α and TGF-ß signaling pathways and dampened IL2-STAT5 signaling in T cells, which finally resulted in the significant up-regulation of a BCL6 transcriptional repressor, a known amplifier of the proliferative capacity of central memory T cells and mediator of a progenitor fate in antigen-specific T cells. In the present study, high levels of BCL6 transcripts negatively correlated with the expression of several exhaustion markers (CTLA4, KLRG1, PTGER2, IKZF2, TIGIT). Therefore, Bcl6 seems to promote a progenitor fate for cancer-experienced T cells from AGI-101H-vaccinated patients by repressing the exhaustion markers.

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.

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 ALDH1A188-96ex 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.

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 ß-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-CD41a(high)CD61(high) 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.

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

BACKGROUND: Interleukin 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 ß (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. RESULTS: The 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). CONCLUSIONS: The biologically active H11 may be potentially useful for treatment of thrombocytopenia, infertility, multiple sclerosis, cardiovascular diseases or inflammatory disorders.

Expression of PiT1 and PiT2 retroviral receptors and transduction efficiency of tumor cells.

Recombinant retroviral vectors are still the most common gene delivery vehicles for gene therapy purposes, especially for construction of genetically modified tumor vaccines (GMTV). However, these vehicles are characterized by relatively low titre and in the case of many tumor cell lines, low transduction efficiency. We constructed bicistronic retroviral vector pseudotypes of amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV), encoding enhanced green fluorescent protein (EGFP) as a rapid and easily detectable reporter gene. Transduction of five different human melanoma and four renal carcinoma cell lines by these two virus pseudotypes revealed differences in transduction efficiency, which wase markedly lower for the renal carcinoma cell lines. Stimulation of retroviral receptor expression (PiT1 and PiT2) by phosphate depletion induced a limited increase of receptor mRNA levels, but did not improve the gene transfer efficiency. In contrast, simultaneous transduction with both vector pseudotypes markedly increased the transduction efficiency, compared to GaLV or A-MuLV alone. The same effect could be achieved by several repeated exposures of target cells to fresh vector preparation. Overexpression of GaLV receptor (PiT1) in target cells significantly increased the transduction rate and enabled retrovirus mediated gene transfer into the cells which normally are not transducible by GaLV pseudotypes. We demonstrated that, using different transduction strategies, the relatively inefficient, widely used retroviral vector systems could be significantly improved.