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In spite of improvements in the last decade, relapses still occurs in the majority of ALL patients, with a long-term survival rate of only 30–40%. Recently, small-molecule inhibitors have been developed for targeting deregulated signal transduction pathways involved in proliferation and apoptosis. Current evidence identifies the Raf/MEK/ERK, the PI3K/AKT/mTOR, and the Bcl-2 pathways as potentially relevant targets for therapeutic intervention. We have previously demonstrated that constitutive ERK phosphorylation is an independent predictor of failure to achieve complete remission in adult ALL (Blood2007;109:5473) and thus we evaluated the in vitro activity of MEK-inhibitors. However, neither PD98059 nor PD0325901 affected cell growth, cell cycle distribution, and/or apoptosis in ALL cell lines (IC50 >1 μM for PD0325901). These results were confirmed in primary ALL samples, in which PD98059 significantly (P=0.012) inhibited ERK phosphorylation in 8/12 samples, without inducing cell cycle changes or apoptosis. We next investigated the activity of the mTOR inhibitor Temsirolimus. Temsirolimus displayed a biphasic dose-response in a panel of different ALL cell lines, with a flat curve (35–55% of inhibition) at concentrations ranging between 1 and 5,000 nM and more pronounced growth inhibition at concentrations ≥ 10 μM. The CEM cell line was the most sensitive (IC50: 7 nM), Jurkat showed intermediate sensitivity (IC50: 200 nM), while MOLT-4 were resistant (IC50 > 20,000 nM). Cell growth inhibition was associated with inhibition of cell cycle progression, while apoptosis induction was observed in less than 15% of the cells even at the highest concentration of Temsirolimus (20 μM). We then investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories to A.T.), a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor, in ALL cell lines and in 10 primary samples. ABT-737 showed a potent dose- and time-dependent growth-inhibitory activity in MOLT-4 (IC50: 198 nM), associated with loss of mitochondrial membrane potential, caspase activation, Bcl-2 cleavage, and ultimately apoptosis induction. Conversely, CEM cells proved resistant (IC50: 12,000 nM). We also found that ABT-737 was highly effective in primary adult and childhood ALL, independent of their chromosomal abnormalities, with a significant decrease in viability (p=0.008) and a remarkable induction of apoptosis (from a mean baseline value of 16.8±8.8% to 43.6±22.8%, p=0.04) at 10 nM ABT-737. A dose-dependent down-regulation of Bcl-2 and Bcl-xL expression was observed in sensitive samples, but not in the only resistant one. In summary, our study shows that ALL cells, do not undergo apoptosis in response to single-pathway inhibition, suggesting the presence of multiple, redundant pathways that preserve leukemic cell survival. The only notable exception was the Bcl-2/Bcl-xL inhibitor ABT-737. Studies are ongoing to identify mechanism-driven combinations of agents that would disrupt multiple signal transduction pathways, resulting in synergistic killing and, ultimately, in novel therapeutic strategies for ALL.

T cells are probably one of the most pivotal cell types in the human adaptive immune system. They have the capability to eradicate primary, metastatic, relapsed tumours and can ameliorate otherwise fatal viral infections. Not surprisingly therefore, activation and expansion of T cells has become one of the main focuses for immunotherapy and immune gene therapy. Sufficient T cells numbers however, are required to deliver a significant clinical impact to patients, and rapid reproducible expansion of viable T cells still remains one of the main challenges for significant improvement. One of the main concerns with adoptive immunotherapy is that it relies on one critical factor: ex-vivo cell manipulation; the problem with this is that the longer the in-vitro T cell culture, the shorter the in-vivo T cell survival after infusion. In-vivo artificial expansion systems for active immunotherapy would clearly circumvent this problem. Therefore ideally a flexible system should be constructed in order to performed both adoptive and/or active immunotherapy depending on the patients requirements. Currently there is no comprehensive artificial Antigen Presenting Cell system (aAPC) for both effective ex-vivo and in-vivo antigen specific T cell expansion. In order to address this, using nanotechnology, we have constructed a nano sized super-para-magnetic artificial targeted and traceable in-vivo APC system by coating liposomes (approved for human use) with an optimised number of MHC Class I / peptide complexes and a specific selected range of ligands for adhesion (anti LFA1), early activation (anti CD28, anti CD27), late activation (anti 4-1BB) and survival (anti CD40L) T cell receptors in the form of Fab antibody regions. We have constructed targeted liposomes (immuno-liposomes), which are also traceable in-vivo via fluorescent and Magnetic Resonance Imaging (MRI). Ex-vivo (human) and in-vivo (animal) models have been investigated showing firstly that these super-para-magnetic immuno-liposomes circulate the body safely and facilitate their own focusing to specific organs, tumour sites or body areas by applying external magnetic attraction. Secondly, in a viral (CMV) antigen specific model and measured by relevant and irrelevant tetramers, the system is capable of activating and expanding antigen specific T cells at greater levels (200 fold) than standard methods from CMV positive (memory) individuals. The system has also been able to accomplish a small successful level of T cell priming from naive CMV negative individuals. The T cells are phenotypicaly relevant and fully functional in terms of degranulation and cytokine production when specifically challenged. As mechanisms of action, we have demonstrated that the system functions directly on T cells as micro APCs and also semi-directly on the surface of natural APCs following a similar exosomes kinetics. The system is generated in less that 48 hr. Once the aAPC is created and it remains viable and stable for 7 days minimum. We have established optimal conditions for an efficient artificial APC, which embodies a superior and controllable approach and platform with enormous potential for cancer nanotechnology and T cell mediated immunotherapy.

One of the main problems of T cell mediated immunotherapy in delivering significant clinical impact and benefit to patients with malignant diseases and life threatening viral infections is the expansion of adequate numbers of functional antigen specific cytotoxic T cells. The current approaches for expanding T cells possess significant drawbacks in terms of timing, reproducibility and reliability. Many if not all these approaches rely on ex-vivo cell manipulation, which often leads to short T cell survival in-vivo after infusion. In-vivo artificial systems should be the ideal. There is no artificial APC system capable of both ex-vivo and, more importantly, in-vivo antigen specific T cell expansion. In order to address this we have developed a novel artificial nanotechnology system capable of priming and expanding antigen specific T cells in-vivo. As defined by the NIH, nanotechnology uses nanoscale injectable, targeted and traceable devices capable of important immunological/clinical functions. This nano-system was constructed using the latest generation of nanoscale immuno liposomes (100nm; 50 times smaller than average cells and same size as most human viruses), approved for in-vivo human use since they are non-toxic, biodegradable, avoid fast recognition by the reticulo-endothelial system, are safe in terms of size, have good stability and favourable pharmacokinetic behaviour for safe in-vivo trafficking. We have coated these liposomes with an optimised number of MHC Class I / peptide complexes and a specific and selected range of ligands for adhesion (ICAM-1), early activation (CD28, CD27), late activation (4-1BB) and survival receptors (CD40L). We have made these immuno liposomes traceable, either via a fluorescent lipid or iron oxide nano particles (13nm each), which make them traceable in vivo using Magnetic Resonance Imaging. Production of this system in a ready to use form is achievable in less than 48 hrs. We are currently working on an HLA-A2 transgenic mouse model to validate in-vivo behaviour of the system. After ex-vivo stimulation with this artificial system (using CMV pp65 as model antigen), we have measured successful expansions of high antigen specific T cell numbers (55 to 80 fold) in CMV positive individuals, which are superior when compared with other systems such as DCs (30 fold), beads (non antigen specific) and soluble tetramers and antibodies (30 fold). Expanded T cells are functional; they produce INF-γ and are predominantly of effector-memory and memory phenotype. We have demonstrated by double fluorescent staining that these liposomes are recognised directly on CD8+ T cells in an antigen specific fashion and also indirectly by being incorporated on the surface of the natural APCs as exosomes do. When tested in naive individuals, this system is also capable of priming naive T cells without additional adjuvants, as other APC systems use. In conclusion we have established the optimal conditions for an efficient artificial APC system, which embodies a powerful, controllable and superior approach with enormous potential for T cell immunotherapy in vivo. Figure Figure

The mitogen-activated protein kinase (MAPK) pathway contributes to the proliferative and differentiative control of hematopoietic cells. Extracellular signal-regulated kinase-1/2 (ERK) is one of the members of the MAPK pathway; its constitutive activation (p-ERK) has been found in a broad spectrum of hematological malignancies, particularly in acute myeloid leukemia (AML). A molecular therapeutic approach based on MAPK inhibitors has been therefore attempted. The aim of this study was to assess the expression and the clinical role of p-ERK in adult acute lymphoblastic leukemia (ALL) evaluating, in addition, the in vitro effects of the MEK inhibitor PD98059, used alone or in combination with chemotherapy, on the proliferation and apoptosis of ALL blast cells. Primary samples from 131 uniformly treated de novo patients enrolled in the GIMEMA LAL 2000 protocol were evaluated, using a p-ERK1/2-specific MoAb (clone E10), by Western blot analysis and flow cytometric assay which allowed single cell phospho-protein determination by the Kolmogorov-Smirnov statistic test (D-value). Lymphoid cell lines (Jurkat, CEM, RAJI and RPMI8866), as well as normal PMA-activated PBL expressed p-ERK, whereas resting PBL exhibited minimal levels (D-value 50x109/L; P=0.0128) and lower RNA-index of G1 phase (P=0.0296). Age, leukemia phenotype and molecular characteristics did not associate with p-ERK expression, while phospho-protein levels, measured as a continuous variable, correlated with failure to achieve CR (P=0.069). According to cell availability, we evaluated the effects of the MEK inhibitor PD98059 at 25μM on 30 fresh ALL samples, cultured in vitro for 24 hours. Eight of them were characterized by constitutive ERK activation, which was efficiently abrogated in 6; however, this effect did not induce cell cycle changes or apoptosis either alone or in combination with chemotherapeutic agents (Ara-C and/or Dexametasone). In summary, our study demonstrated that p-ERK is expressed in one third of adult ALL samples at presentation. Patients with p-ERK expression are characterized by higher leukemic mass, with a trend to induction treatment failure. MEK inhibition by PD98059 is capable of modulating p-ERK in primary ALL cells, though it does not result in cell cycle/apoptotic changes. In conclusion, ERK signaling appears only partially involved in the control of proliferative signals in adult ALL and modulation of additional pathways may result in more effective therapeutic strategies.