Multicenter phase II trials with Gemtuzumab Ozogamicin (GO/ Mylotarg s ), consisting of a CD33 antibody linked to the cytotoxic drug calicheamicin, have shown a 30% overall response rate in relapsed acute myeloid leukemia patients. However, no clear correlation was observed between CD33 expression on leukemic blasts and response to GO therapy. We analyzed the CD33 specificity of GO-induced cell death and the effect of GO on CD33-negative malignancies. We demonstrate that lysis induced by clinically relevant GO concentrations is partially CD33 mediated, and that efficient non-CD33-mediated GO uptake can occur via endocytosis. In agreement with these results, we observed GO-mediated death of human CD33-negative acute lymphoblastic leukemia cells both in vitro and in vivo in an NOD/SCID mouse model. Finally, sensitivity to GOinduced cell death was at least partially determined by the activation status of leukemic cells, with cells in activated phases of the cell cycle being most effective in both CD33-specific GO internalization, renewed expression of CD33 molecules, and non-CD33-mediated GO uptake via endocytosis. In conclusion, these data provide mechanistic insight into the efficacy of GO in CD33-positive as well as in CD33-negative malignancies with endocytic capacity, and provide a rationale for the use of GO in the treatment of malignancies with endocytic capacity.

Currently, individuals with pre-existing neutralizing antibodies (NABs) against adeno-associated virus (AAV) above titer of 5 are excluded from systemic AAV-based clinical trials. In this study we explored the impact of pre-existing anti-AAV5 NABs on the efficacy of AAV5-based gene therapy. AMT-060 (AAV5-human FIX) was evaluated in 10 adults with hemophilia B who tested negative for pre-existing anti-AAV5 NABs using a GFP-based assay. In this study, using a more sensitive luciferase-based assay, we show that 3 of those 10 patients tested positive for anti-AAV5 NABs. However, no relationship was observed between the presence of pre-treatment anti-AAV5 NABs and the therapeutic efficacy of AMT-060. Further studies in non-human primates (NHPs) showed that AAV5 transduction efficacy was similar following AMT-060 treatment, irrespective of the pre-existing anti-AAV5 NABs titers. We show that therapeutic efficacy of AAV5-mediated gene therapy was achieved in humans with pre-existing anti-AAV5 NABs titers up to 340. Whereas in NHPs circulating human factor IX (hFIX) protein was achieved, at a level therapeutic in humans, with pre-existing anti-AAV5 NABs up to 1030. Based on those results, no patients were excluded from the AMT-061 (AAV5-hFIX-Padua) phase IIb clinical trial (n = 3). All three subjects presented pre-existing anti-AAV5 NABs, yet had therapeutic hFIX activity after AMT-061 administration.

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PurposeAberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established.Experimental DesignWe compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative.ResultsDual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2.ConclusionsInhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.

Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry.