Showing papers by "Stephan A. Grupp published in 2011"

T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia

Abstract: Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non–cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR+ T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex–independent approach for the effective treatment of B cell malignancies.

Treatment of Advanced Leukemia in Mice with mRNA Engineered T Cells

Abstract: Cytotoxic T lymphocytes (CTLs) modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in preclinical models and are being tested in several clinical trials. Although CTLs bearing stably expressed CARs generated by integrating viral vectors are efficacious and have potential long-term persistence, this mechanism of CAR expression can potentially result in significant toxicity. T cells were electroporated with an optimized in vitro transcribed RNA encoding a CAR against CD19. These RNA CAR CTLs were then tested in vitro and in vivo for efficacy. We found that T cells expressing an anti-CD19 CAR introduced by electroporation with optimized mRNA were potent and specific killers of CD19 target cells. CD19 RNA CAR T cells given to immunodeficient mice bearing xenografted leukemia rapidly migrated to sites of disease and retained significant target-specific lytic activity. Unexpectedly, a single injection of CD19 RNA CAR T cells reduced disease burden within 1 day after administration, resulting in a significant prolongation of survival in an aggressive leukemia xenograft model. The surface expression of the RNA CARs may be titrated, giving T cells with potentially tunable levels of effector functions such as cytokine release and cytotoxicity. RNA CARs are a genetic engineering approach that should not be subject to genotoxicity, and they provide a platform for rapidly optimizing CAR design before proceeding to more costly and laborious stable expression systems.

Phase I Study of Temsirolimus in Pediatric Patients With Recurrent/Refractory Solid Tumors

Abstract: Purpose To determine dose-limiting toxicities, maximum-tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of weekly intravenous temsirolimus, a mammalian target of rapamycin (mTOR) signaling pathway inhibitor, in pediatric patients with recurrent or refractory solid tumors. Patients and Methods Cohorts of three to six patients 1 to 21 years of age with recurrent or refractory solid tumors were treated with a 1-hour intravenous infusion of temsirolimus weekly for 3 weeks per course at one of four dose levels: 10, 25, 75, or 150 mg/m2. During the first two courses, pharmacokinetic and pharmacodynamic evaluations (phosphorylation of S6, AKT, and 4EBP1 in peripheral-blood mononuclear cells) were performed. Results Dose-limiting toxicity (grade 3 anorexia) occurred in one of 18 evaluable patients at the 150 mg/m2 level, which was determined to be tolerable, and an MTD was not identified. In 13 patients evaluable for response after two courses of therapy, one had complete response (CR; neuroblastoma) ...

Statins are active in acute lymphoblastic leukaemia (ALL): a therapy that may treat ALL and prevent avascular necrosis.

Abstract: The prognosis for children with acute lymphoblastic leukaemia (ALL) has substantially improved with the use of multi-agent therapy over the last few decades. Unfortunately, two opposing challenges remain in childhood ALL treatment: relapse and toxicity. One in five children with ALL relapse and many of these succumb to disease. One toxicity from ALL therapy that is particularly debilitating is avascular necrosis (AVN). A recent study suggested over 70% of children with ALL develop AVN, including 15–20% who develop symptomatic AVN (Kawedia et al, 2011). While the cause of AVN is multi-factorial, significant high risk factors include high cholesterol and treatment with corticosteroids. An ideal new agent for ALL treatment would be a drug that has single agent activity, can be effectively combined with existing cytotoxics, and can alleviate the toxic effects of existing agents. Statins, a class of cholesterol lowering agents, may fit this model. Statins are effective in preclinical models of a number of malignancies, including acute myeloid leukaemia (Burke & Kukoly, 2008). Pravastatin and simvastatin have been studied in preclinical models, demonstrating the ability to prevent steroid-induced AVN (Iwakiri et al, 2008). Based on these data, clinicians are using statins in patients off-label and multi-institutional cooperative group clinical trials are under development, moving pravastatin forward as an AVN preventing agent (Sala et al, 2007). We investigated six different statins for their effects against ALL in vitro, including their ability to inhibit proliferation, induce apoptosis, and be combined with cytotoxics. Statins used were atorvastatin, simvastatin, pravastatin, and mevastatin (LKT Laboratories, St. Paul, MN, USA), as well as, lovastatin and fluvastatin (Calbiochem, Gibbstown, NJ, USA). The following human ALL cell lines were used for these experiments: precursor-B cell ALL (Reh, Nalm 6, 380, and RS4-11) and precursor-T ALL (Jurkat, Molt4, CEM). All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) or Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). In order to determine the activity of statins against ALL cells, the seven different ALL cell lines were treated with six different statins at doses from 1 to 20 μmol/l. Dose ranges were based on other published work treating malignant cell lines with statins, targeting levels obtainable in patients (Burke & Kukoly, 2008; Gbelcova et al, 2008). Response to treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) as previously described (Teachey et al, 2008). ALL cells were responsive to most agents (Fig 1A–D). Every ALL cell line was sensitive to fluvastatin, lovastatin, atorvastatin, and simvastatin, however, the degree of sensitivity varied between lines. All cell lines were resistant to pravastatin. The majority of cell lines (five of seven) were resistant to mevastatin. Fig 1 Statins inhibit ALL-cell proliferation. ALL cell lines were treated with six different statins for 72 h and proliferation was determined by MTT assay (Panels A–D and data not shown). ALL cells from all lines were sensitive to simvastatin, fluvastatin, ... The majority of statins, including fluvastatin, lovastatin, atorvastatin, and simvastatin are highly lipophilic substances. Pravastatin, in contrast, is hydrophilic (Gbelcova et al, 2008). Mevastatin is only slightly lipophilic (Neuhaus et al, 2002). Lipophilic statins can non-selectively diffuse into most cell types. Hydrophilic statins require active transport to enter a cell, limiting activity primarily to hepatocytes. Mevastatin's permeability lies in between as it can diffuse into some cell types and not others; it is unable to diffuse into lymphocytes (Neuhaus et al, 2002). The differences in response of ALL cells to statins could be explained by lipid solubility. Simvastatin and pravastatin are the only agents proven to reverse AVN in preclinical models (Iwakiri et al, 2008). Other statins could have a similar benefit; however, they are untested. As simvastatin was one of the most potent statins against the ALL cell lines and pravastatin had no activity, we focused on simvastatin, assessing whether it could eliminate ALL cells as opposed to stopping cell growth, using four cells lines (Nalm6, CEM, Jurkat, and Molt4). We found that simvastatin caused cell death by inducing apoptosis in each cell line (Fig 2A, B). In order to confirm that these findings were not limited to cell lines, primary human ALL cells collected from three patients with pre-B ALL were treated with simvastatin, using a bone marrow stromal culture system supplemented with stem cell factor as previously described (Teachey et al, 2006). All three samples were sensitive to simvastatin with a dose response similar to the cell lines, as determined by 7-aminoactinomycin D staining. After treatment with 20 μmol/l simvastatin for 48 h, there were 52–58% fewer viable CD19+ lymphoblasts in treated wells as compared to control. After treatment for 7 d with 5 or 20 μmol/l of simvastatin, there were 70–81% and 94–98% fewer blasts in treated wells as compared with control, respectively. Using this culture system, it cannot formally be excluded that the statins do not have effects on the stromal cells as well. Fig 2 Mechanistic effects of statins on ALL. Cells were cultured with combinations of simvastatin (20 μmol/l) and mevalonolactone (10 μmol/l) for 72 h. Mevalonolactone converts to mevalonate once added to solution. ALL blasts were plated at ... In order to determine whether statins can be effectively combined with cytotoxic chemotherapy, ALL cells from the seven cell lines were tested with simvastatin in combination with three cytotoxic agents used to treat human ALL, vincristine (Hospira Inc., Lake Forest, IL, USA), doxorubicin (Bedford Labs, Bedford, OH, USA), and dexamethasone (American Reagent Inc., Shirley, NY, USA) (Fig 1E, F and data not shown). Using median effects analysis, we found that the majority of combinations were synergistic; however, some were additive in certain cell lines. Very slight antagonism was found when doxorubicin was combined with simvastatin at very high doses in one of six cell lines and when vincristine was combined with simvastatin at lower doses in one of six cell lines. Overall, these data suggest that simvastatin can be safely combined with cell cycle dependent and independent cytotoxic agents. Statins are 3-hydroxyl-3-meythlglutaryl-CoA (HMG-CoA) reductase inhibitors. To determine if the effects of statins against ALL cells were dependent on inhibiting HMG-CoA reductase, ALL cells were treated with simvastatin with and without mevalonate (mevalonic acid, mevalonolactone) (Gbelcova et al, 2008). HMG-CoA reductase converts HMG-CoA into mevalonate as part of the HMG-CoA reductase pathway in cholesterol biosynthesis. Providing mevalonolactone (Sigma-Aldrich), which converts to mevalonate in solution, directly to the ALL cells in vitro bypasses HMG-CoA reductase. Mevalonate was able to reverse the effects of simvastatin on ALL cells (Fig 2A, B). In AML, statins were demonstrated to have some anti-tumour activity through down-regulation of signalling through the Raf/MEK/ERK pathway (Wu et al, 2004). As targeting the Raf/MEK/ERK pathway is effective against ALL in preclinical models and as ERK-mediated phosphorylation of BIM can reduce ALL blast responsiveness to steroids, the effects of statins on ERK signalling were assessed (Rambal et al, 2009). Treatment with simvastatin decreased phosphorylation of ERK in a dose-dependent manner and had no effect on total ERK (Fig 2C). Mevalonate reversed these effects. In summary, statins have preclinical activity against ALL cells and can be effectively combined with chemotherapy. As this class of drug has the potential ability to reverse avascular necrosis, a devastating side effect of ALL therapy, statins hold the unique promise of treating a disease and reversing the side effects of other medications used in therapy. Nevertheless, not all statins are the same, and careful consideration should be made regarding the best agent to move forward in clinical trials.