Genetically Modified T-Cells Expressing Chimeric Antigen Receptors in the Treatment of Cancer

Dr. Carl June and his colleagues at the University of Pennsylvania have succeeded in treating patients with Chronic Lymphocytic Leukemia using gene therapy. Two of the three patients treated sustained a complete remission and one a partial remission. The procedure involved transducing the patients’ T cells to express chimeric antigen receptors which target a particular protein found on both healthy and cancerous B cells. Following infusion of the newly transduced T cells, each patient developed clinical symptoms associated with an intense immune response. Shortly thereafter, tumors were completely eliminated in two of the patients and partially eliminated in the third. All three patients were pre-treated with conventional therapies and responded poorly. This study coalesces volumes of research in genetics, immunology, and molecular biology in what might just be the future of cancer treatment

Amplified inhibition of stellate cell activation pathways by PPAR-γ, RAR and RXR agonists.

Peroxisome proliferator activator receptors (PPAR) ligands such as 15-Δ12,13-prostaglandin L(2) [PJ] and all trans retinoic acid (ATRA) have been shown to inhibit the development of liver fibrosis. The role of ligands of retinoic X receptor (RXR) and its ligand, 9-cis, is less clear. The purpose of this study was to investigate the effects of combined treatment of the three ligends, PJ, ATRA and 9-cis, on key events during liver fibrosis in rat primary hepatic stellate cells (HSCs). We found that the anti-proliferative effect of the combined treatment of PJ, ATRA and 9-cis on HSCs was additive. Further experiments revealed that this inhibition was due to cell cycle arrest at the G0/G1 phase as demonstrated by FACS analysis. In addition, the combined treatment reduced cyclin D1 expression and increased p21 and p27 protein levels. Furthermore, we found that the three ligands down regulated the phosphorylation of mTOR and p70(S6K). The activation of HSCs was also inhibited by the three ligands as shown by inhibition of vitamin A lipid droplets depletion from HSCs. Studies using real time PCR and western blot analysis showed marked inhibition of collagen Iα1 and αSMA by the combination of the three ligands. These findings suggest that the combined use of PJ, ATRA and 9-cis causes inhibition of cell proliferation by cell cycle arrest and down-regulation of fibrotic markers to a greater extent compared to each of the ligands alone

Combined treatment of PJ, ATRA and 9-<i>cis</i> led to cell cycle arrest in primary HSCs.

<p>HSCs were incubated 24 hrs with 30 ng/ml PDGF, PJ, ATRA and 9-<i>cis</i> at a dose of 10^-5 M. Cellular DNA was stained with propidium iodide and flow cytometric analysis was preformed.</p

The combined anti-proliferative effect of PJ, ATRA and 9-<i>cis</i> is additive.

<p>HSCs were plated in 96 well plates. After 24 hrs, the medium was changed to starvation medium (DMEM with 0.5% FCS) overnight. HSCs were incubated for 24 hrs with 30 ng/ml PDGF, PJ, ATRA and 9-<i>cis</i> at doses of 10^-5-10^-4M. Cells were quantified by crystal violet staining. The inhibition of cell proliferation was expressed as % inhibition and calculated according to the following equation: Inhibitory effect (%) = 100 X (OD_control – OD_treatment) / (OD_control – OD_bl). The interaction is synergistic when the experimentally observed effect is larger than the calculated additive effect. When the experimentally observed effect is smaller the calculated additive effect, the interaction is antagonistic, and the interaction is additive when there is no difference between the two effects. Histogram showing average ±SE of densitometry results from 3 independent experiments.</p

Suppression of collagen Iα1 protein expression and mRNA by combined treatment of PJ, ATRA and 9-<i>cis</i>.

<p>Western blot analysis of primary cultured HSCs that were incubated for 24 hrs with 10 ng/ml TGF-β, PJ, ATRA and 9-<i>cis</i> at a concentration of 10^-5 M (A). Total RNA was isolated from HSCs treated overnight with 10 ng/ml TGF-β, PJ, ATRA and 9-<i>cis</i> at a concentration of 10^-5 M and analyzed by quantitative real-time PCR using primers specific to collagen Iα1. The results were normalized to β-actin mRNA expression levels. Data are expressed as mean <u>+</u> SE ^*<i>p</i><0.05 vs. TGF-β (B).</p

PJ, ATRA and 9-<i>cis</i> inhibit proliferation of rat primary HSCs in the presence of PDGF.

<p>HSCs were plated in 96 well plates. After 24 hrs, the medium was changed to starvation medium (DMEM with 0.5% FCS) overnight. HSCs were incubated for 24 hrs with 30 ng/ml PDGF, PJ, ATRA and 9-<i>cis</i> at a dose of 10^-5 M. After incubation, cell proliferation was assessed using the BrdU assay and plates read using an ELISA reader at 450 nm. Histogram showing average ±SE of densitometry results from 5 independent experiments. ^*<i>P</i><0.05 vs. control, ^**<i>P</i> <0.05 vs. PDGF.</p

Combined treatment of PJ, ATRA and 9-<i>cis</i> inhibit mTOR.

<p>(A) and p70^S6K (B) phosphorylation in primary HSCs. HSCs were incubated for 10, 20, 30 and 60 mins with 30 ng/ml PDGF, PJ, ATRA and 9-<i>cis</i> at a dose of 10^-5 M. Total lysate was separated by western blot analysis. Histogram showing average ±SE of densitometry results from 3 independent experiments. ^*<i>P</i><0.05 vs. PDGF.</p

Combined treatment of PJ, ATRA and 9-<i>cis</i> effect cell cycle protein expression in primary HSCs.

<p><b>A</b>. Combined treatment of PJ, ATRA and 9-<i>cis</i> decreased cyclin D1 expression in primary rat HSCs. <b>B</b>. Combined treatment of PJ, ATRA and 9-<i>cis</i> increased P21 expression in primary rat HSCs. <b>C</b>. Combined treatment of PJ, ATRA and 9-<i>cis</i> increased P27 expression in primary rat HSCs. HSCs were incubated for 24 hrs with 30 ng/ml PDGF, PJ, ATRA and 9-<i>cis</i> at a dose of 10^-5 M. Total lysate was separated by western blot analysis. Histogram showing average ±SE of densitometry results from 3 independent experiments. ^*<i>P</i><0.05 vs. PDGF.</p

Disposal of iron by a mutant form of lipocalin 2.

Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe-/-) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway