Ex vivo culture of chimeric antigen receptor T cells generates functional CD8 T cells with effector and central memory-like phenotype

The anti-tumor efficacy of adoptively transferred T cells requires their in vivo persistence and memory polarization. It is unknown if human chimeric antigen receptor (CAR)-expressing T cells can also undergo memory polarization. We examined the functional status of CAR CD8 T cells, re-directed to Lewis Y antigen (LeY-T), throughout a period of ex vivo expansion. Immediately before culture CD8 T cells comprised a mixture of phenotypes including naive (CD45RA /CCR7 /CD27 /CD28 /perforin), central memory (CM, CD45RA /CCR7 lo /CD27 /CD28 /perforin lo), effector memory (EM, CD45RA /CCR7 /CD27 /CD28 /perforin mod) and effector (Eff, CD45RA /CCR7 /CD27 /CD28 /perforin hi) cells. After transduction and expansion culture of peripheral blood mononuclear cells from normal donors or multiple myeloma patients, CD8 LeY-T cells polarized to EM- and CM-like phenotype. CD8 LeY-T cells differed from starting CD8 CM and EM T cells in that CD27, but not CD28, was downregulated. In addition, CD8 LeY-T cells expressed high levels of perforin, similar to starting CD8 Eff. CD8 LeY-T cells also showed hallmarks of both memory and Eff function, underwent homeostatic proliferation in response to interleukin (IL)-15, and showed interferon (IFN)-γ production and cytotoxicity in response to Le-Y antigen on OVCAR-3 (human ovarian adenocarcinoma) cells. This study confirms CD8 LeY-T cells have a CM- and EM-like phenotype and heterogeneous function consistent with potential to persist in vivo after adoptive transfer

Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis y antigen

We have evaluated the carbohydrate antigen Lewis (Le ) as a potential target for T-cell immunotherapy of hematological neoplasias. Analysis of 81 primary bone marrow samples revealed moderate Le expression on plasma cells of myeloma patients and myeloblasts of patients with acute myeloid leukemia (AML) (52 and 46% of cases, respectively). We developed a retroviral vector construct encoding a chimeric T-cell receptor that recognizes the Le antigen in a major histocompatibility complex-independent manner and delivers co-stimulatory signals to achieve T-cell activation. We have shown efficient transduction of peripheral blood-derived T cells with this construct, resulting in antigen-restricted interferon-γ secretion and cell lysis of Le-expressing tumor cells. In vivo activity of gene-modified T cells was demonstrated in the delayed growth of myeloma xenografts in NOD/SCID mice, which prolonged survival. Therefore, targeting Le-positive malignant cells with T cells expressing a chimeric receptor recognizing Le was effective both in vitro and in a myeloma mouse model. Consequently, we plan to use T cells manufactured under Good Manufacturing Practice conditions in a phase I immunotherapy study for patients with Le-positive myeloma or AML

Can burning restrict eucalypt invasion on grassy balds? .

Abstract: Eucalyptus tereticornis seedlings occurring on the edges of grassy balds on the Bunya Mountains were burnt by four separate fires. From the results, a logistic model demonstrated that lignotuber size was positively related and fire temperature negatively related to survivorship. While mortality was high for young seedlings there was no mortality of 5-year old survivors from these trials subject to repeat burning. The model predicted that burning every 2 years will not substantially limit seedling establishment. This prediction was strengthened by results verifying that management fires on the grassy balds are generally of low intensity. Fire intensity is weakly related to a Fire Danger Index, indicating that the timing of burning in relation to weather conditions will not substantially enhance opportunities for more intense fires. Thus, even with biennial burning under optimal conditions eucalypt forest will replace grassy balds where they adjoin. Regular burning by aborigines may have maintained grassy bald-rainforest boundaries, but not boundaries with eucalypt forest. Seed dispersal and migration barriers may have limited the expansion of eucalypt forest. It is concluded that under current conditions the long-term preservation of the grassy balds is only possible where they are entirely surrounded by rainforest and are regularly burnt

The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death

Persistence and efficacy of second generation CAR T Cell against the LeY Antigen in acute myeloid leukemia

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence