Case Report of a Fatal Serious Adverse Event Upon Administration of T Cells Transduced With a MART-1-specific T-cell Receptor.

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion.

Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.

MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue.

The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage.

DNA double-strand breaks (DSBs) are biologically one of the most important cellular lesions and possess varying degrees of chemical complexity. The notion that the repairability of more chemically complex DSBs is inefficient led to the concept that the extent of DSB complexity underlies the severity of the biological consequences. The repair of DSBs by non-homologous end joining (NHEJ) has been extensively studied but it remains unknown whether more complex DSBs require a different sub-set of NHEJ protein for their repair compared with simple DSBs.

Monoclonal TCR-redirected tumor cell killing.

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs).

Participation of DNA-PKcs in DSB repair after exposure to high- and low-LET radiation.

Cellular lesions (e.g. DSBs) are induced into DNA upon exposure to radiation, with DSB complexity increasing with radiation ionization density.

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using gammaH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle.

Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks.

DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle.

Biochemical kinetics model of DSB repair and induction of gamma-H2AX foci by non-homologous end joining.

We developed a biochemical kinetics approach to describe the repair of double-strand breaks (DSBs) produced by low-LET radiation by modeling molecular events associated with non-homologous end joining (NHEJ). A system of coupled nonlinear ordinary differential equations describes the induction of DSBs and activation pathways for major NHEJ components including Ku70/80, DNA-PKcs, and the ligase IV-XRCC4 heterodimer. The autophosphorylation of DNA-PKcs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled.

Induction and quantification of gamma-H2AX foci following low and high LET-irradiation.

Purpose: To investigate quantitatively the induction and rejoining of DNA double strand breaks (DSB) in V79-4 and xrs-5 Chinese hamster cells and HF19 human fibroblast cells, using the phosphorylation of the histone protein H2AX (gamma-H2AX) as an indicator of DSB, exposed to low doses of either low linear energy transfer (LET) (60)Co gamma-rays or high LET a-particles.

Materials And Methods: Cells were irradiated with low or high LET (20 - 2000 mGy). The gamma-H2AX foci were detected using immunohistochemistry and quantified by image analysis.

Synchronization of cell populations in G1/S and G2/M phases of the cell cycle.

The method described in the following chapter utilizes a double thymidine block (an inhibitor of DNA synthesis) followed by treatment of cells with nocodazole (a mitotic inhibitor) to obtain large cell populations at distinct phases of the cell cycle. Treatment with double thymidine results in a G1/S-phase arrested cell population, and the use of flow cytometry allows progression of the cells through the cell cycle to be monitored. Flow cytometry enables the calculation of timings for collection of cells at distinct cell cycle phases from G1/S (following treatment with thymidine) through to G2/M (owing to the presence of nocodazole).

The mammalian cell cycle: an overview.

In recent years, we have witnessed major advances in our understanding of the mammalian cell cycle and how it is regulated. Normal mammalian cellular proliferation is tightly regulated at each phase of the cell cycle by the activation and deactivation of a series of proteins that constitute the cell cycle machinery. This review article describes the various phases of the mammalian cell cycle and focuses on the cell cycle regulatory molecules that act at each stage to ensure normal cellular progression.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle.

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown.