Integrating a 19F MRI Tracer Agent into the Clinical Scale Manufacturing of a T-Cell Immunotherapy.

Leukocyte immunotherapies have made great progress in the treatment of cancer. Recent reports on the treatment of B-cell malignancies using Chimeric Antigen Receptor and affinity enhanced T-Cell Receptor therapies have demonstrated encouraging clinical results. As investigators begin to explore the treatment of solid tumors with these cells, the hurdle of evaluating T-cell homing to and persistence at the site of disease remain. Significant challenges regarding the GMP manufacture and administration of a therapeutic dose of millions to billions of transduced T-cells remain. Here we report on the application of a clinically authorized 19F MRI tracer agent to human T-cells, employing state-of-the-art methods and equipment in the manufacture of a cellular therapy. Using a general T-cell expansion protocol and clinical scale industrial bioreactors, we show 19F labeling without detriment to the product +/- cryopreservation. While the incorporation of the 19F tracer is not trivial, it is just one of the many steps that can aid in progression of a therapeutic to and though the clinic. Combining the MRI tracking capabilities, safety profiles, and clinical sensitivity of this method, this application demonstrates the ability of 19F MRI to be used in industrial scale applications to visualize the spatial fate of cellular therapeutics.

Apoptosis in the skeletal muscle of untreated children with juvenile dermatomyositis: impact of duration of untreated disease.

Juvenile dermatomyositis (JDM) is the most common myopathy in children with characteristic skin rash and muscle weakness, in which longer duration of untreated disease was associated with less muscle weakness. The duration of untreated inflammation may alter the apoptotic pathways involved in skeletal muscle damage. Diagnostic muscle biopsies from 14 untreated patients were stained for apoptosis markers. TUNEL-positive nuclei and caspase 3 were detected within the laminin layer, indicating apoptosis of skeletal muscle nuclei. Untreated JDM disease duration greater than 2 months ("long"), was associated with higher Fas-positive cell counts in the perivascular region compared with the "short" disease duration group, 2 months or less. Within the "long" duration group, higher Fas-positive cell counts were positively associated with increased TUNEL-positive nuclei and caspase 3. We conclude that the duration of untreated disease (chronic inflammation) influences the mode of continuing cell damage and death in children with JDM.

Increased plasma thrombospondin-1 (TSP-1) levels are associated with the TNF alpha-308A allele in children with juvenile dermatomyositis.

Vascular occlusion is more frequent in children with juvenile dermatomyositis (JDM) who have the TNF alpha-308A allele. One of the potent anti-angiogenic factors is thrombospondin-1 (TSP-1). This study investigated the association of the TNF alpha-308A allele with circulating levels of angiogenic mediators, TSP-1, and platelet factor 4 (PF4) using fresh, platelet-poor plasma (PPP). The TNF alpha-308A allele was characterized by PCR amplification and NcoI digestion. Concentrations of TSP-1 and PF4 in PPP from 31 JDM patients and 25 matched pediatric controls were determined by ELISA. The majority of the JDM children with the TNF alpha-308A allele (7/12) produced more TSP-1 than their TNF alpha-308G counterparts (P < 0.05), and their TSP-1 values were inversely related to those for PF4 (P < 0.0006). We conclude that the increased circulating concentrations of TSP-1 associated with the TNF alpha-308A allele suggest that this anti-angiogenic regulator may play a significant role in the augmented vascular occlusion observed in JDM children with this genetic marker.

Gene expression profiling in DQA1*0501+ children with untreated dermatomyositis: a novel model of pathogenesis.

Juvenile dermatomyositis (JDM), the most common pediatric inflammatory myopathy, is a systemic vasculopathy affecting young children. Epidemiology studies documenting an antecedent illness in the 3 mo before the first definite symptom (rash and/or weakness) of JDM are supported by immunologic data that suggest that the disease pathophysiology is Ag driven. The purpose of this study was to compare the gene expression profiles in muscle biopsies of four untreated DQA1*0501(+) JDM children with profiles from children with a known necrotizing myopathy (Duchenne muscular dystrophy), as well as an in vitro antiviral model (NF90), and healthy pediatric controls. Nearly half (47%) of the dysregulated genes in JDM were associated with the immune response. In particular, increased expression of IFN-alphabeta-inducible genes 6-16, myxovirus resistance protein p78, latent cytosolic transcription factor, LMP2, and TAP1 was observed. This profile is consistent with an IFN-alphabeta transcription cascade seen in the in vitro viral resistance model. The IFN-alphabeta-inducible profile was superimposed on transcription profiles reflective of myofiber necrosis and regeneration shared with Duchenne muscular dystrophy. Expressed genes were confirmed by quantitative real-time PCR (6-16), immunofluorescence (thrombospondin 4), and immunolocalization (IFN-gamma, p21). We hypothesize that these data support a model of Ag (?viral) induction of an apparent autoimmune disease based on dynamic interaction between the muscle, vascular, and immune systems in the genetically susceptible (DQA1*0501(+)) child.