Targeting CD123 in blastic plasmacytoid dendritic cell neoplasm using allogeneic anti-CD123 CAR T cells.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.

Allogeneic TCRαß deficient CAR T-cells targeting CD123 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Hematopoietic stem cells can be distinguished from LSCs by an array of cell surface antigens such as CD123, thus a candidate to eliminate LSCs using a variety of approaches, including CAR T cells. Here, we evaluate the potential of allogeneic gene-edited CAR T cells targeting CD123 to eliminate LSCs (UCART123). UCART123 cells are TCRαßneg T cells generated from healthy donors using TALEN® gene-editing technology, decreasing the likelihood of graft vs host disease. As safety feature, cells express RQR8 to allow elimination with Rituximab. UCART123 effectively eliminates AML cells in vitro and in vivo with significant benefits in overall survival of AML-patient derived xenograft mice. Furthermore, UCART123 preferentially target AML over normal cells with modest toxicity to normal hematopoietic stem/progenitor cells. Together these results suggest that UCART123 represents an off-the shelf therapeutic approach for AML.

Screening of Lipopeptide-Producing Strains of Bacillus sp. Using a New Automated and Sensitive Fluorescence Detection Method.

Lipopeptides, such as surfactins are important biosurfactants produced by Bacillus sp. that find applications in many areas (environment, medicine, and food industries). Giving their importance, the use of simple detection methods will facilitate screening and quantification. In the present work, the authors describe a completely automated workflow for the screening of lipopeptide-producing strains, including quantification. First, isolated colonies from environmental samples are automatically picked and inoculated in 96 wells growth plate. After overnight incubation, surfactin produced in the broth is quantified, using a new sensitive fluorescent method. The method uses fluorescein (FL), which is an anionic dye at neutral to alkaline pH and forms a stable complex with the cationic surfactant cetylpiridinium chloride (CPC), quenching fluorescence. Upon addition of surfactin or other lipopeptides, fluorescein is released from the CPC-FL complex and quantified. The robustness of this method is assessed by comparing the quantification results to those conventionally measured by RP-UPLC and the results of strain screening are confirmed by MALDI-ToF analysis. The authors report for the first time the successful application of this analytical method for high-throughput screening of novel lipopeptide-producing strains.

Age-related changes in rat myocardium involve altered capacities of glycosaminoglycans to potentiate growth factor functions and heparan sulfate-altered sulfation.

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF(165) was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.

Matrix therapy with RGTA OTR4120 improves healing time and quality in hairless rats with deep second-degree burns.

BACKGROUND: ReGeneraTing Agents (RGTAs) are biodegradable polymers engineered to mimic heparan-sulfate in the extracellular matrix of damaged tissue. RGTAs improve tissue healing in several animal models by stabilizing and protecting heparin-binding growth factors and matrix proteins. RGTA restores the normal matrix architecture and supports tissue regeneration. In this study, the authors evaluated the effects of RGTA on epidermal repair and dermal remodeling in a rat burn model. METHODS: Deep second-degree burns were induced in 156 hairless rats, of which half (n = 78) received topical and intramuscular RGTA immediately after the burn followed by intramuscular RGTA weekly for 1 month. The controls (n = 78) received saline according to the same protocol. Rats were killed starting on each day of the first week and on days 14, 28, 60, 120, 240, and 365. The burns were evaluated by photography, histology, and immunohistochemistry. RESULTS: Coagulation necrosis involved the entire epidermis and superficial adnexa. Compared with the controls, speed of epidermal repair, as assessed between days 3 and 7 based on cell-layer number and anticytokeratin-14 staining, was faster in the RGTA group; and the zone of stasis, as assessed based on secondary vascular lesions in the dermis, was smaller. On day 7, reepithelialization was complete in both groups. On days 14 and 28, the remodeled dermal zone was smaller in the RGTA group. CONCLUSION: RGTA accelerated epidermal repair and protected the dermis from secondary effects of heat as quantified by zone-of-stasis size and extent of dermal remodeling.