Detection and quantification of integrated vector copy number by multiplex droplet digital PCR in dual-transduced CAR T cells.

The success of chimeric antigen receptor (CAR) T cell therapies in refractory hematologic malignancies has prompted investigation of their efficacy in solid tumors. AUTO6NG is a dual-transduced GD2-targeting CAR that encodes distinct modules designed to enhance T cell activity in relapsed/refractory neuroblastoma. The ability to detect and precisely quantify vector copy number (VCN) for each integrated vector is essential for assessing the effect of each module on T cell tumor infiltration, persistence, and clinical activity. Droplet digital PCR (ddPCR) enables accurate, sensitive, and absolute quantification of specific nucleic acid sequences. Compared to standard detection of two targets, multiplex ddPCR assays allow simultaneous detection of up to four targets by selective modulation of signal amplitude while retaining the ability to quantify the target. We have developed a multiplex assay based on the two-channel system for simultaneous detection and quantification of three targets in AUTO6NG CAR T cells. The assay was highly specific, sensitive, accurate, and reproducible across time and samples. No differences were observed in measuring VCN between standard duplex and multiplex assays. Our results demonstrate that ddPCR is an accurate and cost-effective method for simultaneous detection of multiple targets in genomic DNA derived from engineered CAR T cells.

Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses In Vivo.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.

Super oblique tendon elongation with fascia lata.

PURPOSE: Superior oblique lengthening with a silicone retinal band is used to treat superior oblique overaction (SOOA); however, secondary infection, implant extrusion, orbital cellulitis, and adhesion may occur. We present a method of superior oblique tendon elongation in which autogenous fascia lata is used to decrease the likelihood of these complications. METHODS: Six patients (5 female, 1 male) aged 7-22 years (mean, 17 years) with 40-85(Delta) exotropia and SOOA (range, +2 to +4; mean, +3.5) underwent bilateral superior oblique lengthening with insertion of fascia lata. In the last 2 cases, the values of elongation were augmented by 2 mm. Fascia lata was harvested through a linear incision on the lateral aspect of the patient's thigh. RESULTS: Postoperatively, correction of A-pattern exotropia to within 10(Delta) was achieved in 66% of the cases as well as correction of SOOA to within +1 in 58% of the cases, with a follow-up of 9 months. All patients with +2 to +3 SOOA (3 cases) were fully corrected, whereas those with +4 SOOA (9 eyes) had residual overaction of +1 to +3. In the 4 eyes with augmented elongation, residual SOOA was between 0 and +2. No patient developed superior oblique palsy. CONCLUSIONS: Autogenous fascia lata may be used as an alternative to a silicone band for superior oblique lengthening. Our results were comparable with published results for the silicone band, with a lower rate of overcorrection. The improved biocompatibility makes it likely that autogenous fascia lata will have a lower complication rate than with a silicone band.

Cell survival in a three-dimensional matrix.

Integrin-mediated adhesion acts as a pluripotent mediator of cell signaling, triggering many pathways that promote proliferation and permit them to resist exogenous proapototic insults. To date, most studies have focused on apoptosis among cells adherent to rigid tissue-culture plastic substrates that tends to maximize integrin survival signaling. The physiological interpretation of such studies remains unclear. Here we describe methods to study integrin-mediated cell survival using matched cell populations that differ only in integrin expression, using a three-dimensional (3D) extracellular matrix culture. The preparation of appropriate cell types as well as the use of 3D collagen and fibrin gels is described. Methods to assess apoptosis and their application in the model are detailed. These techniques will offer an opportunity to study cell survival in the context of a non-rigid 3D extracellular matrix.

Chemoresistance of endothelial cells induced by basic fibroblast growth factor depends on Raf-1-mediated inhibition of the proapoptotic kinase, ASK1.

Genotoxic stress induced by anticancer drugs can lead to apoptosis of both angiogenic endothelial cells (ECs) and proliferating tumor cells. However, growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) present within the tumor microenvironment can promote chemoresistance by suppressing apoptotic mechanisms in these cells. Here, we have identified apoptosis signal-regulating kinase 1 (ASK1), a proapoptotic member of the MAP3K family, as a target of bFGF-mediated survival signaling in ECs. Evidence is provided that ASK1 is required for EC apoptosis in response to the genotoxic chemotherapeutic agent doxorubicin, and that bFGF, but not VEGF, neutralizes the death-promoting activity of ASK1. Specifically, bFGF stimulation promotes the formation of a Raf-1/ASK1 complex at the mitochondria, inhibits ASK1 kinase activity, and protects ECs from genotoxic stress. Mutation of the Raf-1 activation domain (SS338/9AA) not only prevents Raf-1/ASK1 complex formation but abolishes bFGF-mediated EC protection from genotoxic stress. In line with these observations, bFGF, but not VEGF, neutralizes the antiangiogenic effects of doxorubicin in vivo. These findings reveal a new pathway of EC survival signaling and define a molecular mechanism for chemoresistance induced by bFGF.

Role of Raf in vascular protection from distinct apoptotic stimuli.

Raf kinases have been linked to endothelial cell survival. Here, we show that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) differentially activate Raf, resulting in protection from distinct pathways of apoptosis in human endothelial cells and chick embryo vasculature. bFGF activated Raf-1 via p21-activated protein kinase-1 (PAK-1) phosphorylation of serines 338 and 339, resulting in Raf-1 mitochondrial translocation and endothelial cell protection from the intrinsic pathway of apoptosis, independent of the mitogen-activated protein kinase kinase-1 (MEK1). In contrast, VEGF activated Raf-1 via Src kinase, leading to phosphorylation of tyrosines 340 and 341 and MEK1-dependent protection from extrinsic-mediated apoptosis. These findings implicate Raf-1 as a pivotal regulator of endothelial cell survival during angiogenesis.