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.

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.