B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

NOV-002 is a novel glutathione disulfide mimetic that when administered in combination with standard chemotherapeutic regimens has resulted in increased efficacy (survival, tumor response) and improved tolerance to chemotherapy (e.g., hematologic recovery) in advanced non-small cell lung cancer patients. We show that NOV-002, which is not cytotoxic as a single agent, generated time-and concentration-dependent oxidative signals at the cell surface (reduction in protein thiols) and intracellularly [altered oxidized glutathione (GSSG) and reduced glutathione levels and ratio; increased reactive oxygen species] in the premyeloid HL-60 cell line and that this was associated with an increase in S-glutathionylation of cell proteins, particularly actin. Commensurate with these effects, NOV-002 activated p38, c-Jun-NH 2 -kinase, and extracellular signal-regulated kinase and caused a dosedependent increase in phosphorylation of three proteins that have previously been linked with hematopoiesis, AKT, JAK2, and STAT5. The effect of NOV-002 on enzymes involved in glutathione metabolism was evaluated. Relative to oxidized glutathione, NOV-002 was an equivalent substrate for glutathione reductase and was an inhibitor of protein disulfide isomerase, one of the components of the redox-sensitive unfolded protein response pathway. These redox-stimulated cell signaling actions occurred in the context of increased HL-60 cell proliferation after treatment with NOV-002. Overall, the pleiotropic pharmacologic effects of NOV-002 can be attributed to the GSSG component of the drug, and modulation of cellular redox balance is a feature central to the mechanism of action of NOV-002. Such modulation may underlie its clinical actions, including hematologic recovery and immunostimulation in the face of chemosuppression.

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Background Clinical application of adoptive T cell therapy (ACT) has been hindered by an inability to generate adequate numbers of non-tolerized, functionally active tumor-specific T cells which can persist in vivo. In order to address this, we evaluated the impact of IL-12 signaling during tumor-specific CD8+ T cell priming in terms of persistence and anti-tumor efficacy using an established B16 melanoma tumor adoptive therapy model. Study Design B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, i.p.), and one day later, treated by adoptive transfer of tumor-specific pmel-1 CD8+ T cells primed ex vivo 3 days earlier with (i) both IL-12 and antigen (hGP10025–33 peptide) or (ii) antigen only. Tumors were measured biweekly and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function. Results Adoptive transfer of tumor-specific CD8+ T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced anti-tumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ). Conclusions Our findings demonstrate that ex vivo priming of tumor-specific CD8+ T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability upon transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.

Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivo culture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivo culture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivo activity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106 Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106 CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105 CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitro culture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivo efficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p < 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Disclosures Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.