Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34 hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E (PGE) as a positive mediator of lentiviral transduction of CD34 cells. Supplementation with PGE increased lentiviral vector (LVV) transduction of CD34 cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34 cells from multiple normal human donors and from patients with β-thalassemia or sickle cell disease. Notably, PGE increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE may be useful in clinical gene therapy applications using lentivirally modified HSPCs.
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.
Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.
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 for treatment requires ex vivo culture to facilitate CAR gene transfer and to achieve a therapeutic dose of the modified cells. Recent data suggests that specific T cell subtypes can provide enhanced anti-tumor efficacy, spurring efforts to optimize the production of therapeutic T cells via the cumbersome physical isolation of central memory T cells or culture in cytokines such as IL-7 and IL-15. Here we explored the potential for a simple culture modification to improve the therapeutic potential of CAR T cells without adding manufacturing complexity. To this end, we produced CAR T cells specific to B cell maturation antigen (BCMA) using standard IL-2 culture conditions supplemented with a PI3K inhibitor, or with IL-7 and IL-15 in place of IL-2. The in vivo activity of CAR T cells was studied in mouse models of human Burkitt's lymphoma (Daudi) and multiple myeloma (RPMI-8226), both of which express BCMA. In the Daudi model, NSG mice were injected intravenously with 2 × 106 tumor cells and allowed to accumulate a large tumor burden to model late stage disease observed in relapsed and refractory lymphoma. In this advanced disease model, anti-BCMA CAR T cells (4 × 106/mouse) cultured either in IL-2 or IL-7 and IL-15 had little or no effect on tumor growth (p = 0.22 and 0.23, respectively) and all mice succumbed to tumors within two weeks of treatment. In contrast, all animals treated with the same number of anti-BCMA CAR T cells cultured with PI3K inhibition survived and had complete long-term tumor regression (p = 0.003). The same anti-BCMA CAR T cells were studied in a model of multiple myeloma. NSG mice were injected subcutaneously with 107 RPMI-8226 cells and 22 days later received a single administration of anti-BCMA CAR T cells (4 × 105/mouse) cultured under various conditions. In this model, tumor regression occurred regardless of in vitro culture conditions. To model tumor relapse and evaluate CAR T cell durability, surviving animals were re-challenged with RPMI-8226 cells on the opposite flank two weeks after initial tumor clearance. In contrast to other conditions, all animals treated with anti-BCMA CAR T cells cultured with PI3K inhibition were protected against subsequent tumor challenge (p = 0.005). This improved therapeutic activity of anti-BCMA CAR T cells cultured with PI3K inhibition was associated with an increased frequency of CD62L+ CD8+ T cells in the drug product (p < 0.001) suggesting enrichment of this distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate an improved anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Citation Format: Shannon Grande, Molly R. Perkins, Amanda Hamel, Holly M. Horton, Fay Eng, Claire J. Rhodes, Tracy E. Garrett, Sara M. Miller, John W. Evans, Howard J. Latimer, Christopher Horvath, Michael Kuczewski, Kevin Friedman, Richard A. Morgan. Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2296.
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