Axicabtagene ciloleucel in relapsed or refractory indolent non-Hodgkin lymphoma (ZUMA-5): a single-arm, multicentre, phase 2 trial.

BACKGROUND: Most patients with advanced-stage indolent non-Hodgkin lymphoma have multiple relapses. We assessed axicabtagene ciloleucel autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in relapsed or refractory indolent non-Hodgkin lymphoma. METHODS: ZUMA-5 is a single-arm, multicentre, phase 2 trial being conducted at 15 medical cancer centres in the USA and two medical cancer centres in France. Patients were eligible if they were aged 18 years or older, with histologically confirmed indolent non-Hodgkin lymphoma (follicular lymphoma or marginal zone lymphoma), had relapsed or refractory disease, previously had two or more lines of therapy (including an anti-CD20 monoclonal antibody with an alkylating agent), and an Eastern Cooperative Oncology Group performance score of 0 or 1. Patients underwent leukapheresis and received conditioning chemotherapy (cyclophosphamide at 500 mg/m2 per day and fludarabine at 30 mg/m2 per day on days -5, -4, and -3) followed by a single infusion of axicabtagene ciloleucel (2 × 106 CAR T cells per kg) on day 0. The primary endpoint was overall response rate (complete response and partial response) assessed by an independent review committee per Lugano classification. The primary activity analysis was done after at least 80 treated patients with follicular lymphoma had been followed up for at least 12 months after the first response assessment at week 4 after infusion. The primary analyses were done in the per-protocol population (ie, eligible patients with follicular lymphoma who had 12 months of follow-up after the first response assessment and eligible patients with marginal zone lymphoma who had at least 4 weeks of follow-up after infusion of axicabtagene ciloleucel). Safety analyses were done in patients who received an infusion of axicabtagene ciloleucel. This study is registered with ClinicalTrials.gov, NCT03105336, and is closed to accrual. FINDINGS: Between June 20, 2017, and July 16, 2020, 153 patients were enrolled and underwent leukapheresis, and axicabtagene ciloleucel was successfully manufactured for all enrolled patients. As of data cutoff (Sept 14, 2020), 148 patients had received an infusion of axicabtagene ciloleucel (124 [84%] who had follicular lymphoma and 24 [16%] who had marginal zone lymphoma). The median follow-up for the primary analysis was 17·5 months (IQR 14·1-22·6). Among patients who were eligible for the primary analysis (n=104, of whom 84 had follicular lymphoma and 20 had marginal zone lymphoma), 96 (92%; 95% CI 85-97) had an overall response and 77 (74%) had a complete response. The most common grade 3 or worse adverse events were cytopenias (104 [70%] of 148 patients) and infections (26 [18%]). Grade 3 or worse cytokine release syndrome occurred in ten (7%) patients and grade 3 or 4 neurological events occurred in 28 (19%) patients. Serious adverse events (any grade) occurred in 74 (50%) patients. Deaths due to adverse events occurred in four (3%) patients, one of which was deemed to be treatment-related (multisystem organ failure). INTERPRETATION: Axicabtagene ciloleucel showed high rates of durable responses and had a manageable safety profile in patients with relapsed or refractory indolent non-Hodgkin lymphoma. FUNDING: Kite, a Gilead Company.

Associação Brasileira de Hematologia, Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. Review article: Cell therapy in solid tumors.

The use of immunotherapy in cancer treatment over the past decade has resulted in significant advances and improvements in cancer patients survival with the use of checkpoint inhibitors. Nevertheless, only a fraction of solid tumors responds to this immunotherapy modality. Another modality of immunotherapy consists of employing cell-based therapy as an adoptive therapeutic modality. That involves distinct modalities of cellular therapies such as CAR T cells (chimeric antigen receptor T cell), TILs (tumor-infiltrating lymphocytes), and TCR T cells. Those treatments have proven effective in hematologic tumors and could have an impact in tumors that do not respond to checkpoint inhibitors. This review aims to outline the rationale, operation, clinical applicability, and results of adoptive cell therapy for patients with solid tumors.

Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System.

Chimeric antigen receptor (CAR) T cell therapy has proven clinically beneficial against B cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, suboptimal clinical outcomes have been associated with decreased expansion and persistence of adoptively transferred CAR T cells, antigen-negative relapses, and impairment by an immunosuppressive tumor microenvironment. Improvements in CAR T cell design are required to enhance clinical efficacy, as well as broaden the applicability of this technology. Here, we demonstrate that interleukin-18 (IL-18)-secreting CAR T cells exhibit enhanced in vivo expansion and persistence and significantly increase long-term survival in syngeneic mouse models of both hematological and solid malignancies. In addition, we demonstrate that IL-18-secreting CAR T cells are capable of modulating the tumor microenvironment, as well as enhancing an effective endogenous anti-tumor immune response. IL-18-secreting CAR T cells represent a promising strategy to enhance the clinical outcomes of adoptive T cell therapy.

Emerging Role of CAR T Cells in Non-Hodgkin's Lymphoma.

Adoptive T-cell therapy with chimeric antigen receptor T cells (CAR-Ts) has produced impressive clinical responses among patients with B-cell malignancies, and several groups have published positive results using anti-CD19 CAR-Ts for the treatment of B-cell acute lymphoblastic leukemia. Recently, new data from clinical trials have demonstrated the benefits of CAR-T therapy in the non-Hodgkin's lymphoma (NHL) setting. This review describes some of the most recent and promising advances in engineered T-cell therapy, with particular emphasis on the clinical benefits of NHL treatment.

A novel bioreactor and culture method drives high yields of platelets from stem cells.

BACKGROUND: Platelet (PLT) transfusion is the primary treatment for thrombocytopenia. PLTs are obtained exclusively from volunteer donors, and the PLT product has only a 5-day shelf life, which can limit supply and result in PLT shortages. PLTs derived from stem cells could help to fill this clinical need. However, current culture methods yield far too few PLTs for clinical application. To address this need, a defined, serum-free culture method was designed using a novel bioreactor to increase the yield of PLTs from stem cell-derived megakaryocytes. STUDY DESIGN AND METHODS: CD34 cells isolated from umbilical cord blood were expanded with a variety of reagents and on a nanofiber membrane using serum-free medium. These cells were then differentiated into megakaryocytic lineage by culturing with thrombopoietin and stem cell factor in serum-free conditions. Polyploidy was induced by addition of Rho kinase inhibitor or actin polymerization inhibitor to the CD41 cells. A novel bioreactor was developed that recapitulated aspects of the marrow vascular niche. Polyploid megakaryocytes that were subjected to flow in the bioreactor extended proPLTs and shed PLTs, as confirmed by light microscopy, fluorescence imaging, and flow cytometry. RESULTS: CD34 cells were expanded 100-fold. CD41 cells were expanded 100-fold. Up to 100 PLTs per input megakaryocyte were produced from the bioreactor, for an overall yield of 10(6) PLTs per input CD34 cell. The PLTs externalized P-selectin after activation. DISCUSSION: Functional PLTs can be produced ex vivo on a clinically relevant scale using serum-free culture conditions with a novel stepwise approach and an innovative bioreactor.

Actin inhibition increases megakaryocyte proplatelet formation through an apoptosis-dependent mechanism.

BACKGROUND: Megakaryocytes assemble and release platelets through the extension of proplatelet processes, which are cytoplasmic extensions that extrude from the megakaryocyte and form platelets at their tips. Proplatelet formation and platelet release are complex processes that require a combination of structural rearrangements. While the signals that trigger the initiation of proplatelet formation process are not completely understood, it has been shown that inhibition of cytoskeletal signaling in mature megakaryocytes induces proplatelet formation. Megakaryocyte apoptosis may also be involved in initiation of proplatelet extension, although this is controversial. This study inquires whether the proplatelet production induced by cytoskeletal signaling inhibition is dependent on activation of apoptosis. METHODS: Megakaryocytes derived from human umbilical cord blood CD34+ cells were treated with the actin polymerization inhibitor latrunculin and their ploidy and proplatelet formation were quantitated. Apoptosis activation was analyzed by flow cytometry and luminescence assays. Caspase activity was inhibited by two compounds, ZVAD and QVD. Expression levels of pro-survival and pro-apoptosis genes were measured by quantitative RT-PCR. Protein levels of Bcl-XL, Bax and Bak were measured by western blot. Cell ultrastructure was analyzed by electron microscopy. RESULTS: Actin inhibition resulted in increased ploidy and increased proplatelet formation in cultured umbilical cord blood-derived megakaryocytes. Actin inhibition activated apoptosis in the cultured cells. The effects of actin inhibition on proplatelet formation were blocked by caspase inhibition. Increased expression of both pro-apoptotic and pro-survival genes was observed. Pro-survival protein (Bcl-xL) levels were increased compared to levels of pro-apoptotic proteins Bak and Bax. Despite apoptosis being activated, the megakaryocytes underwent minimal ultrastructural changes during actin inhibition. CONCLUSIONS: We report a correlation between increased proplatelet formation and activation of apoptosis, and that the increase in proplatelet formation in response to actin inhibition is caspase dependent. These findings support a role for apoptosis in proplatelet formation in this model.

Ex vivo production of platelets from stem cells.

Stem cell technology holds great promise for transfusion medicine, and generation of platelets from stem cells would be transformative. Platelet transfusions are life saving for millions of people and the clinical demand for platelets continues to increase: there is a real need to increase the supply of platelets. Accordingly, there is great interest in the potential of producing platelets from stem cells for clinical use. There has been initial success in ex vivo generation of platelets from stem cells using cord blood stem cells, embryonic stem cells and induced pluripotent stem cells. However, the platelet yields achieved by these strategies have not been sufficient for clinical purposes. This review provides updated information about the current strategies of ex vivo generation of platelets. Megakaryocytopoiesis and platelet generation, along with the importance of genetic determinants of these processes, are reviewed in the context of efforts to generate these products from stem cells. Current challenges and rate-limiting steps in ex vivo platelet generation are discussed, together with strategies to overcome them. While much work remains, great progress has been made, moving ex vivo generation of platelets ever closer to the clinic.

Rho kinase inhibition drives megakaryocyte polyploidization and proplatelet formation through MYC and NFE2 downregulation.

The processes of megakaryocyte polyploidization and demarcation membrane system (DMS) formation are crucial for platelet production, but the mechanisms controlling these processes are not fully determined. Inhibition of Rho kinase (ROCK) signalling leads to increased polyploidization in umbilical cord blood-derived megakaryocytes. To extend these findings we determined the effect of ROCK inhibition on development of the DMS and on proplatelet formation. The underlying mechanisms were explored by analysing the effect of ROCK inhibition on the expression of MYC and NFE2, which encode two transcription factors critical for megakaryocyte development. ROCK inhibition promoted DMS formation, and increased proplatelet formation and platelet release. Rho kinase inhibition also downregulated MYC and NFE2 expression in mature megakaryocytes, and this down-regulation correlated with increased proplatelet formation. Our findings suggest a model whereby ROCK inhibition drives polyploidization, DMS growth and proplatelet formation late in megakaryocyte maturation through downregulation of MYC and NFE2 expression.

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes. STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy. RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization. CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.