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The recent success of the commercialization of CAR-T and other immune effector cells has led to the rapid expansion of clinical trials using cellular therapy products. The expansion of both investigational and commercially available cell therapies has been driven largely by products that are manufactured outside the point-of-care medical center by industry partners or other third parties. The delivery of externally manufactured products to patients requires a coordinated effort with the medical center, as it may be responsible for collection/processing of starting material, shipping, receipt, storage and release for administration of the drug product. As medical centers are grappling with increasing demands for supporting externally manufactured products, they have been forced to modify their processes to handle this demand in reactive rather than proactive fashion. The cell processing facility (CPF) plays a critical role to ensure proper handling and safety of the product as it is transported from the medical center to the manufacturer and back to the patient for infusion. In this mini-series, we have invited several CPFs from medical centers to share their experience, including how they have implemented processes and procedures to successfully ensure product integrity as they cooperate with industry/third parties to deliver novel cell therapy products. For the purpose of this mini-series, we focus on externally manufactured products that fall into the category of biologics that comprise human cells, tissues or cellular and tissue-based products (HCT/Ps) and are regulated under section 351 of the Food and Drug Administration's Public Health Service Act, or advanced therapy medicinal products (ATMPs) governed by regulatory bodies such as the European Medicines Agency. The goal of this collection of articles is to engage professionals in the discussion about issues related to externally manufactured products and, together, define best practices and potential standards for the cell therapy community to streamline the safety and delivery of externally manufactured products to our patients.

There is considerable interest in the next generation of personalized medicine, especially cell and gene therapy products such as chimeric antigen receptor T cells (CAR-Ts). Unlike other small molecules or pharmacologic drugs, most existing cell or cell-based gene therapy products (CGTs) require apheresis collection of the patient or donor, subsequent manufacture of the product, and final shipment of the product to the clinical site for infusion. Whereas traditional pharmaceutical drugs have involved the drug sponsor and the clinical site and clinical pharmacy, this new manufacturing paradigm has evolved, in many cases, to include an apheresis center, a cell processing lab, the sponsor's manufacturing facility, and a clinical site with or without a pharmacy. Here we report the results of a survey of current practices handling investigational CGTs conducted by the Immuno-Gene Therapy committee of the International Society of Cell and Gene Therapy.

Background & Aim Genetically-modified cellular immunotherapies, including chimeric antigen receptor T-cells (CAR-T) and natural killer cells (CAR-NK), have generated significant clinical and commercial outcomes due to their unparalleled response rates against cancer. However, the development and manufacture of these advanced therapies face a number of translational bottlenecks that must be addressed to ensure long-term commercial viability. Methods, Results & Conclusion A comprehensive analysis of the current clinical pipeline of genetically-modified cellular immunotherapies will be presented to demonstrate the implications of current manufacturing bottlenecks. This analysis will give a deeper understanding into future manufacturing process needs by highlighting clinical pipeline trends such as, autologous v. allogeneic or immune cell type utilized for therapy. The cost and variability associated with these personalized advanced therapies presents a critical manufacturing and translational challenge. The current clinical pipeline in this field shows the imminent need for manufacturing optimization. However, current manufacturing protocols vary widely, including media used, cytokine concentrations, and starting cell densities. This study will highlight the importance of determining critical manufacturing process parameters. The patient-specific nature of the CAR-T therapies currently on the market is a particular challenge for the field, therefore as much of the process must be controlled in order to decrease overall process variability.

Aim: The first review of advanced cell therapy trials with perinatal cells. Materials & methods: We compiled 281 clinical trials of advanced cell therapy with perinatal cells that were registered in 2005–2015. Results: The most common cell source in these trials is cord blood, but the cell type that provides the mechanism of action in the majority of trials is mesenchymal stem/stromal cells. We analyze trends among the 15 parameters we compiled for these trials. Conclusion: Advanced cell therapy with perinatal cells is a new field that covers a wide range of diagnoses but where most of the trials are early Phase. Researchers in different countries tend to work with a preferred cell source and cell type.

Cell-mediated cytotoxicity is a critical function of the immune system in mounting defense against pathogens and cancers. Current methods that allow direct evaluation of cell-mediated cytotoxicity suffer from a wide-range of drawbacks. Here, we present a novel strategy to measure cytotoxicity that is direct, sensitive, rapid, and highly adaptable. Moreover, it allows accurate measurement of viability of both target and effector cells. Target cells are fluorescently labeled with a non-toxic, cell-permeable dye that covalently binds to cell proteins, including nuclear proteins. The labeled target cells are incubated with effector cells to begin killing. Following the killing reaction, the cell mixture is incubated with another dye that specifically stains proteins of dead cells, including nuclear proteins. In the final step, cell nuclei are released by Triton X-100, and analyzed by flow cytometry. This results in four nuclear staining patterns that separate target and effector nuclei as well as nuclei of live and dead cells. Analyzing nuclei, instead of cells, greatly reduces flow cytometry errors caused by the presence of target-effector cell aggregates. Target killing time can often be reduced to 2 h and the assay can be done in a high throughput format. We have successfully validated this assay in a variety of cytotoxicity scenarios including those mediated by NK-92 cells, Chimeric Antigen Receptor (CAR)-T cells, and Tumor Infiltrating Lymphocytes (TIL). Therefore, this technique is broadly applicable, highly sensitive and easily administered, making it a powerful tool to assess immunotherapy-based, cell-mediated cytotoxicity.

MicroRNAs are small noncoding RNAs that regulate cellular development by interfering with mRNA stability and translation. We examined global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were up-regulated and 81 were down-regulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were up-regulated most strongly in murine megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly up-regulated during mouse and human megakaryopoiesis but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR-146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing inflammatory cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously.

Abstract 702 Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. Transcription factors GATA-1 and GATA-2 co-operate with the cofactor FOG-1 to promote erythroid and megakaryocyte differentiation. In contrast, FOG-1 is inhibitory to the function of GATA factors in the context of mast cells. The mechanisms by which FOG-1 functions distinctly as co-activator and co-repressor are incompletely defined. Purification of FOG-1-associated proteins led to the identification of the nucleosome remodeling and histone deacetylase NuRD complex (Hong, et al., EMBO J. 2005). To investigate the in vivo function of this interaction, we generated mice bearing point mutations in FOG-1 that disrupt NuRD binding. The phenotype of homozygous (Fog1ki/ki) mice revealed an essential role of NuRD for normal FOG-1 function in a broad range of cell types, including erythroid cells and megakaryocytes. Here we report that defects in hematopoietic development arise as early as the bipotent megakaryocyte-erythroid progenitor in adult mice. Strikingly, Fog1ki/ki megakaryocyte-erythroid progenitors (MEP) retained multi-lineage capacity, capable of generating mast cells and other myeloid lineages. We did not observe an increase in mast cell potential in E10.5 yolk sacs, as has been previously reported in Fog1null embryos, indicating a stage-specific role for FOG-1's interaction with NuRD. Genome wide transcriptome analysis of purified Fog1ki/ki MEP revealed inappropriate expression of several mast cell-specific genes including Fcer1a, Fcer1b, Mcpt2, and Cpa3. Notably, aberrant mast cell gene expression persisted in committed Fog1ki/ki megakaryocyte and erythroid cells. Thus FOG-1 and NuRD are essential to constrain mast cell gene expression throughout erythroid and megakaryocyte ontogeny. A direct mechanism of repression is suggested as select mast cell genes are occupied by GATA-1 and FOG-1 in erythroid precursor cells. Furthermore, a mutant form of GATA-1 that is impaired for FOG-1 binding (GATA-1-V205M) failed to repress mast cell gene expression. Together, these observations suggest that a simple heritable epigenetic silencing mechanism is insufficient to permanently repress mast cell genes. Instead, the continuous presence of GATA-1, FOG-1 and NuRD is required to maintain lineage fidelity throughout megakaryocyte-erythroid ontogeny. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic stem cell (HSC) homeostasis depends on the balance between self renewal and lineage commitment, but what regulates this decision is not well understood. Using loss-of-function approaches in mice, we found that glycogen synthase kinase-3 (Gsk3) plays a pivotal role in controlling the decision between self renewal and differentiation of HSCs. Disruption of Gsk3 in BM transiently expanded phenotypic HSCs in a betta-catenin-dependent manner, consistent with a role for Wnt signaling in HSC homeostasis. However, in assays of long-term HSC function, disruption of Gsk3 progressively depleted HSCs through activation of mammalian target of rapamycin (mTOR). This long-term HSC depletion was prevented by mTOR inhibition and exacerbated by betta-catenin knockout. Thus, GSK-3 regulated both Wnt and mTOR signaling in mouse HSCs, with these pathways promoting HSC self renewal and lineage commitment, respectively, such that inhibition of Gsk3 in the presence of rapamycin expanded the HSC pool in vivo. These findings identify unexpected functions for GSK-3 in mouse HSC homeostasis, suggest a therapeutic approach to expand HSCs in vivo using currently available medications that target GSK-3 and mTOR, and provide a compelling explanation for the clinically prevalent hematopoietic effects observed in individuals prescribed the GSK-3 inhibitor lithium.

Advanced atherosclerotic lesions are characterized by lipid accumulation, chronic inflammation, and defective efferocytosis; an ideal therapy should address all aspects of this multi-factorial disease. Ixmyelocel-T, an expanded autologous multicellular therapy showing clinical promise in the treatment of severe diseases associated with atherosclerosis, includes a novel population of M2-like macrophages. We examined the properties of these macrophages to determine their potential mechanisms of action in atherosclerotic states. Flow cytometry revealed that ixmyelocel-T macrophages express surface receptors of alternative activation (CD206 and CD163) and scavenger receptors involved in lipid and apoptotic cell uptake (CD36, LDLR, CD91, MSR1, and MerTK). ELISA analysis revealed ixmyelocel-T macrophages secrete substantial levels of IL-10 (1060 278 vs. 1978 313 pg/ml, p < 0.05) and IL-1ra (12673 1242 vs. 37576 3650 pg/ml, p < 0.001) before and after overnight LPS stimulation. Baseline secretion of the pro-inflammatory cytokines IL-1b, TNFa, and IL-12 is minimal and remains low after LPS stimulation. Fluorescent microscopy and flow cytometry revealed that ixmyelocel-T readily phagocytosed apoptotic cells. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T isn’t sensitive to the lipotoxic effects of modified cholesterol (87 10.78 vs. 100 14.58% of control). Real time PCR revealed that the cholesterol transporter genes ABAC1 (1.49 0.32 vs. 0.42 0.11 fold change, p < 0.01) and ABCG1 (0.41 0.13 vs. 0.07 0.03 fold change, p < 0.05) are both significantly upregulated when ixmyelocel-T macrophages are loaded with cholesterol. Ixmyelocel-T also exhibits enhanced ApoAI mediated cholesterol efflux (13.91 1.90 vs. 3.42 0.31%, p < 0.001 compared to THP-1). In addition, ixmyelocel-T effluxed cholesterol in vivo in using a mouse model for reverse cholesterol transport. These biological properties of ixmyelocel-T macrophages suggest that this cellular therapy may exert beneficial effects on atherosclerotic disease by providing macrophages that potentially limit atheroma development through secretion of anti-inflammatory cytokines, removal of apoptotic cells, and promotion of cholesterol efflux.

Abstract 86 Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis and its dysregulation can lead to hematologic malignancies. Recently activating mutations in JAK2 were found in a large fraction of patients with myeloproliferative neoplasms (MPNs). We previously demonstrated that lymphocyte adaptor protein (Lnk) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion (Bersenev et al., JCI, 2008;118:2832-2844). We further showed that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPNs in mice (Bersenev et al., JCI, 2010;120:2058-2069). Lnk directly inhibits oncogenic JAK2 as well as acting through JAK2- independent pathways to constrain MPN development. Consistently, aged Lnk–/– mice spontaneously developed a CML-like MPN (Bersenev et al., JCI, 2010;120:2058-2069). More importantly, loss-of-function mutations in Lnk are found in human MPN patients (Oh, et. al., Blood, 2010, in press). This work suggests that Lnk plays a pivotal role in regulating both normal and malignant HSPC expansion. However, it remains to be determined how Lnk attenuates JAK2 activity since Lnk itself does not possess any enzymatic activity. Therefore, we began exploring Lnk regulatory mechanisms by identifying novel Lnk partners through protein purification and mass spectrometric analysis. This resulted in the identification of a number of novel Lnk binding proteins, which include the 14-3-3 proteins as the most robust interactors. 14-3-3s are a group of scaffold proteins that regulate many disease-relevant gene products and play important functions in many aspects of cellular processes. We found that Lnk is phosphorylated at two serine residues, which serve as the critical binding sites for 14-3-3. 14-3-3 abrogates the Lnk-JAK2 interaction thereby alleviating Lnk inhibitory function in both JAK2 signaling and cell growth. Furthermore, 14-3-3 binding is necessary and sufficient to maintain Lnk in an inactive state. We also investigated the signals that regulate Lnk phosphorylation, revealing Lnk as a signaling nodal point that integrates multiple signaling pathways in controlling HSPC homeostasis. The physiological significance of the Lnk-14-3-3 interaction in HSPC development will be discussed. Thus, our data implicate previously unappreciated serine phosphorylation events in Lnk-dependent hematopoietic function and regulation of JAK2. In addition, identification of novel signaling molecules that influence hematopoiesis might facilitate stem cell therapies and provide novel therapeutic targets for the treatment of MPNs. Disclosures: No relevant conflicts of interest to declare.

Abstract 1437 Poster Board I-460 Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2), which in turn triggers a cascade of downstream signal transduction pathways that regulate key aspects of cell development. Mice that lack the inhibitory adaptor protein Lnk harbor a vastly expanded HSC pool with enhanced self-renewal. We previously demonstrated that Lnk controls HSC self-renewal predominantly through the Mpl/JAK2 pathway. Lnk binds directly to phosphorylated tyrosine 813 in JAK2 upon Tpo stimulation. Moreover, Lnk-deficient HSCs display potentiated JAK2 activation. Dysregulation of cytokine receptor signaling pathways frequently lead to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML) in human patients. Moreover, the JAK2 V617F mutation has been observed at high frequency in several myeloproliferative diseases (MPDs). The JAK2V617F retains Lnk binding, suggesting that alterations in Lnk could influence MPD development. Indeed, we found that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPD in mouse transplant models. Specifically, Lnk deficiency enhanced cytokine signaling, thereby augmenting the ability of oncogenic JAK2 to expand myeloid progenitors. To test whether the interaction between Lnk and JAK2V617F directly constrains MPD development in mice, we transplanted wild-type bone marrow cells expressing the JAK2V617F/Y813F double mutant that does not interact with Lnk (WT;JAK2VF/YF). WT;JAK2VF/YF engrafted mice exhibited increased myeloid expansion when compared to WT;JAK2VF mice, and conferred accelerated polycythemia vera development in secondary transplants. In summary, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may constrain leukemic transformation conferred by oncogenic JAK2. Disclosures No relevant conflicts of interest to declare.

Tissue-specific nuclear factors can establish gene expression patterns in one cell lineage and suppress that of another. GATA-1 and its cofactor FOG-1 (Zfpm1) regulate erythroid and megakaryocyte development by activating and repressing gene transcription. We previously showed that a conserved motif within the N-terminus of FOG-1 binds the Nucleosome Remodeling and Deacetylase (NuRD) co-repressor complex. Here we report that mice bearing FOG-1 point mutations that disrupt the NuRD interaction display mild anemia with splenomegaly and macrothrombocytopenia, a phenotype reminiscent of that observed in animals bearing germline mutations that disrupt the GATA-1/FOG-1 interaction. Microarray studies revealed relatively few changes in gene expression pattern sin mutant erythroid cells and megakaryocytes. Among the most prominent findings was a marked increase in the levels of Gata2, which is normally silenced in mature erythroid cells. Strikingly, mutant erythroid cells also displayed activation of several genes of the mast cell lineage where FOG-1 is normally extinguished. Furthermore, mutant megakaryocytes misexpressed the same set of mast cell genes, suggesting that NuRD binding by FOG-1 is required to suppress mast cell fate throughout the erythro-megakaryocytic ontogeny. In agreement, prospectively isolated megakaryocytic-erythroid progenitors (MEP) not only exhibited elevated Gata2 and mast cell gene expression, but maintained a multilineage capacity, generating both mast cells and other myeloid lineage cells in culture. Upregulation of mast cell-specific genes is likely the combined consequence of the failure of mutant FOG-1 to function as a repressor and the high levels of GATA-2. Together, these results underscore the importance of the FOG-1-NuRD interaction as an effector of GATA-1 activity. In particular, recruitment of NuRD to GATA-1/FOG-1 regulated genes is required to optimize erythroid and megakaryocytic maturation and restrict a mast cell program in those lineages. More generally, recruitment of NuRD by lineage-specific transcription factors may be a common mechanism to narrow and focus gene expression during tissue maturation.

Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.