scholarly journals Identification of Small Molecules That Induce Therapeutic Levels of Fetal Hemoglobin for Treatment of Sickle Cell Disease By Pairing Machine Learning with High-Resolution Single Cell RNA Sequencing Maps of Adult and Fetal Human Erythropoiesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2022-2022
Author(s):  
Mauricio Cortes ◽  
Anthony J Monti ◽  
Sunny Sun ◽  
Orna Lynch ◽  
Youli Xia ◽  
...  
Keyword(s):  
Small Molecules ◽  
Stock Options ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Cell Disease ◽  
Transcriptional Signature ◽  
Single Cell Rna Sequencing ◽  
Resolution Single ◽  
Privately Held

Abstract Induction of fetal hemoglobin (HbF) to treat sickle cell disease (SCD) and beta-thalassemia has been validated as a clinical strategy to ameliorate these diseases. However, despite advances in cell and gene therapy to treat SCD, current approaches still require myeloablation and bone marrow transplantation, which carries risk and limits treatment accessibility. Currently, hydroxy urea (HU) is the only FDA approved small molecule for SCD that acts as a moderate inducer of HbF. Thus, the ability to develop a next generation small molecule with comparable efficacy to gene therapy approaches, but without the existing limitations of HU, would have immense clinical benefit. Towards the goal of identifying novel molecules that can robustly induce HbF, we generated a high-resolution single cell RNA sequencing (scRNAseq) dataset capturing pseudo-trajectories of adult and fetal erythropoiesis. This dataset allowed us to identify cell-states, cell-features, and gene-networks associated with fetal erythropoiesis in a data driven manner. In addition, by applying proprietary algorithms to this dataset, we identified a transcriptional signature targetable with a selected number of small molecules. Evaluation of these predicted small molecules in a 14-day human in vitro erythroid differentiation assay, identified a subset that induced HbF in mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPC) from healthy donors, as measured by % F-cells (flow cytometry) and % HbF (HPLC). CLT-1081 induced HbF (42.3% +17.26, n=4) above HU (17.16% +4.78, n=5) and BCL11A CRISPR knockdown (32.58% +10.66, n=5). We then measured globin gene expression to further characterize how CLT-1081 compared to HU induced changes in fetal and adult globin expression. CLT-1081 induced robust mRNA expression of both HBG1 and HBG2 and concomitant decrease in HBB transcript relative to HU. To determine if CLT-1081 acted by inducing the targeted transcriptional signature, we performed scRNAseq on a number of known HbF inducers, including CLT-1081, HU, and BCL11A CRISPR knockdown. Analysis revealed that CLT-1081 acted as predicted by our algorithms and activated the genes identified as drivers of HbF induction in the human in vitro erythroid differentiation assay. Furthermore, comparison between BCL11A CRISPR knockdown and CLT-1081 demonstrated strong correlation in terms of transcriptional response; CLT-1081 treatment reduced BCL11A expression in addition to other transcriptional changes, which elucidated a partial mechanism of action. In sum, we have used high dimensional data and computational tools in combination with established in vitro cell based assays to identify and validate a cell behavior marked by robust HbF expression and defined by a gene signature identified by our process. These findings demonstrate the power of Cellarity's platform, which augments a systems biology approach with machine learning to identify and develop drug candidates. Disclosures Cortes: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Monti: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Sun: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Lynch: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Xia: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Lin: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Malamas: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Steelman: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Krishnamoorthy: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Stewart: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company. Tozzo: Cellarity, Inc.: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals Whole Blood Adhesion to VCAM-1 and P-Selectin and RBC Mechanical Fragility Can be Compromised in Long COVID-19 Patients with Sickle Cell Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 959-959
Author(s):  
Michael Tarasev ◽  
Marta Ferranti ◽  
Cidney Allen ◽  
Xiufeng Gao ◽  
Kayla Topping ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Whole Blood ◽  
Stock Options ◽  
Membrane Stability ◽  
Cell Disease ◽  
Adhesive Properties ◽  
Rbc Membrane ◽  
Privately Held

Abstract Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe vascular complications associated with endothelial dysfunction and systemic inflammation. COVID19-specific IgG are detectable within a week of infection. Long COVID-19 has been described in patients continuing to exhibit symptoms after the virus is no longer detectable in the respiratory secretions, including fatigue, dyspnea, headache, and brain fog. The recent FAIR Health study reviewed a total of 1,959,982 COVID-19 patients for the prevalence of long COVID symptoms and reported that 23.2% had at least one post-COVID symptom [1]. The underlying biologic mechanisms of long COVID remain unclear, thus treatments are limited to symptomatic relief and supportive care. Many long COVID symptoms are consistent with systemic inflammation and impaired oxygen delivery observed in individuals with sickle cell disease (SCD), in turn associated with elevated blood cell adhesion and decreased red blood cell (RBC) stability. The aim of this study was to determine if deleterious changes in in blood cell properties related to adhesion and membrane stability under stress can be associated with the symptoms of long COVID-19. In this work we evaluated 7 SCD patients that were diagnosed with SARS-Cov-2 and tracked their recovery using semiquantitative IgG and blood cell function assays. Methods: Blood samples were collected by the Foundation for Sickle Cell Disease (SCD) Research from SCD (homozygous SS, n=6) patients coming for regular or urgent clinic visit with SARS-CoV-2 serological and blood cell functions tests performed per the standard of care. Semiquantitative IgG assay was performed using DXi-80 (Beckman Coulter). Flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM)and P-Selectin (FA-WB-Psel) substrates were determined by counting the cells that remain adherent in a microfluidics channel after perfusion with whole blood 1:1 diluted with HBSS buffer and washed by reversed flow at 1 dyne/cm 2. Red blood cell mechanical fragility (RBC MF) was measured as hemolysis induced by an oscillating cylindrical magnet with periodic non-invasive probing of cell-free hemoglobin fraction. Six individuals with SCD recovering from SARS-Cov-2 with biomarker data available both before and for more than 3 months after the infection (179±62 days) were included in the study. Results: IgG levels varied from less than 0.1 to 37, with positive values being defined as IgG > 1. The median estimated half-life of IgG decline was 53 days ranging from 25 to 90 days (the last, for the hospitalized patient). Averaged for IgG positive (IgG+) and IgG negative (IgG-) conditions, combining pre- and post-infection IgG- conditions, values of patient hemoglobin (Hb), FA-WB-VCAM, FA-WB-Psel, and RBC MF cell properties lacked statistical significance (under both a paired t-test and population statistics). Hb levels remained essentially unchanged regardless of the time from infection or IgG status. However, FA-WB-VCAM, FA-WB-Psel, and RBC MF were all significantly elevated after SARS-Cov-2 seroconversion and remained elevated despite declining IgG levels (e.g., Fig. 1). These increases in biomarker values were statistically significant for both FA-WB-VCAM and RBC MF, and were approaching significance for FA-WB-Psel (p<0065). These increases were highly patient-specific with potential return to pe-infection values observed in some cases at about 5-6 months after the infection. A qualitative review of the medical records indicated a new subjective report of fatigue in 5 of 6 patients. Longer observations are required to determine if abnormal blood cell adhesive properties and RBC membrane instability are mechanisms of long-COVID-19 pathophysiology. Conclusions: Whole blood adhesion to both p-selectin and VCAM-1 as well as RBC membrane stability can be significantly impaired in convalescent SARS-Cov-2 patients suggesting an association with long COVID-19. New and emerging treatments that modify whole blood adhesive properties and RBC membrane stability should be investigated for their potential to accelerated recovery from long COVID-19. Health F. A Detailed Study of Patients with Long-Haul COVID: An Analysis of Private Healthcare Claims; White Paper. June 15, 2021 Disclosures Tarasev: Functional Fluidics: Current holder of stock options in a privately-held company. Ferranti: Functional Fluidics: Current holder of stock options in a privately-held company. Allen: Functional Fluidics: Current Employment. Gao: Functional Fluidics: Current Employment. Topping: Functional Fluidics: Current Employment. Ferranti: Functional Fluidics: Current Employment. Makinde-Odesola: Functional Fluidics: Other: conduct research for academic program. Hines: Functional Fluidics: Current holder of stock options in a privately-held company.

scholarly journals Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Sara El Hoss ◽  
Sylvie Cochet ◽  
Auria Godard ◽  
Hongxia Yan ◽  
Michaël Dussiot ◽  
...  
Keyword(s):  
Cell Death ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Ineffective Erythropoiesis ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
F Cells

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

Preclinical Evaluation for Engraftment of Gene-Edited CD34+ Cells with a Sickle Cell Disease Mutation in a Rhesus Transplantation Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 609-609
Author(s):  
Naoya Uchida ◽  
Linhong Li ◽  
Tina Nassehi ◽  
Morgan Yapundich ◽  
Jackson Gamer ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Genome Editing ◽  
Erythroid Differentiation ◽  
Cell Disease ◽  
Gene Correction ◽  
Guide Rna ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Before And After

Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene. State-of-the-art genome editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. We have demonstrated efficient gene correction in SCD CD34+ cells with SCD mutation-specific guide RNA, Cas9 mRNA/protein, and single strand donor DNA, resulting in ~30% gene correction and ~50% indels at the DNA level, and ~60% normal β-globin production at the protein level in in vitro erythroid differentiation (ASH 2018). Gene correction by homology directed repair is thought to be enhanced by cell proliferation; however, cell proliferation might reduce stemness of HSCs. To investigate this hypothesis, we sought to evaluate engraftment of gene-edited CD34+ HSCs in a non-human primate model. To model SCD gene correction, a β-to-βs globin conversion was designed in rhesus macaques. Mobilized rhesus CD34+ cells (n=2) were electroporated using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver rhesus β-globin-targeting guide RNA (the same target site as the SCD mutation-specific guide RNA), SpCas9 protein, and single strand donor DNA including a SCD mutation (20A>T). We also added an adjuvant to improve gene conversion efficiencies. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. We observed high-efficiency genome editing without the adjuvant (20-30% gene conversion and 61-64% indels), and further enhanced genome editing with the adjuvant (51-59% gene conversion and 36-39% indels). After erythroid differentiation, we observed production of βs-globin protein (~100%) but not normal β-globin in gene-edited cells. We then evaluated engraftment of gene-edited rhesus CD34+ cells with β-to-βs globin conversion (n=2, 13U005 and 12U011). Mobilized rhesus CD34+ cells (3.4-3.8e7) were pre-stimulated for 2 days, and edited cells were cryopreserved after electroporation with editing tools. Small aliquots of edited cells (before and after cryopreservation) were differentiated into erythroid cells in vitro, resulting in 17-26% of gene conversion and 57-71% of indels at the DNA level and 50-100% of β-globin production at the protein level, with no difference observed between aliquots taken before and after cryopreservation. Following 9.5 Gy total body irradiation, the frozen edited CD34+ cells (1.6-2.2e7) were injected into autologous macaques. We observed robust recovery of blood counts in 13U005, while peripheral blood recovery was delayed in 12U011, who was supported by serial whole blood transfusion. We observed 7-11% of gene conversion and 44-54% of indels in both granulates and lymphocytes in 13U005 1 month post-transplant. Around 15% sickle Hb production in red blood cells was detected by Hb electrophoresis in 13U005 three months post-transplant and ~7% in 12U011 two months post-transplant. Interestingly, ~10% of fetal Hb production was observed in 12U001, likely due to stress hematopoiesis. In summary, we developed a rhesus β-to-βs globin conversion model with HSC-targeted genome editing strategies. The gene-edited rhesus CD34+ cells are engraftable for at least 3 months post-transplant. Although further follow-up is necessary for transplanted animals, these findings are helpful in designing HSC-targeted gene correction trials. Figure Disclosures Li: MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Peshwa:MaxCyte, Inc: Employment.

scholarly journals Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1729-1729
Author(s):  
Luis Borges ◽  
Mark A Wallet ◽  
Chiamin-Liao Bullaughey ◽  
Michael F Naso ◽  
Buddha Gurung ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Nk Cells ◽  
Stock Options ◽  
Tumor Growth Inhibition ◽  
B Cell Malignancies ◽  
Privately Held

Abstract Induced-pluripotent stem cells (iPSCs) can be differentiated into various somatic cells, including different immune cell types. We have engineered iPSC-derived NK cells with multiple features to generate therapeutic candidates designed to eliminate cancer cells while avoiding recognition by the host immune system. The unlimited replication capacity of iPSCs facilitates the engineering of several genetic modifications without the risk of driving cells to exhaustion as in the case of cell products derived from fully differentiated immune cells. Once all edits are completed, our cells are single-cell cloned and each clone is genetically characterized to select clones without off-target insertions or deletions. Following the genetic characterization, selected clones are differentiated and tested in vitro and in vivo to identify the final clinical candidate. The use of a single-cell iPSC clone enables the generation of a master cell bank producing a highly uniform cell product that can be made available off-the-shelf at any clinical site. CNTY-101 is an iPSC-derived CAR-NK clinical candidate for the treatment of B-cell malignancies. It incorporates six gene edits designed to improve persistence and functionality as well as safety. These modifications include edits to reduce graft rejection due to alloreactivity, the expression of a homeostatic cytokine to improve functionality and persistence, the introduction of a chimeric antigen receptor (CAR) targeting CD19 to mediate tumor cell engagement and killing, as well a safety switch to eliminate the cells, if ever necessary. To prevent rejection by the patient's CD8 T cells, the beta-2-microbulin (ß2M) gene was disrupted with simultaneous insertion of a transgene encoding the HLA-E protein tethered with ß2M and a peptide. HLA-E was introduced to prevent NK cell cytotoxicity against the engineered cells, which lack HLA-I. For resistance to CD4 T cell-mediated allogenic immune rejection, the class II major histocompatibility complex transactivator (CIITA) gene was disrupted with simultaneous insertion of a transgene encoding the extra-cellular and transmembrane domains of EGFR, and the NK cell growth factor IL-15. EGFR provides an elimination tag that can be engaged by clinically approved anti-EGFR antibodies, such as cetuximab. Finally, the CAR transgene targeting the CD19 antigen was inserted into the AAVS1 safe harbor locus. Our data indicates that CNTY-101 iNK cells have strong antitumor activity against lymphoma cell lines both in vitro and in vivo. In vitro, CNTY-101 eliminates lymphoma cell lines through multiple rounds of killing without reaching exhaustion. Clones expressing higher levels of IL-15 tend to have better persistence and functionality, with some clones showing robust cytotoxicity for over fifteen rounds of serial killing. In vivo, the clones that demonstrated better in vitro serial killing tend to mediate the best anti-tumor activity in lymphoma xenograft models. Upon 3 weekly doses, the most active candidate clone demonstrated significant tumor growth inhibition after administration of fresh (91 % tumor growth inhibition) or cryopreserved cells (76 % tumor growth inhibition). The efficacy of the EGFR-safety switch was also investigated both in vitro and in vivo. In vitro, addition of cetuximab to co-cultures of IL-2-activated PBMC and cells mediated antibody-dependent cellular cytotoxicity (ADCC) in a concentration-dependent fashion, with an EC50 of 2 ng/ml. In vivo, there was a 96% reduction in the number of iPSC-derived CAR-NK cells in the lungs and a 95% reduction in the number of CAR-NK cells in the blood of mice that received cetuximab versus PBS-treated mice. In summary, CNTY-101 is a novel, multi-engineered, allogeneic CAR-iNK product candidate for the treatment of B-cell malignancies. It includes multiple immune evasion features to prevent recognition by the patient's immune system and expression of IL-15 to facilitate persistence and functionality. We have initiated GMP manufacturing of CNTY-101 and plan to enter clinical trials in 2022. Disclosures Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bullaughey: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Naso: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Gurung: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Keating: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Carton: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Wheeler: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Campion: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Jessup: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Chin: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals Development of Chimeric Antigen Receptor-Expressing iPSC-Derived Macrophages with Improved Anti-Tumor Activity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1693-1693
Author(s):  
Somayeh Pouyanfard ◽  
Manuel Fierro ◽  
Dan S Kaufman
Keyword(s):  
Ovarian Cancer ◽  
Stem Cells ◽  
Tumor Cells ◽  
Pluripotent Stem Cells ◽  
Stock Options ◽  
Ovarian Cancer Cells ◽  
Anti Tumor Activity ◽  
Privately Held

Abstract Previous studies by our group demonstrate the ability to routinely derive hematopoietic and immune cells from human pluripotent stem cells. Here, we demonstrate the efficient derivation of macrophages from human induced pluripotent stem cells (iPSCs). These macrophages have phenotypic and genotypic characteristics similar to monocytes/macrophages isolated from human peripheral blood. We also demonstrate the ability to polarize these iPSC-derived macrophages (iPSC-Macs) to M1 and M2 populations. Specifically, M1 iPSC-Macs have pro-inflammatory characteristics including expression of CD40 and CD80 on the cell surface, produce increased amounts of TNF-a and IL-6 detected in the supernatant, as well have increased expression of inflammatory cytokines/chemokines (TNF-a, IL-6, IL-1b, IL-12, CCL2, CCL3 and TRAIL) and increased expression of matrix metalloproteases (MMPs). Function of these iPSC-Macs was initially assessed by phagocytosis of fluorescently-labeled beads. These studies demonstrated both the iPSC-M1 and M2 macrophages efficiently phagocytized these beads, and at similar amounts as their peripheral blood counterparts. Next, we tested the ability of the iPSC-Macs to phagocytize human tumor cells. Using A1847 ovarian tumor cells, we found while the iPSC-Macs alone had limited ability to phagocytize the tumor cells (9%), addition of either an anti-CD47 mAb (41%) or anti-EGFR (41%) lead to markedly increased phagocytosis, with the combination of the 2 antibodies being even better (55% phagocytosis). We then tested iPSC-Macs in vivo against luciferase (luc)-expressing A1847 ovarian cancer cells as a xenograft model in NSG-SGM3 mice that express human IL3, GM-CSF and SCF. Using bioluminescent imaging, we found that the combination of iPSC-Macs with both anti-CD47 and anti-EGFR demonstrated significantly improved anti-tumor activity, with median survival of 75 days, compared to 50-60 days for mice treated with only iPSC-Macs, only mAbs or with iPSC-Macs combined either single mAb. Next, we aimed to use the iPSC platform to produce iPSC-Macs engineered to express chimeric antigen receptors (CARs) to further improve their anti-tumor activity. Here, we developed and tested novel macrophage specific CARs that were stably expressed in undifferentiated iPSCs using transposon-mediated gene transfer, similar to our previous studies to derive iPSC-derived CAR-expressing NK cells that have now been translated into clinical trials. We used an anti-mesothelin (meso) scFv combined with 8 different CAR constructs with distinct intracellular signaling components. We found that the iPSC-Macs could express good levels of the CARs (iPSC-CarMacs). Function was again tested in vitro by phagocytosis of the Meso+ A1847 ovarian cancer cells. The iPSC-CarMacs with a Bai1 stimulatory domain consistently demonstrated the best activity in this assay system. We next tested the anti-meso-iPSC-CarMacs in vivo using the A1847 cells. Again, we demonstrate the iPSC-CarMacs combined with anti-CD47 mAb mediate significantly improved anti-tumor activity using this in vivo model compared to the non-CAR-iPSC-Macs + anti-CD47, p <0.005 (Figure). Survival studies are still ongoing. Together, these studies demonstrate that iPSCs can be used to routinely and efficiently derive macrophages with potent anti-tumor activity. Additionally, CARs that are optimized for macrophage-mediated activity can be expressed to generate iPSC-CarMacs that effectively kill tumor cells in vitro and in vivo. These iPSC-CarMacs provide another approach to provide a standardized, targeted, off-the-shelf cell therapy product that can be used to treat both hematological malignancies as well as diverse solid tumors. Figure 1 Figure 1. Disclosures Kaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

Increased GM-CSF Levels in Sickle Cell Disease Are Associated with Increased Leukocyte Counts and Are Reversed by Hydroxyurea.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3573-3573
Author(s):  
Nicola Conran ◽  
Tohru Ikuta ◽  
Sara T.O. Saad ◽  
Fernando F. Costa
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Gm Csf ◽  
Cell Counts ◽  
Ion Exchange Hplc ◽  
Csf Levels ◽  
White Blood Cell Counts

Abstract The hematopoietic cytokine, GM-CSF, stimulates the proliferation and differentiation of progenitor cells to macrophages, eosinophils and megakaryocytes. Increased levels of GM-CSF have previously been reported in sickle cell disease (SCD) patients with low fetal hemoglobin (HbF) levels (Croizat, Br J Haematol. 87:592; 1994); however, a clearcut association between GM-CSF and HbF levels in SCD has yet to be established, since there is some evidence that levels may depend on other factors (Haider et al., Acta Haematol. 102:140; 1999). To further investigate the possible role of GM-CSF in SCD, plasma samples were collected from normal individuals (AA), steady-state sickle cell anemia patients (SS) and sickle cell patients on hydroxyurea therapy (SSHU; 20-30 mg/kg/day). Plasma GM-CSF levels were measured using a specific immunoenzymatic assay. HbF levels were quantified by ion-exchange HPLC and hematological measurements obtained using an Advia Hematology System. Plasma GM-CSF levels seem to be higher in SS patients than in normal controls (0.828 ± 0.215 pg/ml, n=39; 0.230 ± 0.081 pg/ml, n=9; respectively). In contrast, GM-CSF was significantly lower in SSHU patients than in SS without HU (0.288 ± 0.104 pg/ml; n=14; P = 0.045 compared to SS). A negative correlation between plasma GM-CSF and HbF levels was seen in patients (r = −0.332; P = 0.026; n=45; SS and SSHU groups) and, notably, a positive correlation between GM-CSF and white blood cell counts was observed (r = 0.302; P = 0.037; n= 48; SS and SSHU groups). Furthermore, GM-CSF (2.5 ng/ml) abolished the production of γ globin during erythropoietin-stimulated differentiation of TF-1 hematopoietic cells. These results provide further support to the hypothesis that plasma GM-CSF correlates negatively with HbF in sickle cell disease. Interestingly, in vitro data suggest that increased GM-CSF may, in fact, diminish HbF levels rather than reflect increased haematopoietic stress as a consequence of low HbF. Importantly, GM-CSF appears to correlate with leukocyte numbers in SCD, and levels were significantly decreased in patients taking HU, indicating that this cytokine may contribute to the leukocytosis seen in some SCD patients and may play a role in leukocyte count reduction by HU.

scholarly journals Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 411-420 ◽  
Author(s):  
Rodwell Mabaera ◽  
Michael R. Greene ◽  
Christine A. Richardson ◽  
Sarah J. Conine ◽  
Courtney D. Kozul ◽  
...  
Keyword(s):  
Posttranscriptional Regulation ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Dna Demethylation ◽  
Globin Genes ◽  
Dna Hypomethylation ◽  
Globin Mrna ◽  
Potent Inducer ◽  
Maximal Induction

5-azacytidine (5-Aza) is a potent inducer of fetal hemoglobin (HbF) in people with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first is based on the drug's ability to inhibit global DNA methylation, including the fetal globin genes, resulting in their activation. The second is based on 5-Aza's cytotoxicity and observations that HbF production is enhanced during marrow recovery. We tested these models using human primary cells in an in vitro erythroid differentiation system. We found that doses of 5-Aza that produce near maximal induction of γ-globin mRNA and HbF do not alter cell growth, differentiation kinetics, or cell cycle, but do cause a localized demethylation of the γ promoter. However, when we reduced γ promoter methylation to levels equivalent to those seen with 5-Aza or to the lower levels seen in primary fetal erythroid cells using DNMT1 siRNA and shRNA, we observed no induction of γ-globin mRNA or HbF. These results suggest that 5-Aza induction of HbF is not the result of global DNA demethylation or of changes in differentiation kinetics, but involves an alternative, previously unrecognized mechanism. Other results suggest that posttranscriptional regulation plays an important role in the 5-Aza response.

Fetal Hemoglobin Expression Is Differentially Affected by Inhibition of the Proposed Dred Complex Constituents, LSD1 and DNMT1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3263-3263 ◽  
Author(s):  
Tara L Arvedson ◽  
Lynn Tran ◽  
Sandra L Ross ◽  
Sean Yoder ◽  
Alexandra Hertz ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Beta Thalassemia ◽  
Cell Disease ◽  
Hematopoietic Progenitor ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Effect Of Treatment ◽  
Globin Promoter

Abstract Abstract 3263 Introduction Sickle cell disease and beta thalassemia are disorders caused by mutations in adult hemoglobin (HbA) or defects in HbA expression. A potential therapeutic solution is reactivation of fetal hemoglobin (HbF) expression. Although HbF, comprising two alpha and two gamma globin chains, is the primary form of hemoglobin expressed in utero, gamma globin expression is silenced in adults. One proposed mechanism of gamma globin silencing involves binding of the direct repeat erythroid definitive (DRED) repressor complex to sequences in the gamma globin promoter. The DRED complex is reported to include the orphan nuclear hormone receptors TR2 and TR4, lysine specific demethylase (LSD1) and DNA methyltransferase (DNMT1). As both LSD1 and DNMT1 are epigenetic modifiers, gamma globin repression is proposed to be mediated by LSD1- and DNMT1-induced epigenetic changes. To investigate the role of DNMT1 and LSD1 in HbF silencing, HbF expression was evaluated in an erythroid differentiation model where hematopoietic progenitor cells were treated with either DNMT1 or LSD1 small molecule inhibitors or siRNA. Methods Human hematopoietic progenitor cells from healthy donors were induced to become erythroid using a two step protocol including erythropoietin, SCF, IL-3 and hydrocortisone for days 1–7 and erythropoietin and SCF for days 8–14. Cultures were treated with a range of concentrations of either tranylcypromine or S2101 (LSD1 inhibitors) or 5-azacytidine (DNMT1 inhibitor) and compared to HbF-inducing, positive control small molecules pomalidomide and lenalidomide. Cultures were also treated with LSD1 siRNAs and compared to controls. The effect of treatment on gamma, beta and alpha globin transcription was determined by qRT-PCR. The effect of treatment on HbA and HbF levels was determined by ELISA, HPLC, flow cytometry and imaging. Differentiation was characterized by morphology and flow-based detection of CD34 and glycophorin. Effects on viability were characterized by ViCell and flow cytometry. Results Treatment with a concentration range of 5-azacytidine increased the rate of red blood cell differentiation as measured by daily changes in CD34 and glycophorin and hemoglobinization. Quantitative ELISA demonstrated that HbF expression increased two-fold. In contrast, LSD1 inhibition reduced both the rate of proliferation and differentiation of erythroid progenitors. Consistent with impaired differentiation, both beta globin transcription and HbA expression were reduced by up to 84% (qRT-PCR) and 65% (quantitative ELISA), respectively. No increase in gamma globin transcription or HbF expression was observed in response to LSD1 inhibition. Control cultures differentiated as expected: after 14 days of treatment the majority of vehicle-, lenalidomide- or pomalidomide-treated cells were glycophorin-positive and enucleation was readily apparent. Both lenalidomide and pomalidomide treatment induced a two-fold increase in HbF expression, as previously reported. Conclusions Although both LSD1 and DNMT1 are reported to be components of the DRED complex and are proposed to be jointly responsible for epigenetically modifying the gamma globin promoter to silence HbF expression, inhibition of the two proteins had different outcomes on HbF expression. DNMT1 inhibition upregulated HbF expression to a similar extent as pomalidomide (currently in Phase 1 clinical trials for HbF induction), whereas LSD1 inhibition impaired erythroid differentiation and hemoglobinization. These results suggest that the mechanism of gamma globin silencing and the proposed role of the DRED complex require further evaluation. Furthermore, this work also suggests that LSD1 inhibition is not a therapeutic strategy for HbF induction in patients with sickle cell disease or beta thalassemia. Disclosures: Arvedson: Amgen: Employment. Tran:Amgen: Employment. Ross:Amgen: Employment. Yoder:Amgen: Employment. Hertz:Amgen: Employment. Hale:Amgen: Employment. Eschelbach:Amgen: Employment. Dineen:Amgen: Employment. Matyas:Amgen: Employment. Hartley:Amgen: Employment. Morgenstern:Amgen: Employment. Winters:Amgen: Employment. Cindy:Amgen: Employment. Molineux:Amgen: Employment. Coxon:Amgen: Employment.

scholarly journals Anti-FLT3 CAR T Cells in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1703-1703
Author(s):  
Sara Sleiman ◽  
Olga Shestova ◽  
Francisco Santiago ◽  
Elina Shrestha ◽  
Raymond Liang ◽  
...  
Keyword(s):  
T Cells ◽  
Stock Options ◽  
Research Funding ◽  
Short Term ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T ◽  
Privately Held

Abstract INTRODUCTION In patients with AML who are eligible for intensive therapy, the goal of treatment is the achievement of complete response followed by consolidation chemotherapy (in favorable risk disease) or hematopoietic stem cell transplantation (in intermediate or adverse risk disease). Patients who do not attain this initial goal lack effective therapeutic options. Extensive experience with chimeric antigen receptor (CAR) T cells in B-ALL has shown that CART cells can deliver potent and durable antigen-specific leukemia control, and that targeting a single antigen (CD19 for B-ALL) is associated with antigen-negative relapse. In this context, we sought to expand the existing preclinical CART armamentarium in AML by developing FLT3-specific CART cells and comparing them to our existing gold standard CD123-specific CART cells. Since activating mutations in FLT3 occur commonly in AML, we reasoned that this molecule would serve as an "Achilles heel" in AML immunotherapy. METHODS Novel fully humanized anti-human FLT3 receptor single chain variable fragments (scFV) were fused to CD28 and CD137 (41BB) costimulatory molecules and the CD3zeta signaling domain and cloned into a lentiviral expression vector. Based on recently published data, we tested linker lengths ranging from 5 to 20 amino acids between the light and heavy chains of the CAR. We used a FLT3-ITD mutated AML cell line (MOLM14) expressing luciferase for in vitro function studies including an exhaustion assay. For in vivo function studies, we engrafted MOLM14 expressing luciferase into NSG mice and treated with CART-FLT3 or untransduced T cells (negative control). RESULTS All FLT3 and CD123-specific CART cells degranulated and produced the effector cytokines IL-2, INFg, TNF and GM-CSF in an antigen-specific manner, with some variability between the different linker lengths and with some superiority of the CAR123 likely resulting from the higher expression of CD123 compared with FLT3 in this model (p < 0.0001, one way ANOVA) (Figure 1). Short-term killing assays (24 hours) revealed that all CART cells killed MOLM14 with equivalent efficiency at low effector:target ratios (Figure 2A). Since short-term killing assays likely do not replicate the physiological situation in vivo wherein CART cells encounter cancer cells repeatedly over many days, we next developed an in vitro exhaustion assay. We incubated MOLM14 cells with CAR T cells at 1:10 E:T ratio and added MOLM 14 tumor cells along with fresh media every other day. Killing was quantified every 48 hours. Interestingly, all CAR constructs showed equivalently efficient cytotoxicity from days 5-15. However, after day 15 there was progressive dysfunction and loss of cytotoxic activity. This exhaustion "stress test" revealed some superiority of the FLT3 CAR 10AA construct (p = 0.042 on day 17, two way ANOVA) (Figure 2B). NOD/SCID gamma chain KO (NSG) mice were then engrafted with 1x10 6 luciferized MOLM14 cells and treated with 0.5x10 6 CAR T cells 7 days later, randomized to treatment groups based on tumor burden. CAR T cells expansion was monitored in peripheral blood by flow cytometry. (Fig 3A). Serial BLI revealed prompt and durable leukemia remissions and survival (Figure 3B,C). CONCLUSIONS We have developed CART-FLT3 for AML using novel human anti-FLT3 targeting domains and demonstrated preclinical efficacy similar to that of CART-123 in an AML model with substantially lower expression of FLT3 compared to CD123 (data not shown). Since inhibition of FLT3 leads to upregulation of surface FLT3 expression, future experiments will explore combinatorial FLT3 inhibition with CART-FLT3. If successful, these experiments will provide a strong rationale for a combination clinical trial in AML where leukemia control by small molecules is followed by a coup-de-grace delivered by CART cells. Figure 1 Figure 1. Disclosures Sleiman: Hemogenyx Pharmaceuticals LLC: Research Funding. Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Santiago: Hemogenyx Pharmaceuticals LLC: Research Funding. Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang: Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda: Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler: Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

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