SummaryCell banking, disease modeling, and cell therapy applications have placed increasing demands on hiPSC technology. Specifically, the high-throughput derivation of footprint-free hiPSCs and their expansion in systems that allow scaled production remains technically challenging. Here, we describe a platform for the rapid, parallel generation, selection, and expansion of hiPSCs using small molecule pathway inhibitors in stage-specific media compositions. The platform supported efficient and expedited episomal reprogramming using just OCT4/SOX2/SV40LT combination (0.5%–4.0%, between days 12 and 16) in a completely feeder-free environment. The resulting hiPSCs are transgene-free, readily cultured, and expanded as single cells while maintaining a homogeneous and genomically stable pluripotent population. hiPSCs generated or maintained in the media compositions described exhibit properties associated with the ground state of pluripotency. The simplicity and robustness of the system allow for the high-throughput generation and rapid expansion of a uniform hiPSC product that is applicable to industrial and clinical-grade use.
Umbilical cord blood (UCB) offers many potential advantages as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation, including ease of collection, rapid availability, flexibility of HLA-matching, lower rates of GvHD and potentially lower relapse rates. However, the low HSC content of UCB compared to other graft sources results in a prolonged time to engraftment, and higher rates of graft failure and early mortality. Pulse ex vivo exposure of HSCs to 16,16-dimethyl PGE2 (FT1050) has been demonstrated to enhance HSC engraftment potential, which could benefit clinical UCB transplant. FT1050 modulation promotes multiple mechanisms, including increased proliferation, reduced apoptosis, and improved migration and homing [North 2007&2009; Hoggatt 2009]. Improved HSC homing is mediated by induction of CXCR4 gene expression leading to increased cell surface CXCR4. Further optimization of the UCB modulation process demonstrated that incubation with 10µM FT1050 for 2 hrs at 37C resulted in a maximal biological response of the FT1050-UCB (ProHema®). A Phase 1 trial was performed to evaluate the safety of FT1050-UCB paired with an unmanipulated UCB unit in reduced-intensity double UCBT (dUCBT) [Cutler 2013]. We observed durable, multi-lineage engraftment of FT1050-UCB with acceptable safety. Earlier neutrophil engraftment was observed relative to historical controls (median 17.5 vs. 21 days (historical control), p=0.045), coupled with preferential engraftment of the FT1050-UCB unit in 10 of 12 subjects. A Phase 2 multi-center clinical trial of FT1050-UCB in adult patients undergoing dUCBT for hematologic malignancies was then initiated. Subjects are randomized 2:1 to FT1050-UCB-containing vs. standard dUCBT after high-dose conditioning. The primary endpoint is a categorical analysis of neutrophil engraftment using a pre-specified control median. Data on the initial 11 subjects, of which 8 were randomized to receive FT1050-UCB, continue to demonstrate acceptable safety with adverse events attributed to FT1050-UCB limited primarily to common infusion-related side effects. Of the 8 FT1050-UCB subjects, 1 died prior to neutrophil engraftment, with the remaining 7 subjects engrafting at a median of 28 days vs. 31 days for the 3 control subjects. With median overall follow-up of 16.1 months, 4 of 8 subjects on the FT1050-UCB arm are alive with a median survival not reached (> 11.0 months). 1 of 3 control subjects is alive with median survival of 6.0 months. During the clinical translation process, the media used during FT1050 modulation of UCB was identified as a key variable. Standard UCB washing media, consisting of a nutrient-free saline solution of low molecular weight dextran and human serum albumin (LMD/HSA), is used clinically to stabilize fragile cells post-thaw by reducing lysis. This media was used in the Phase 1 trial and to initiate Phase 2. Early during the Phase 2 trial, we identified a novel cell-stabilizing nutrient-rich formulation (NRM), containing glucose, amino acids and other HSC-supporting nutrients that promoted full FT1050 modulation of UCB and increased cell viability. The expression of key FT1050-pathway genes was significantly higher with NRM compared to intermediate levels observed with LMD/HSA. Modulation of human CD34+ (hCD34+) cells with FT1050 in NRM led to an 8-fold increase over LMD/HSA in induced CXCR4 gene expression (20-fold total), which translated to significantly increased surface CXCR4 protein. In vivo homing models demonstrated that UCB CD34+ cells modulated with FT1050 in NRM resulted in a 2.2-fold homing increase relative to vehicle (p < 0.001) compared to a 1.6-fold increase with LMD/HSA (p = 0.002), with a significant difference between the two media conditions (p = 0.04). A xenotransplantation study in NSG mice with hCD34+ cells modulated with FT1050 in either NRM or LMD/HSA demonstrated a 2-fold increase in circulating hCD45+ cells 12-weeks post-transplant with NRM (p = 0.007; unpaired t-test). These findings supported the incorporation of NRM into the FT1050-UCB manufacturing process in order to further improve its clinical engraftment potential. Enrollment of a 60-patient Phase 2 trial has been initiated that incorporates this manufacturing change. Disclosures Shoemaker: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment. Guerrettaz:Fate Therapeutics: Employment. Robbins:Fate Therapeutics: Employment. Medcalf:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment, Equity Ownership. Ferraro:Fate Therapeutics: Employment. Multani:Fate Therapeutics: Employment.
BackgroundHuman Epidermal Growth Factor Receptor 2 (HER2), is a receptor tyrosine kinase that is highly expressed on the surface of many solid tumors. While many patients derive meaningful benefit from the approved HER2-directed therapies, most will eventually suffer relapse or progression of their disease highlighting the need for additional treatment options. Currently there are no FDA-approved cellular therapies targeting HER2. Over the past decade, however, cellular therapy has been shown to be a viable treatment option in different cancer types. Here we present AB-201, an off-the-shelf, cryopreserved cord blood (CB)-derived HER2 chimeric antigen receptor (CAR)-natural killer (NK) cell therapy as a safe, active, and readily available option for patients with HER2+ solid tumors.MethodsAB-201 is comprised of ex vivo expanded allogeneic CB-derived NK cells that have been genetically modified to express a HER2-directed CAR and presented as a cryopreserved infusion-ready product. The manufacturing process utilizes a feeder-cell line engineered to express factors specifically identified as supportive to NK cell expansion and a lentiviral transduction step to introduce the HER2 CAR construct. In vitro characterization of AB-201 included evaluation of the purity and expression of cell surface markers by flow cytometry and short- (4 hour) and long-term (over 5 days) cytotoxicity assays in the presence of HER2+ tumor cell lines at various effector to target ratios. In addition, AB-201 efficacy was assessed in vivo in established ovarian (intraperitoneal, SKOV-3), breast (intraperitoneal, HCC1954) and gastric (subcutaneous, N87) xenograft models in NSG mice.ResultsHER2 CAR expression was detected in 93.1% of AB-201 cells. AB-201 is 97.9% CD3-/CD56+ cells and 94.6% CD56+/CD16+. Further characterization of AB-201 demonstrated high expression of NK activating receptors such as NKG2D, NKp30, NKp46, and DNAM-1 and expression of the chemokine receptor, CXCR3. AB-201 demonstrated concentration-dependent and HER2 targeted short-term cytotoxic activity and sustained long-term cell killing against the tumor cell lines SKOV-3, HCC1954, and NCI-N87. Efficacy, as evidenced by a significant reduction in bioluminescent signal or tumor volume, was observed in all xenograft models. A significant survival benefit over non-transduced NK cells or trastuzumab controls was demonstrated in the HCC1954 model.ConclusionsData presented herein suggests that AB-201, a highly pure and readily expandable HER2-directed CAR NK cell product, has potential to be an effective therapy in the treatment of HER2+ tumors.Ethics ApprovalThe animal studies were conducted in accordance with an Institutional Animal Care and Use Committee-approved protocol and with the approval of an IACUC committee at each center where the studies took place
Adoptive transfer of autologous T cells expressing chimeric antigen receptor (CAR) has shown great promise in the treatment of blood malignancies. Challenges for the application of current CAR T cell therapies to broader and more diverse patient populations include inherent variability, cost of manufacture, and the requirement for precise genetic engineering to generate a highly homogenous and consistent CAR T cell product. We have previously reported pre-clinical data supporting the development of FT819, a first-of-kind off-the-shelf CAR T cell product candidate. FT819 is generated from a renewable clonal master human induced pluripotent stem cell (hiPSC) line derived from a single cell engineered to contain bi-allelic disruption of the T cell receptor (TCR) and a novel CD19 CAR targeted into the T cell receptor α constant (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease. For the manufacture of a clinical-grade FT819 clonal master hiPSC line, we sourced peripheral blood mononuclear cells from a fully consented and eligible donor with protocol overseen by an independent Institutional Review Board. Sourced T cells were enriched (>98%) through positive selection for TCRαβ, and cryopreserved cells were confirmed to have stable genome by karyotyping. Using our proprietary non-integrating cellular reprogramming platform, αβ T cells were reprogrammed into hiPSCs. Concurrently with the reprogramming process, reprogrammed cells received nuclease and donor template to mediate targeting of CD19 CAR into the TRAC locus with bi-allelic knockout of the TCR. To generate clonal lines, engineered cells were sorted by flow cytometry for various markers and single cells were seeded into individual wells of feeder-free 96-well plates. hiPSC clones were screened for bi-allelic integration of CAR into the TRAC locus by amplifying the genomic DNA flanking the homologous recombination site and confirmed by a SNP phasing assay. Clones were further screened for random integration of donor template by quantitative PCR (qPCR), and the CAR copy number was confirmed by droplet digital PCR. Out of 545 hiPSC clones screened, 27 clones (5%) had bi-allelic TRAC targeting with no detectable random integration. Maintenance of pluripotency was confirmed in 19 out of the 27 engineered hiPSC clones (70%). Seventeen clones were further tested and were confirmed to be footprint-free of transgenic reprogramming factors. Of the 18 clones tested for genomic stability, 12 clones had normal karyotypes (67%). Validated, TRAC-targeted hiPSC clones were cryopreserved (~150 vials per clone) and are currently being assessed for off-target editing, differentiation propensity into highly-functional T cells, genomic stability, clone identity, sterility and lack of mycoplasma detection. In summary, using our novel iPSC technology platform for reprogramming, single cell engineering and multiplex high-throughput screening of hiPSCs, we have generated clinical-grade clonal master hiPSC lines in support of our first-of-kind clinical trials evaluating FT819 allogenic off-the-shelf hiPSC-derived TCR-less TRAC-CAR19 T cells for the treatment of blood malignancies. Citation Format: Ramzey Abujarour, Yi-Shin Lai, Mochtar Pribadi, Tom Lee, Megan Robinson, Chelsea Ruller, Sjoukje Van der Stegen, Xiuyan Wang, Jolanta Stefanski, Juan Zhen, Jason Dinella, Greg Bonello, Janel Huffman, Helen Chu, Raedun Clarke, Alec Witty, Amanda Medcalf, Jaeger Davis, Stacey Moreno, Pieter Lindenbergh, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. Generation of novel single cell-derived engineered master pluripotent cell line as a renewable source for off-the-shelf TCR-less CAR T cells in support of first-of-kind clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-073.
ProTmune is an ex vivo pharmacologically-modulated, next-generation mobilized peripheral blood (mPB) cell graft with FDA fast track designation for the reduction of incidence and severity of acute graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic cell transplantation (HCT). Here we report the 1-year safety and efficacy data from the Phase 1 stage of PROTECT, an ongoing Phase 1 open-label / Phase 2 blinded, randomized and controlled trial of ProTmune in adult subjects with hematologic malignancy undergoing matched unrelated donor HCT following myeloablative conditioning and receiving standard GvHD prophylaxis of methotrexate and tacrolimus. Seven subjects were administered ProTmune in the Phase 1 stage of PROTECT. Underlying hematologic diseases included ALL (n=3), AML (n=3) and MDS (n=1). Subjects were predominantly female (n=5) with a median age of 56 (range 34 to 69) and a median weight of 79 kg (range 53 to 133). As of July 23, 2018, five subjects remain on study with a median follow up of 390 days (range 342 to 490 days). ProTmune was manufactured on-site on the day of HCT by pharmacologically-modulating a conventional mobilized peripheral blood (mPB) cell graft ex vivo with 16,16-dimethyl prostaglandin E2 and dexamethasone, to enhance the biologic properties and therapeutic function of the graft. All ProTmune units were manufactured successfully with a mean of 6.6 (range 3.0 to 11.0) x106 CD34+ cells/kg and 2.3 (range 1.2 to 3.1) x108 CD3+ cells/kg. ProTmune was well tolerated. ProTmune-related AEs were all mild to moderate and consisted of nausea, vomiting and non-cardiac chest pain on the day of administration. No ProTmune-related SAEs have been reported. All subjects engrafted with no primary or secondary graft failure. Average time to neutrophil engraftment was 17 days (range 14 to 22). Day 100 acute GvHD assessment revealed three of the seven subjects had steroid responsive acute CIBMTR Grade 2-4 GvHD with median time of seven days [range 5 to 8 days] to resolution of the maximum GvHD grade. Since therapeutic approaches intended to reduce acute GvHD may increase disease relapse risk, composite endpoints are being explored by the HCT community to more thoroughly define transplant success. 1-year current GvHD-free, relapse-free survival (CGRFS) is one endpoint under study measuring proportion of subjects that are alive without relapse and without active moderate or severe chronic GvHD per NIH consensus criteria at Day 365. We intend to present final 1-year CGRFS on the seven Phase 1 subjects at the ASH Annual Meeting. To date, non-relapse mortality, deemed not attributable to ProTmune, occurred in two subjects (Subject 1 on Day 228 from pulmonary edema; Subject 3 on Day 151 from cardiac arrhythmia). There has been no disease relapse, and no moderate / severe chronic GvHD at Day 365. Two subjects are pending the 1-year assessment. The Phase 2 stage of PROTECT is currently enrolling a planned 60 subjects at 15 U.S. centers. Subjects with hematologic malignancies undergoing matched unrelated donor HCT following myeloablative conditioning are randomized 1:1 to receive in a blinded manner, either ProTmune or a conventional matched unrelated donor mPB unit as the cell graft. Table Table. Disclosures Maziarz: Athersys, Inc.: Patents & Royalties; Incyte: Consultancy, Honoraria; Kite Therapeutics: Honoraria; Juno Therapeutics: Consultancy, Honoraria; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Saad:Actinium: Consultancy. Diaz:Fate Therapeutics, Inc.: Employment. Medcalf:Fate Therapeutics, Inc.: Employment. Storgard:Fate Therapeutics, Inc.: Employment, Equity Ownership. Deol:Kite Pharmaceuticals: Consultancy; Novartis: Consultancy.
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