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This supplementary file contains Supplemental Figures 1-6. Supplemental Figure 1 shows that 7-day ex vivo culture of NK cells with IL-15 and 5 μM CHIR99021 leads to an increase in the frequencies of NK cells with heterogeneous adaptive NK cell phenotypes (defined by expression of CD57, PLZF, SYK and FcεR1γ) relative to DMSO controls. Supplemental Figure 2 contains a detailed phenotypic characterization of receptor expression and cytotoxic granule component levels in sorted CD3-CD56dimCD57- and CD3-CD56dimCD57+ NK cells cultured for 7 days with IL-15 and either DMSO or 5 μM CHIR99021. Supplementary Figure 3 shows an analysis of NK cell phenotype, viability and proliferation after 7-day culture with IL-15 and either DMSO or CHIR99021 at several concentrations (1 μM, 3 μM and 5 μM). Supplemental Figure 4 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and NKG2C. Supplemental Figure 5 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and KIR. Supplemental Figure 6 shows the frequency of ex vivo expanded NK cells expressing CD57 and/or NKG2C 14 days after adoptive transfer into NSG mice.

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664–75. ©2017 AACR.

Background: The autologous cell therapy field has investigated numerous novel strategies and processes to improve response rates and expand the use of adoptive cell therapies (ACT) to patient suffering from a broad range of cancers. Tumor infiltrating lymphocytes (TIL) can be harvested from tumors, expanded in vitro, and infused to patients leading to substantial clinical benefits in some patient populations. Tumor reactivity of TIL can vary greatly, representing a key limiting factor of bulk TIL therapies. The ability to generate an autologous cell product enriched in tumor reactive cells while limiting the presence of potentially detrimental and/or competitive non-reactive cells is highly desirable for next generation TIL products. Here we demonstrate that direct selection of antigen experienced TIL from tumor dissociates is feasible and allows to obtain functional cells enriched for tumor reactivity from various tumor indications. Methods: Patient-derived tumor material from various indications, including ovarian, kidney, colorectal cancer and lung tumors were first processed into single cell suspensions, maximizing TIL recovery and viability. Antigen experienced TIL were immediately selected based on the co-expression of PD-1 and CD39, using fluorescence-activated single cell sorting (FACS). The sorted cells or the unselected counterparts were expanded in a rapid expansion protocol (REP), then analyzed for phenotypic and functional characteristics and reactivity against autologous tumor cells. Results: The PD-1+ CD39+ selection strategy consistently allowed sorting of a population of viable TIL that was amenable to in vitro culture from tumors of various cancer indications. The selected TIL successfully expanded in a rapid expansion protocol. TCR sequencing analysis revealed that the PD-1+ CD39+ sorted populations were enriched in unique clones compared to the unselected populations. Expanded PD-1+ CD39+ selected cells demonstrated the ability to produce key effector cytokines upon restimulation in polyclonal assays. Importantly, the PD-1+ CD39+ expanded TIL were enriched for tumor reactive T cells and showed improved cytotoxic activity against autologous tumors. Conclusions: PD-1+ CD39+ selected TIL can be successfully isolated and expanded in vitro, generating a TIL product of superior reactivity in multiple cancer indications. PD-1+ CD39+ selected TIL showed increased cytokine secretion and cytotoxic activity against autologous material, indicating that this selection strategy enriches for functional tumor-reactive lymphocytes, which is likely to be a key feature of successful ACT. Citation Format: Christophe Pedros, Larissa Pikor, Anna Fritzsche, Zachary K. Jilesen, Bethany Macleod, Niloufar Khojandi, Bryant Thompson, Matthew Thayer, Nikolas Bryan, Emily Carron, Sowbarnika S Ratliff, April Fraley, Jake Nikota, Robert Fisher, Sebastien Delpeut, Anna Kluew, Madysson Scott, Christian Laing, Leo He, Antoine Bernard, Nathalie Brassard, Simon Turcotte, Timothy J Langer, David Stojdl, Stewart Abbot, Barbara Sennino. Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4052.

Background: Tumor infiltrating lymphocyte (TIL) therapy is capable of mediating durable complete responses in melanoma. While solid tumors such as colorectal cancer (CRC), non-small cell lung cancer (NSCLC), ovarian and breast have been shown to contain neoantigen reactive TIL, the success of bulk TIL therapy in these tumors has been limited. Enhancing tumor reactivity through the selective expansion of neoantigen-reactive subpopulations, has demonstrated success in cancers outside of melanoma underscoring the potential of a neoantigen selected TIL approach in indications with lower tumor mutational burdens. Here we demonstrate that the TIDAL-01 process, which utilizes tumor-specific mutation containing peptides to select neoantigen reactive TIL produces TIL products significantly enriched in neoantigen reactivity. Methods: Fresh tumors were cut into fragments or dissociated and cultured in a primary expansion (preREP). Antigen presenting cells (APCs) were isolated and expanded from patient matched blood. Whole exome and RNA sequencing was performed on tumor tissue and autologous PBMCs and used to predict and prioritize neoantigen mutations. Peptides encoding the mutations were synthesized, loaded onto APCs and co-cultured with autologous TIL. Neoantigen reactive TIL were selected by fluorescence activated cell sorting (FACS), based on the upregulation of the activation markers CD134 and CD137 and expanded with a rapid expansion protocol (REP). Bulk and unselected TIL were expanded alongside for comparison. Neoantigen reactivity was quantified and deconvoluted by cytokine secretion, degranulation, upregulation of CD134/CD137 by flow and when practical, killing of autologous tumor cell lines or organoids. Results: Successful TIL expansion was achieved in 31/34 (91%) tumors (14/17 CRC, 10/10 NSCLC, 3/3 ovarian and 3/3 melanoma) using both tumor fragments and dissociated tumors. CRC tumors accounted for half of the samples (17/34), and the tumor mutational burden within these samples varied substantially, ranging from 229 to 5436 mutations. Upregulation of CD134 and CD137 and increased IFN-γ production was observed in all samples upon co-culture with peptide loaded APCs. Peptide restimulation and deconvolution revealed that the TIDAL-01 process is capable of enriching for both CD4 and CD8 reactivities. Selected TIL products produced up to 50x more IFN-γ, TNF-α and Granzyme B than bulk TIL and at least 2x higher levels of degranulation, indicative of greater killing potential. Conclusions: TIL from metastatic CRC, melanoma, NSCLC and ovarian tumors were successfully expanded from the majority of patients. Co-culture of TIL and peptide loaded APCs followed by FACS significantly enriched for neoantigen reactivity compared to bulk TIL, demonstrating the potential of the TIDAL-01 process to produce selected TIL products for the treatment of non-melanoma tumors. Citation Format: Larissa A. Pikor, Antoine Bernard, Nathalie Brassard, Anna Fritzsche, Anna Kluew, Zachary K. Jilesen, Jake Nikota, Rohan Bareja, Christian Laing, David F. Stojdl, TJ Langer, Stewart Abbot, Barbara Sennino, Simon Turcotte. TIDAL-01: A selected TIL process that enriches for neoantigen reactive TIL in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4049.

Background Autologous chimeric antigen receptor (CAR) T cells have been shown to be efficacious for the treatment of B cell malignancies; however, widespread adoption and application of CAR T cell products still face a number of challenges. To overcome these challenges, Adicet Bio is developing an allogeneic γδ T cell-based CAR T cell platform, which capitalizes on the intrinsic abilities of Vδ1 γδ T cells to recognize and kill transformed cells in an MHC-unrestricted manner, to migrate to epithelial tissues, and to function in hypoxic conditions. To gain a better understanding of the requirements for optimal intratumoral CAR Vδ1 γδ T cell activation, proliferation, and differentiation, we developed a three-dimensional (3D) tumor spheroid assay, in which tumor cells acquire the structural organization of a solid tumor and establish a microenvironment that has oxygen and nutrient gradients. Moreover, through the addition of cytokines and/or tumor stromal cell types, the spheroid microenvironment can be modified to reflect hot or cold tumors. Here, we report on the use of a 3D CD20+ Raji lymphoma spheroid assay to evaluate the effects of IL-2 and IL-15, positive regulators of T cell homeostasis and differentiation, on the proliferative and antitumor capacities of CD20 CAR Vδ1 γδ T cells. Methods Molecular, phenotypic, and functional profiling were performed to characterize the in vitro dynamics of the intraspheroid CD20 CAR Vδ1 γδ T cell response to target antigen in the presence of IL-2, IL-15, or no added cytokine. Results When compared to no added cytokine, the addition of IL-2 or IL-15 enhanced CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and cytokine production in a dose-dependent manner but were only able to alter the kinetics of Raji cell killing at low effector to target ratios. Notably, differential gene expression analysis using NanoString nCounter® Technology confirmed the positive effects of IL-2 or IL-15 on CAR-activated Vδ1 γδ T cells as evidenced by the upregulation of genes involved in activation, cell cycle, mitochondrial biogenesis, cytotoxicity, and cytokine production. Conclusions Together, these results not only show that the addition of IL-2 or IL-15 can potentiate CD20 CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation into antitumor effectors but also highlight the utility of the 3D spheroid assay as a high throughput in vitro method for assessing and predicting CAR Vδ1 γδ T cell activation, proliferation, survival, and differentiation in hot and cold tumors.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

BackgroundGlypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in hepatocellular carcinoma (HCC). Autologous chimeric antigen receptor (CAR) αβ T cell therapies have established significant clinical benefit in hematologic malignancies, although efficacy in solid tumors has been limited due to several challenges including T cell homing, target antigen heterogeneity, and immunosuppressive tumor microenvironments. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells through major histocompatibility complex (MHC)-independent antigens upregulated under stress. The Vδ1 subset is preferentially localized in peripheral tissue and engineering with CARs to further enhance intrinsic antitumor activity represents an attractive approach to overcome challenges for conventional T cell therapies in solid tumors. Allogeneic Vδ1 CAR T cell therapy may also overcome other hurdles faced by allogeneic αβ T cell therapy, including graft-versus-host disease (GvHD).MethodsWe developed the first example of allogeneic CAR Vδ1 T cells that have been expanded from peripheral blood mononuclear cells (PBMCs) and genetically modified to express a 4-1BB/CD3z CAR against GPC-3. The CAR construct (GPC-3.CAR/secreted interleukin-15 (sIL)-15) additionally encodes a constitutively-secreted form of IL-15, which we hypothesized could sustain proliferation and antitumor activity of intratumoral Vδ1 T cells expressing GPC-3.CAR.ResultsGPC-3.CAR/sIL-15 Vδ1 T cells expanded from PBMCs on average 20,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like memory phenotype with limited exhaustion marker expression and displayed robust in vitro proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low (PLC/PRF/5) and high (HepG2) GPC-3 levels. In a subcutaneous HepG2 mouse model in immunodeficient NSG mice, GPC-3.CAR/sIL-15 Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose efficiently controlled tumor growth without evidence of xenogeneic GvHD. Importantly, compared with GPC-3.CAR Vδ1 T cells lacking sIL-15, GPC-3.CAR/sIL-15 Vδ1 T cells displayed greater proliferation and resulted in enhanced therapeutic activity.ConclusionsExpanded Vδ1 T cells engineered with a GPC-3 CAR and sIL-15 represent a promising platform warranting further clinical evaluation as an off-the-shelf treatment of HCC and potentially other GPC-3-expressing solid tumors.

BackgroundGlypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors including hepatocellular carcinoma (HCC) but is rarely expressed in healthy adult tissues and serves as a therapeutic target of interest. Autologous αβ chimeric antigen receptor (CAR) T cell therapy has established clinical benefit in hematologic malignancies but limited success in solid tumors due to numerous challenges including poor T cell homing, heterogenous antigen expression, and hostile tumor microenvironments.1 These challenges may be overcome by the Vδ1 subset of gamma delta T cells due to their natural peripheral tissue tropism and ability to recognize and kill tumor cells through MHC-independent antigens upregulated under stress.2 Allogeneic Vδ1 T cells engineered with CARs can have enhanced intrinsic antitumor activity and overcome challenges of allogeneic αβ T cells, including graft-versus-host disease (GvHD). Here, we describe the first preclinical evaluation of ADI-002, a next-generation allogeneic CAR Vδ1 T cell therapy targeting GPC-3 and armored with IL-15, for the treatment of solid tumors.MethodsVδ1 T cells were expanded from healthy donor PBMCs and transduced to express a 4–1BB/CD3z CAR against GPC-3 that encodes constitutively-secreted IL-15 (sIL-15), which we hypothesized could sustain proliferation and antitumor activity of intratumoral GPC-3.CAR Vδ1 T cells. In vitro characterization included co-culture with HCC targets expressing high (HepG2) and low (PLC/PRF/5) GPC-3, phenotypic analysis by flow cytometry, and cytokine production by multiplexed immunoassay. For in vivo assessment, immunodeficient NSG mice were subcutaneously injected with HepG2 tumor cells and treated with a single dose of GPC-3.CAR Vδ1 T cells. Tissues were harvested 7 days post transfer and analyzed for Vδ1 T cell tissue homing and proliferation, or at study end and analyzed for GvHD by immunohistochemistry.ResultsGPC-3.sIL-15.CAR Vδ1 T cells expanded over 10,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like phenotype (CD45RA+CD27+) with minimal exhaustion receptor expression and displayed robust proliferation, cytokine production, and cytotoxic activity against HCC cell lines in vitro. In vivo, GPC-3.sIL-15.CAR Vδ1 T cells primarily accumulated and proliferated in tumors, and a single dose could efficiently control tumor burden without causing GvHD. When compared to GPC-3.CAR Vδ1 T cells lacking sIL-15, GPC-3.sIL-15.CAR Vδ1 T cells displayed greater tumor-specific proliferation that resulted in enhanced tumor control (figure 1).Abstract 119 Figure 1In vivo antitumor efficacy in a subcutaneous HepG2 tumor model in NSG miceConclusionsExpanded Vδ1 T cells engineered with GPC-3.CAR and sIL-15 represent a promising approach for safe and effective off-the-shelf treatment of HCC and support further investigation in the clinical setting.ReferencesLabanieh L, Majzner RG, Mackall CL. Programming CAR-T cells to kill cancer. Nat Biomed Eng 2018;2(6):377–91.Sebestyen Z, Prinz I, Déchanet-Merville J, Silva-Santos B, Kuball J. Translating gammadelta (γδ) T cells and their receptors into cancer cell therapies. Nat Rev Drug Discov 2020;19(3):169–84.Ethics ApprovalAll mouse experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and followed all institutional and national guidelines and after appropriate approvals.

e14511 Background: Autologous αβ chimeric antigen receptor (CAR) T cell therapy has shown promising clinical results in hematologic malignancies but limited success in solid tumors. Allogeneic αβ T cell therapy may overcome several challenges faced by autologous therapy but carries the risk of graft-versus-host disease (GvHD) and does not readily recognize multiple tumor-associated antigens. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells in an MHC-unrestricted manner without causing GvHD. The Vδ1 subset is preferentially localized in peripheral tissue and is critical for tumor immunosurveillance. Engineering Vδ1 T cells with CARs can further enhance antitumor activity and represents an attractive and safe approach to treating solid tumors. However, their clinical use has been hindered by the limited number of circulating Vδ1 T cells. Here, we describe the development of the first allogeneic Vδ1 T cells that have been expanded from healthy donor PBMCs and genetically modified to secrete IL-15 (sIL15) and express a CAR targeting glypican-3 (GPC3), a rational target for hepatocellular carcinoma (HCC). Methods: Vδ1 T cells in healthy donor PBMCs were activated by a Vδ1-specific monoclonal antibody and transduced with 41BBζ or 41BBζ-sIL15 GPC3-CARs prior to cell expansion, αβ T cell depletion and cryopreservation. In vitro characterization included: 1) co-culture assays with GPC3-expressing HCC targets HepG2 and PLC/PRF/5, 2) phenotypic analysis by flow cytometry, and 3) cytokine production by multiplexed immunoassay. For in vivo assessment of tumor control, immunodeficient NSG mice were subcutaneously injected with HepG2 cells and treated with a single dose of 41BBζ or 41BBζ-sIL15 GPC3-CAR Vδ1 T cells. Additionally, tissues were harvested 7 days post transfer and analyzed by flow cytometry for Vδ1 T cell tissue homing and proliferation, or at end of study and analyzed for GvHD by immunohistochemistry. Results: Vδ1 T cells expanded over 10,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like phenotype (CD45RA+CD27+) with minimal exhaustion receptor expression and displayed robust proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low and high GPC3 levels in vitro. In a HepG2 mouse model, GPC3-CAR Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose was able to efficiently control tumor burden without causing GvHD. Importantly, 41BBζ-sIL15 GPC3-CAR Vδ1 cells displayed enhanced tumor-specific proliferation that resulted in better tumor control without any toxicity. Conclusions: Our results show that expanded Vδ1 T cells engineered with GPC3-CAR and sIL-15 represent a promising platform for safe and effective off-the-shelf treatment of HCC.

Summary The aging population in the U.S. and other developed countries has led to a large increase in the number of patients suffering from degenerative diseases. Transplantation surgery has been a successful therapeutic option for certain patients; however, the availability of suitable donor organs and tissues significantly limits the number of patients who can benefit from this approach. Regenerative medicine has witnessed numerous recent and spectacular advances, making the repair or replacement of dysfunctional organs and tissues an achievable goal. Public-private partnerships and government policies and incentives would further catalyze the development of universally available donor tissues, resulting in broad medical and economic benefits. This article describes a Regenerative Medicine Grand Challenge that the Alliance for Regenerative Medicine recently shared with the White House's Office of Science and Technology Policy in response to a White House call to action in scientific disciplines suggesting that the development of “universal donor tissues” should be designated as a Regenerative Medicine Grand Challenge. Such a designation would raise national awareness of the potential of regenerative medicine to address the unmet needs of many diseases and would stimulate the scientific partnerships and investments in technology needed to expedite this goal. Here we outline key policy changes and technological challenges that must be addressed to achieve the promise of a major breakthrough in the treatment of degenerative disease. A nationalized effort and commitment to develop universal donor tissues could realize this goal within 10 years and along the way result in significant innovation in manufacturing technologies. Significance Regenerative therapies, in which dysfunctional or degenerating cells, tissues, or organs are repaired or replaced, have the potential to cure chronic degenerative diseases. Such treatments are limited by a shortage of donor organs and tissues and the need for immune suppression to prevent rejection. This article proposes a 21st Century Grand Challenge that would address this significant medical need by coordinating a national effort to convene the multidisciplinary expertise needed to manufacture functional and engraftable cells, tissues, or organs that could be made available to any patient without significant risk of rejection—so-called universal donor tissues.

Continuous production of red blood cells (RBCs) in an automated closed culture system using hematopoietic stem cell (HSC) progenitor cell populations is of interest for clinical application because of the high demand for blood transfusions. Previously, we introduced a four-compartment bioreactor that consisted of two bundles of hollow fiber microfiltration membranes for transport of culture medium (forming two medium compartments), interwoven with one bundle of hollow fiber membranes for transport of oxygen (O(2)), carbon dioxide (CO(2)), and other gases (forming one gas compartment). Small-scale prototypes were developed of the three-dimensional (3D) perfusion cell culture systems, which enable convection-based mass transfer and integral oxygenation in the cell compartment. CD34(+) HSC were isolated from human cord blood units using a magnetic separation procedure. Cells were inoculated into 2- or 8-mL scaled-down versions of the previously designed 800-mL cell compartment devices and perfused with erythrocyte proliferation and differentiation medium. First, using the small-scale 2-mL analytical scale bioreactor, with an initial seeding density of 800,000 cells/mL, we demonstrated approximately 100-fold cell expansion and differentiation after 7 days of culture. An 8-mL laboratory-scale bioreactor was then used to show pseudocontinuous production by intermediately harvesting cells. Subsequently, we were able to use a model to demonstrate semicontinuous production with up to 14,288-fold expansion using seeding densities of 800,000 cells/mL. The down-scaled culture technology allows for expansion of CD34(+) cells and stimulating these progenitors towards RBC lineage, expressing approximately 40% CD235(+) and enucleation. The 3D perfusion technology provides an innovative tool for studies on RBC production, which is scalable.

To predict the timing and nature of future changes in the practice of blood transfusion, several factors must be considered. The historical rate of change of a scientific field can often provide a rough guide to the rate of future progress. To improve the accuracy of these predictions, historical rates must be adjusted to take into account the decelerating effects of technological or methodological barriers to progress, together with the potentially accelerating effects of transformative technology breakthroughs and unmet needs in the field that act as drivers for change. The cumulative impact of unpredictable and, often, limited availability of traditional blood donors, increasingly elderly populations, the potential for storage-associated adverse events, and increasingly prevalent transfusion-transmittable diseases is likely to provide significant drive to develop transformational alternatives to current transfusion practices. Considering the current stage of development of stem cell-based therapeutics and the rates of change in clinically compatible bioreactors and cell sorting systems, it is reasonable to believe that stem cell-based ex vivo manufacture of blood components will become routine, robust, and reliable within the next decade.

Natural killer (NK) cells represent a lineage of immune cells capable of direct cytotoxicity against tumor cells and are a critical source of key inflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF). NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system. The unique biological attributes of NK cells, including multifaceted effector function, tumor cell recognition independent of antigen presentation and target cell selectivity independent of HLA-matching, has enabled NK cells from a donor to be adoptively transferred to a patient for the treatment of cancer. This safe and effective administration of donor NK cells to patients validates their potential for broad use as part of an off-the-shelf cancer immunotherapy strategy, including in combination with monoclonal antibody and checkpoint inhibitor therapies. We have previously shown that human induced pluripotent stem cells (hiPSC) can be clonally-selected, cryopreserved and banked, and that these master pluripotent cell lines (MPCLs) can be used to renewably generate large clonal populations of NK cells. The use of MPCLs represents a highly-promising, off-the-shelf approach to cell-based cancer immunotherapy, with the potential to overcome many of the challenges and limitations of patient-sourced and donor-derived cell therapies. However, to clinically and commercially enable this off-the-shelf strategy, it is essential to efficiently and reproducibly differentiate MPCLs to fully-functional NK cells using a robust and scalable process that meets regulatory requirements. Here we describe a novel paradigm for the manufacture of hiPSC-derived NK (iNK) cells consisting of a well-defined, small molecule-driven, staged protocol that enables clinical translation and is compatible with current good manufacturing practice (cGMP) requirements. The manufacturing protocol is currently being transferred from the laboratories of Fate Therapeutics to Molecular and Cellular Therapeutics at the University of Minnesota, which is a state-of-the-art GMP/GTP compliant, full-service developer and manufacturer of cell- and tissue-based products. iNK cell therapy manufacture consists of four unique steps including: 1) the derivation and master cell banking of a clonal pluripotent cell line (> 95% SSEA4+/TRA181+ hiPSCs); 2) differentiation of the clonal pluripotent cell line towards hematopoietic progenitor cells (enriched for > 80% CD34+ cells); 3) differentiation and expansion of iNKs (Figure 1A, approximately 1,000-fold expansion in 14 days); and 4) freeze and thaw of drug product, comprised of a sufficiently pure homogenous population of iNKs (Figure 1B, > 95% CD45+, > 90% CD56+, minimal CD3+ T cells). Importantly, testing at both the molecular and culture stages demonstrate that no hiPSCs exist in the final drug product (limit of detection 1 hiPSC in 1.25 million iNK cells). This novel manufacturing paradigm supports the generation of significant numbers of iNK cells: approximately 1 million-fold cell expansion is achieved in less than 50 days, such that a very small population of hiPSCs can readily produce 1x1012 iNK cells. We estimate that this represents hundreds of doses of drug product per each manufacturing run (Figure 1A). The iNK cells display markedly augmented effector function relative to ex vivo expanded primary peripheral blood or cord blood NK cells with respect to cytokine release (IFN-γ and TNF) and cellular cytotoxicity against various leukemic and solid tumor-derived target cells including K562, Raji, A549 and SKOV3 (Figure 1C). To enable centralized manufacturing, we established a freeze and thaw strategy that supports greater than 85% viability with a recovery of greater than 80% iNK cells at twenty-four hours post-thaw. Because the freeze process uses an infusible medium formulation, we demonstrated in vitro and in vivo that the iNK cells maintain their efficacy post-thaw and can be immediately infused into patients. The manufacturing data presented herein support the filing of an Investigational New Drug application for an off-the-shelf iNK cell therapy product to treat advanced hematologic and solid tumor malignancies alone or in combination with monoclonal antibody and checkpoint inhibitor therapies. Disclosures Bjordahl: Fate Therapeutics: Employment, Equity Ownership. Gaidarova: Fate Therapeutics Inc.: Employment, Equity Ownership. Rogers: Fate Therapeutics Inc.: Employment, Equity Ownership. Clarke: Fate Therapeutics Inc.: Employment, Equity Ownership. Groff: Fate Therapeutics Inc.: Employment. Moreno: Fate Therapeutics Inc.: Employment. Abujarour: Fate Therapeutics Inc.: Employment. Robinson: Fate Therapeutics Inc.: Employment. Bonello: Fate Therapeutics Inc.: Employment. Lee: Fate Therapeutics Inc.: Employment, Equity Ownership. Lan: Fate Therapeutics Inc.: Employment, Equity Ownership. Rezner: Fate Therapeutics, Inc.: Employment. Abbot: Fate Therapeutics Inc.: Employment. Wolchko: Fate Therapeutics Inc.: Employment. Kaufman: Fate Therapeutics: Consultancy, Research Funding. Valamehr: Fate Therapeutics: Employment, Equity Ownership. Miller: Oxis Biotech: Consultancy; Celegene: Consultancy; Fate Therapeutics: Consultancy, Research Funding.

Human embryonic stem cells (hESCs) can differentiate into a variety of specialized cell types. Thus, they provide a model system for embryonic development to investigate the molecular processes of cell differentiation and lineage commitment. The development of the cardiac lineage is easily detected in mixed cultures by the appearance of spontaneously contracting areas of cells. We performed gene expression profiling of undifferentiated and differentiating hESCs and monitored 468 genes expressed during cardiac development and/or in cardiac tissue. Their transcription during early differentiation of hESCs through embryoid bodies (EBs) was investigated and compared with spontaneously differentiating hESCs maintained on feeders in culture without passaging (high-density (HD) protocol). We observed a larger variation in the gene expression between cells from a single cell line that were differentiated using two different protocols than in cells from different cell lines that were cultured according to the same protocol. Notably, the EB protocol resulted in more reproducible transcription profiles than the HD protocol. The results presented here provide new information about gene regulation during early differentiation of hESCs with emphasis on the cardiomyogenic program. In addition, we also identified regulatory elements that could prove critical for the development of the cardiomyocyte lineage.

Housekeeping genes (HKGs) are involved in basic functions needed for the sustenance of the cell and are assumed to be constitutively expressed at a constant level. Based on these features, HKGs are frequently used for normalization of gene expression data. In the present study, we used the CodeLink Gene Expression Bioarray system to interrogate changes in gene expression occurring during differentiation of human ESCs (hESCs). Notably, in the three hESC lines used for the study, we observed that the RNA levels of 56 frequently used HKGs varied to a degree that rendered them inappropriate as reference genes. Therefore, we defined a novel set of HKGs specifically for hESCs. Here we present a comprehensive list of 292 genes that are stably expressed (coefficient of variation

We describe a development of a novel high-throughput phagocytosis assay based on a pH-sensitive cyanine dye, CypHer5E™, which is maximally fluorescent in an acidic environment. This dye is ideally suited for the study of phagocytosis because of the acidic conditions generated in the intracellular phagocytic vesicles after particle uptake. Use of CypHer5E-labeled particles results in greatly reduced background from noninternalized particles and makes the assay more robust. Additionally, CypHer5E-labeled particles are resistant to fluorescence quenching observed in the aggressive and acidic environment of the phagosome with traditional dyes. The CypHer5E-based assay has been shown to work reliably in a variety of cell types, including primary human monocytes, primary human dendritic cells, primary human endothelial cells, human monocytic THP-1 cell line, and human/mouse hybrid macrophage cell line WBC264-9C. Inhibition of CypHer5E bead uptake by cytochalasin D was studied, and the 50% inhibition concentration (IC50) was determined. The assay was performed in 96- and 384-well formats, and it is appropriate for high-throughput cellular screening of processes and compounds affecting phagocytosis. The CypHer5E phagocytosis assay is superior to existing protocols because it allows easy distinction of true phagocytosis from particle adherence and can be used in microscopy-based measurement of phagocytosis.

The unique attributes of a combinatorial tumor recognition system, diminished off-tumor cytotoxicity, and multifaceted effector function make natural killer (NK) cells a prime candidate for a universal approach to cancer immunotherapy. In addition, NK cells are the principal mediator of antibody-directed cellular cytotoxicity (ADCC). However, NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system and the therapeutic efficacy of monoclonal antibodies. To address the need for advanced and combinatorial cancer therapies, we developed a unique and effective strategy to create a renewable source of engineered “off-the-shelf” NK cells with augmented function, including enhanced ADCC and persistence. Key challenges associated with genetic editing, limited expansion, persistence and variability of peripheral blood (PB)-derived NK cells were overcome by utilizing our induced pluripotent stem cell (iPSC) technology as the unlimited starting material for the reproducible and consistent derivation of engineered NK cells. Through targeted transgene integration, we produced a clonal iPSC master cell line to continuously produce NK cells engineered to uniformly express a high affinity, non-cleavable version of CD16 (hnCD16-NK). In directed differentiation, the hnCD16-NK cells displayed homogeneous expression of CD16 (>95%) and a mature CD56+ NK cell phenotype, as exhibited by expression of KIR, NCRs, DNAM-1, and NKG2D. In contrast to endogenous CD16 expression, the engineered hnCD16 molecule was shown to be cleavage resistant upon NK cell activation (>95% CD16+ hnCD16-NK vs. Citation Format: Ryan Bjordahl, Frank Cichocki, Raedun Clarke, Svetlana Gaidarova, Brian Groff, Paul Rogers, Stacey Moreno, Ramzey Abujarour, Greg Bonello, Tom Lee, Weijie Lan, Matthieu Bauer, Dave Robbins, Betsy Rezner, Sarah Cooley, Bruce Walcheck, Stewart Abbot, Bruce Blazar, Scott Wolchko, Daniel Shoemaker, Jeffrey S. Miller, Bahram Valamehr. Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3755. doi:10.1158/1538-7445.AM2017-3755

Natural killer (NK) cells are innate lymphoid cells that mediate immune responses against pathogens and cancer. Human NK cells are distinguished by the surface phenotype CD3-CD56+, and maturation of CD56dim NK cells is associated with acquisition of CD57. Rather than being an immunosenescence marker, CD57 acquisition represents a shift toward greater effector function, including increased CD16 signaling (Fc receptor responsible for triggering antibody-dependent cellular cytotoxicity), more potent cytotoxicity and enhanced inflammatory cytokine production after target cell engagement. The main challenge in enriching for CD57+ NK cells using current ex vivo expansion protocols is that interleukin (IL)-15, the cytokine that drives NK cell proliferation and is critical for NK cell survival, preferentially expands less mature NK subsets that fail to terminally differentiate in culture. Our group has developed a novel NK cell expansion method that overcomes this barrier. Peripheral blood mononuclear cells from are depleted of CD3+ T cells and CD19+ B cells and cultured for 7 days with IL-15 and a small molecule inhibitor of glycogen synthase kinase 3-beta (GSK3β), a multifunctional kinase downstream of the PI(3)K pathway. Compared to vehicle control, addition of the GSK3β inhibitor led to a substantial increase (2.2-fold ± 0.19, n=23, p We have scaled our process to manufacture a GMP product (referred to as FATE-NK100) for clinical use. Using an apheresis product from a donor containing 21.5 x 108 CD57+ NK cells, we achieved 6.4-fold NK cell expansion resulting in a final GMP-grade product containing 158 x 108 CD57+ NK cells. The cytotoxicity of these ex vivo expanded NK cells in response to SKOV-3 cells is superior to that of CD3/CD19-depleted haploidentical NK cells activated overnight with either IL-2 or IL-15 (representing the NK products used in current clinical trials). These data have been presented to the FDA in preparation for a clinical trial of FATE-NK100 in lymphodepleted patients with advanced AML anticipated for Q1 2017. Citation Format: Frank Cichocki, Barham Valamehr, Ryan Bjordahl, Bin Zhang, Dhifaf Sarhan, Sarah Cooley, Bruce Blazar, Betsy Rezner, Paul Rogers, Chad Green, Stewart Abbot, Daniel Shoemaker, Scott Wolchko, Jeffrey S. Miller. FATE-NK100: A novel NK cell-mediated cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3752. doi:10.1158/1538-7445.AM2017-3752

Encouraging clinical outcomes in autologous cellular immunotherapy have garnered hope and excitement. However, limitations of patient-derived cancer immunotherapies remain to be addressed to deliver reliable and efficacious therapies with broader applicability. Induced pluripotent stem cells (iPSCs) are a unique, renewable source for the continuous generation of cellular therapeutics and represent a highly promising approach for overcoming many of the limitations of autologous therapy. To advance the promise of iPSC technology as an “off-the-shelf” (OTS) source of cellular therapeutics, several considerations need to be addressed. Ensuring the persistence of allogeneic OTS therapies after adoptive cell transfer across histocompatibility barriers is a key requirement. Establishing a master cell line from genetically engineered clonal iPSC lines with the capacity to continuously generate homogenous populations of highly functional effector cells will also be necessary. Here we demonstrate a comprehensive approach for the generation of immune tolerant effector cells derived from a genetically engineered iPSC master cell line. We successfully combined deletion of classical human leukocyte antigen molecules with expression of immunosuppressive proteins to generate clonal iPSC lines with the ability to escape immune rejection. Utilizing in vitro quantitative live cell analysis we show that OTS-iPSCs elicit a significantly decreased cytotoxic response from both peripheral blood (PB)-NK cells (47.9 vs. 91.4% survival at 3:1 E:T ratio) and PB-T cells (>2.7-fold greater number of OTS-iPSC derived cells remaining at 88 hrs). Additionally, mixed lymphocyte reactions employing unfractionated PB mononuclear cells resulted in significantly decreased activation and proliferation of CD8+ T cells (63.4 vs. 29.6%), CD4+ T cells (70.9 vs. 17.3%) and NK cells (46.8 vs. 11.6%). In preclinical mouse models we demonstrate that OTS-iPSCs exhibit improved persistence in vivo. Bilateral engraftments were established in non-conditioned, fully immune-competent recipient mice using luciferized wildtype and OTS-iPSCs. Daily bioluminescence imaging revealed a significant increase in persistence of OTS-iPSCs during the 48-196 hour post injection window (>5.5 fold greater luminescence at 96 hrs). Using our potent chemically-defined stage-specific monolayer hematopoietic differentiation platform, we demonstrate that OTS-iPSC derived CD34 expressing hematopoietic cells are reproducibly scaled and readily give rise to functional lymphocytes carrying the engineered targeted modality in a homogenous manner (95 +/- 5%). The outlined preclinical data illustrate that iPSCs are an ideal renewable source for OTS hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive immunotherapy. Citation Format: Raedun L. Clarke, Matthieu Bauer, Ryan Bjordahl, Jeffrey Sasaki, Brian Groff, Svetlana Gaidarova, Tom Tong Lee, Weijie Lan, Michelle Burrascano, Ramzey Abujarour, Greg Bonello, Megan Robinson, Stewart Abbot, Scott Wolchko, Daniel Shoemaker, Bob Valamehr. Overcoming host histocompatibility barrier to create a renewable source of off-the-shelf effector lymphocytes for adoptive immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 609. doi:10.1158/1538-7445.AM2017-609

Objective Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population from full-term placental tissue and were previously shown to possess anti-inflammatory and immunomodulatory properties. PDACs (formulated as PDA-002) are in clinical trials for peripheral arterial disease with diabetic foot ulcer. In the current study, we examined their angiogenic and tissue reparative properties. Methods The effects of PDACs on survival and tube formation of human umbilical vein endothelial cells (HUVECs) were tested using conditioned media and noncontact coculture. Angiogenic effects were assessed in the chick chorioallantoic membrane assay. Hindlimb ischemia (HLI) was induced in mice and rats by femoral artery transection, and blood flow and blood vessel density were monitored in vivo by laser Doppler and angiography in the ischemic and control limbs. Tissue damage and regeneration in HLI were examined in histologic sections of quadriceps muscle stained with hematoxylin and eosin, and newly synthesized blood vessels were detected by indoxyl-tetrazolium staining for alkaline phosphatase. Results PDACs enhanced the survival of serum-starved HUVECs and stimulated HUVEC tube formation, and in the chick chorioallantoic membrane assay, PDACs stimulated blood vessel formation. In HLI, intramuscular administration of PDACs resulted in improved blood flow and vascular density, and in quadriceps muscle, tissue regeneration and increased numbers of blood vessels were observed. Conclusions PDACs exhibited various activities consistent with angiogenesis and tissue repair, supporting the continued investigation of this cell therapy as treatment for vascular disease-related indications.

Encouraging clinical outcomes in autologous cellular immunotherapy have garnered hope and excitement. However, considerable challenges and limitations of patient-derived cancer immunotherapies remain and need to be addressed in order to consistently deliver reliable and efficacious therapies with broadened applicability. Human induced pluripotent stem cells (hiPSCs) are a unique, renewable source for the continuous generation of cellular therapeutics for the treatment of hematological and non-hematological malignancies, and represent a highly promising approach for overcoming many of the limitations of autologous therapy. To advance the promise of hiPSC technology as an "off-the-shelf" source of cellular therapeutics, several considerations need to be addressed. Enabling cell transfer across histocompatibility barriers to permit persistence and therapeutic efficacy in an allogeneic setting is a key requirement. In addition to improving persistence, the ability to overcome histocompatibility barriers may facilitate multi-dosing regimens which may be a requirement in more advanced and complicated disease settings. Genetic incompatibilities between donor and recipient among the classical human leukocyte antigen (HLA) molecules is the leading cause of alloresponse by the host immune system and is currently mitigated by immunosuppressive strategies. Unfortunately, this treatment strategy is not only a stressful event for the patient but also damages the endogenous immune system, compromising the patient's ability to continue to fight the disease and opportunistic infections. Genetic editing of the HLA genes to generate histocompatible universal cell products is a viable opportunity that is currently being investigated. In addition to selective editing of unique genes to avoid a T cell mediated alloresponse, additional considerations such as natural killer (NK) cell-mediated rejection will need to be addressed. We have previously demonstrated that our proprietary reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous renewable population of hiPSCs, our platform is amenable to precise multi-gene and multi-loci targeted engineering at the single cell level, in both nuclease -dependent and -independent strategies. Furthermore, we have shown through small molecule-guided differentiation protocols, these highly-stable pluripotent cell lines can be banked and repeatedly tapped to consistently produce homogenous populations of immune cells with enhanced effector properties. Here we demonstrate a multi-faceted and comprehensive approach for the generation of immune tolerant hiPSCs and hiPSC-derived immune effector cells. We successfully combined deletion of classical HLA molecules with enforced expression of robust immunosuppressive proteins, including non-classical HLA molecules, to generate clonal hiPSC lines with the ability to escape immune rejection for "off-the-shelf" (OTS-hiPSCs) cellular immunotherapy. Utilizing in vitro real-time quantitative live cell analysis we determined that OTS-hiPSCs elicit a significantly decreased cytotoxic response from both activated peripheral blood (PB)-NK cells and primed PB-T cells compared to wildtype controls. Furthermore we demonstrate that OTS-hiPSCs exhibit improved persistence in xenograft studies in vivo. Bilateral teratomas were formed in a non-conditioned, fully immune-competent recipient mice using luciferized wildtype and OTS-hiPSCs. Daily bioluminescence imaging over a period of 7 days revealed a significant increase (>50 fold difference) in persistence of OTS-hiPSCs compared to wildtype hiPSCs during the 60-144 hour post injection window. Lastly we demonstrate that OTS-hiPSCs can successfully differentiate into functional effector lymphocytes using our potent chemically-defined monolayer hematopoietic differentiation platform. Our current studies focus on the functional characterization of OTS-hiPSC-derived effector lymphocytes in humanized mouse models and generating increased potency of OTS-hiPSC-derived effector lymphocytes through precise genetic engineering of antigen targeting and costimulatory proteins to create and optimized source of "off-the-shelf" cell-based immunotherapies. Disclosures Bauer: Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Bjordahl:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics: Employment. Abbot:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Natural Killer (NK) cells play a crucial role in immunosurveillance and form a first line of defense against cancer. In comparison to other lymphocytes, NK cells are unique in their capability to elicit tumoricidal responses without the need for antigen presentation or prior sensitization. Clinical data from bone marrow transplant and allogeneic NK immunotherapy suggest that MHC mismatch is advantageous in promoting graft-versus-leukemia without eliciting graft-versus-host, providing evidence that NK cells hold promisa as an allogeneic, universal immunotherapeutic. Further, the anti-tumor effect of many monoclonal antibodies is mediated through binding of the low-affinity Fc receptor CD16 on NK cells, which induces tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC). Thus, NK cells represent a unique source of effector cells that can be combined with monoclonal antibodies, bispecific engagers or chimeric antigen receptors to direct tumor specificity and enhance cytotoxicity. Despite the significant potential of NK cell therapy, current clinical practices are limited by the need for large numbers of healthy NK cells, lack of in vivo persistence, and a burdensome manufacturing strategy that requires donor cell extraction, modulation, expansion and re-introduction per each patient. The ability to generate universally histocompatible and genetically-enhanced NK cells from continuously renewable human induced pluripotent stem cell (hiPSC) lines offers the potential to develop a true "off-the-shelf" cellular immunotherapy. While NK differentiation from hiPSC has been demonstrated, the clonal derivation of engineered hiPSCs to improve effector function has been challenging and the scalability and robustness of the differentiation method has been limited by skewed development towards primitive hematopoiesis and the cumbersome use of embryoid bodies. Here we highlight our "off-the-shelf" NK cell therapy preclinical program by demonstrating robust and highly scalable generation of functionally mature, genetically targeted and universally histocompatible NK cells. This program utilizes our previously described naïve hiPSC platform where we uniquely create clonal lines of precisely engineered, renewable hiPSCs and drive definitive hematopoiesis in a highly scalable manner. Because hiPSC differentiation is lineage directed, minimal cellular contamination is seen, including the lack of T and B cells, in the final product. Through precise genetic engineering of naïve hiPSC lines, we have engineered HLA-class I deficient NK cells uniformly expressing a high affinity, non-cleavable version of the Fc receptor CD16 (NcHaCD16-NK). The hiPSC-derived NcHaCD16-NKs display markers of maturity, including CD16, KIR, NCRs, and CD94. When compared to conventional cord blood and peripheral blood sourced NK cells, NcHaCD16-NKs exhibit superior cytotoxicity and production of effector cytokines in response to both solid and liquid tumor cell challenge in vitro. NcHaCD16-NKs exhibit augmented cytokine response following Fc-mediated stimulation, demonstrating function competence of the engineered CD16 construct. Because surface expression of CD16 is resistant to activation-induced shedding, NcHaCD16-NKs continuously maintain enhanced ADCC while retaining the capacity for general cytotoxicity. Importantly, the hiPSC-derived hematopoietic cells can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Preliminary data also shows NcHaCD16-NKs elicit preferred specificity for cancer stem cells as defined by expression of ALDH1 and surface markers such as CD24. In conclusion, the outlined preclinical data demonstrate the potential therapeutic utility of NK cells developed via precision genetic engineering of a renewable, scalable hiPSC platform, and highlights the therapeutic value of NcHaCD16-NKs as an ideal ADCC-mediated "off-the-shelf" NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity and preclinical efficacy. Disclosures Bjordahl: Fate Therapeutics, Inc: Employment. Clarke:Fate Therapeutics: Employment. Gaidarova:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Abujarour:Fate Therapeutics, Inc.: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics, Inc.: Employment. Kim:Fate Therapeutics, Inc.: Employment. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Cellular immunotherapies are poised to transform the treatment of cancer and immunological disorders. In the most promising setting to date, genetic modification to the T lymphocytes in the form of chimeric antigen receptors (CAR) has dramatically increased therapeutic efficacy with reported initial complete remission rates in acute lymphoblastic leukemia ranging between 80-100%. However, pressing challenges remain to be solved to ensure that engineered T-cell immunotherapies can be cost-effectively and consistently manufactured, and safely and reliably delivered at the scale necessary to support wide patient base commercialization. Human induced pluripotent stem cell (hiPSC) derived T lymphocytes represent a unique, renewable source of genetically engineered T cells for "off-the-shelf" immunotherapy. Through the precise genetic engineering at the hiPSC stage, clonal and uniform populations of modified cell lines can be banked and reliably tapped into on demand to generate highly efficacious T cells for therapeutic applications. Although great progress has been made, several challenges need to be addressed including the ability to enhance effector function through genome-engineering of persistence, targeting, histocompatibility and controlled safety mechanisms at the hiPSC juncture while retaining the capacity to efficiently and reproducibly generate the intricate stages of lymphocyte development in an accurate and scalable process. We have previously demonstrated that our proprietary reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous population of hiPSCs, our platform enables efficient multi-gene and multi-loci targeted engineering at a single cell level resulting in clonal population of pluripotent cell lines with desired genetic attributes. Here we will provide an update on our "off-the-shelf" T-cell immunotherapy preclinical program where engineered hiPSC lines are uniquely used as the renewable starting material. We will also highlight our novel differentiation platform to derive definitive hematopoietic progenitor cells termed hemogenic endothelium (HE); a well-defined, small molecule-driven, staged process that is currently being translated into cGMP (current good manufacturing practice) settings. The highly efficient differentiation system (on average >65% hiPSC to CD34 conversion) delivers approximately 100 CD34+ HE cells per each input hiPSC, representing a highly scalable process that is further expanded during lymphocyte differentiation and maturation. To validate that the iCD34+ HE is definitive in nature we demonstrate that during further hematopoietic differentiation the emerging CD43+ hematopoietic cells exhibit Notch dependency and high expression of key genes such as MYB and the HOXA cluster, found only in definitive hematopoietic progenitors. The hiPSC-derived HE exhibits multi-lineage potential and can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Through genetic modifications at the single cell hiPSC stage, we confer antigen-specificity via the expression of temporally inducible CARs as premature expression of CAR proteins during in vitro differentiation has been found to skew development towards innate-lymphoid like lineages. Utilizing our stage-specific hematopoietic differentiation platform we have identified the optimal developmental window to induce the expression of CAR proteins to maintain optimal differentiation towards functional effector lymphocytes. The hiPSC-derived engineered T lymphocytes are currently under preclinical investigation for in vitro and in vivo effector function including thymic rejuvenation, T cell repertoire repopulation, target specific recognition and enhanced killing potential. Preliminary data suggests that hiPSC-derived lymphocytes are functional and can home to their respective niche to support initial repopulation in vivo. Our study continues to support that naïve hiPSCs are an ideal renewal source for "off-the-shelf" hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive T cell therapy. Disclosures Clarke: Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Foster:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

Human induced pluripotent stem cells (hiPSCs) are a unique population of cells that can serve as an unlimited source for "off-the-shelf" cellular immunotherapeutics. Similar to master cell lines used in the manufacture of monoclonal antibodies, engineered hiPSC lines have the potential to serve as a renewable cell source for the consistent manufacture of homogeneous populations of effector cells for the treatment of thousands of patients. However, the creation of an effective master line is largely dependent on the ability to genetically edit hiPSCs in a precise, efficient and clonal manner. Furthermore, the genetically edited hiPSCs must maintain their inherent ability to continuously self-renew while retaining ability to express engineered modalities upon directed differentiation to the cell type of choice. We have previously reported the use of stage-specific media compositions to enable the footprint-free generation and long-term maintenance of single cell naïve hiPSCs with enhanced clonogenicity, an attribute critical for the derivation of engineered single cell-derived lines. Here we demonstrate the use of our naïve hiPSC platform to precisely introduce, in a site-specific manner, multiple genes into multiple safe harbor loci. By combining our single-cell naïve hiPSC platform with different nuclease-independent and -dependent strategies, we are able to generate large numbers of precisely engineered iPSC clones. The single cell-derived hiPSC clones were subsequently screened in a multiplexed fashion for successful multi-parameter engineering, maintained pluripotency and propensity for differentiation with lack of undesired phenotypes and genomic alterations. Using this approach, we derived individual clones containing a uniform population (>99%) of multi-engineered modalities consisting of tumor targeting, a controllable safety switch and a tracking marker. Moreover, we show that engineered modalities are expressed in undifferentiated and differentiated hiPSCs, including being expressed in >95% of both CD34 positive hematopoietic progenitor cells and CD56 positive natural killer cells. Furthermore, we have generated hiPSC clones with dual suicide genes (inducible Caspase 9 (iCasp9) and Herpes simplex virus thymidine kinase (HSV-TK)) targeted into two independent safe harbor loci, in both mono- and bi-allelic manner. The dual-targeted hiPSC clones were confirmed to have specific insertions into the predicted sites and were screened to exclude random insertions. Concurrent activation of both suicide genes led to complete elimination of engineered hiPSCs and no treatment-refractory cells were observed unlike the case when only one suicide gene was activated. In addition to robust targeted insertions, we were able to generate small insertions and deletions in up to 70% of naïve hiPSCs without selection and homozygous knockout of gene of interest in 100% of cells after selection. Finally, we will discuss efforts to temporally synchronize ectopic gene expression through endogenously regulated promoters by simultaneous endogenous gene disruption and transgene insertion. Overall, we show our naïve hiPSC platform is an ideal renewable source to efficiently generate, genetically engineer, isolate and bank clonally-derived homogenous population of pluripotent cells. These highly-stable pluripotent clonal banks can be repeatedly tapped to facilitate the consistent production of homogenous populations of potent, persistent, scalable and safer off-the-shelf cellular immunotherapeutics. Disclosures Abujarour: Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Meza:Fate Therapeutics: Employment, Equity Ownership. Robinson:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct antitumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3- placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3-, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46 and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile, immunophenotype and greater antitumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.

Clinically available red blood cells (RBCs) for transfusions are at high demand, but in vitro generation of RBCs from hematopoietic stem cells requires significant quantities of growth factors. Here, we describe the production of four human growth factors: erythropoietin (EPO), stem cell factor (SCF), interleukin 3 (IL-3), and insulin-like growth factor-1 (IGF-1), either as non-fused proteins or as fusions with a carrier molecule (lichenase), in plants, using a Tobacco mosaic virus vector-based transient expression system. All growth factors were purified and their identity was confirmed by western blotting and peptide mapping. The potency of these plant-produced cytokines was assessed using TF1 cell (responsive to EPO, IL-3 and SCF) or MCF-7 cell (responsive to IGF-1) proliferation assays. The biological activity estimated here for the cytokines produced in plants was slightly lower or within the range cited in commercial sources and published literature. By comparing EC50 values of plant-produced cytokines with standards, we have demonstrated that all four plant-produced growth factors stimulated the expansion of umbilical cord blood-derived CD34(+) cells and their differentiation toward erythropoietic precursors with the same potency as commercially available growth factors. To the best of our knowledge, this is the first report on the generation of all key bioactive cytokines required for the erythroid development in a cost-effective manner using a plant-based expression system.

Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14(+) monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells.

Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

The propensity of tumours to generate resistant clones is a major barrier to cytotoxic and hormone therapy. The vasculature of malignancies is a more appealing target, since tumour endothelial cells are not known to be tranformed or subject to somatic mutation or random phenotypic variation. Thus an effective anti-angiogenic therapy need not be vitiated by the acquisition of resistance. Although several antiangiogenic agents have been described and used in experimental models, there remain severe problems about optimising their use and about setting up informative clinical trials. These difficulties arise predominantly because of the “invisible” nature of tumour angiogenesis.

Abstract 3717 Human placenta has emerged as a valuable, uncontroversial source of transplantable cells for many cytotherapeutic purposes, including modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDAC) are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Multiple myeloma (MM) is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. The aim of the study was to evaluate the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDAC in the SCID-rab model of MM-associated bone disease. SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which primary human myeloma cells were directly injected (Yatta et al., Leukemia 2004; Yaccoby et al., Blood 2007). Bone disease was evaluated by measurements of bone mineral density (BMD) and X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and histologically. For in vivo tracking PDAC were transduced with a luciferase/GFP reporter in a lentiviral vector. SCID-rab mice engrafted with primary myeloma cells from 2 patients. Upon establishment of MM growth, PDAC (1×106 cells/bone) or vehicle were injected into the implanted myelomatous bone (Patient's 1, 5 mice/group; Patient's 2, 7 mice/group). While BMD of the implanted bones was significantly reduced in control hosts, intralesional PDAC cytotherapy significantly increased BMD of the implanted bones from pretreatment levels by >37% (p Disclosures: Khan: Celgene Cellular Therapeutics: Research Funding. Heidaran:Celgene Cellular Therapeutics: Employment. Pal:Celgene Cellular Therapeutics: Employment. Zhang:Celgene Cellular Therapeutics: Employment. He:Celgene Cellular Therapeutics: Employment. Zeitlin:Celgene Cellular Therapeutics: Employment. Abbot:Celgene Cellular Therapeutics: Employment. Faleck:Celgene Cellular Therapeutics: Employment. Hariri:Celgene Cellular Therapeutics: Employment.