For some reason the links don't work, so I will add the abstracts below.
Development of Master Multiplexed-Engineered iPSC Bank for Off-the-Shelf Cell-Based Cancer Immunotherapy with Reduced Conditioning Chemotherapy
Induced pluripotent stem cells (iPSCs) are a unique renewable starting material for the manufacture of off-the-shelf immune cells and offer the advantages such as enhanced product uniformity, reduced cost, and on-demand availability. Current practice of both autologous and allogeneic cell-based cancer immunotherapy requires administration of systemic conditioning chemotherapy, which suppresses the patient’s immune system in order to potentiate the adoptively-transferred immune cells. However, the intensity and frequency of immune suppression can also lead to increased risk of life-threatening complications, such as severe infections. New therapeutic strategies that enable potentiation of adoptively-transferred immune cells without substantially ablating the patient’s immune system may significantly improve the therapeutic paradigm of cell-based cancer immunotherapy. Here, we describe a novel off-the-shelf, iPSC-derived CAR NK (CAR-iNK) cell derived from a master multiplexed-engineered iPSC line with the potential to maintain functional persistence in the background of an intact host immune system. The multiplexed-engineered iPSC line incorporates six unique functional elements: 1) a CAR for targeting of plasma cells; 2) IL-15/IL-15 receptor fusion protein (IL-15RF) for enhanced NK cell activity; 3) a high-affinity, non-cleavable CD16 (hnCD16) for enhanced antibody-dependent cellular cytotoxicity (ADCC); 4) beta 2 microglobulin (B2M) deletion for resistance to host CD8 T cell-mediated rejection; 5) Class II transactivator (CIITA) deletion for resistance to host CD4 T cell-mediated rejection; and 6) CD38 deletion for resistance to fratricide when combined with anti-CD38 monoclonal antibody (mAb) to eliminate host allo-reactive immune cells. The multiplexed-engineered iPSC line was derived using our proprietary iPSC product platform. In the first stage, a clonal master iPSC line was established incorporating IL-15RF and hnCD16 at the CD38 locus. In the second stage, iPSCs from the clonal master iPSC line were further engineered to knock-out B2M and CIITA and knock-in the CAR. Single-cell selection was performed at each stage to assess for specific targeted integration, biallelic disruption of desired loci, and the lack of random donor template integration. Transgene copy numbers were further confirmed by droplet digital PCR. Importantly, karyotype analysis ensured genome stability of these clonal master iPSC lines, a testament to the robustness of the platform in enabling staged multiplexed engineering and single-cell selection. The master multiplexed-engineered iPSC line incorporating six functional elements was used to produce CAR-iNK cells, which were assessed for persistence and anti-tumor functionality. The successful production of these CAR-iNK cells supported the notion that the engineered modalities did not impact CAR-iNK cell differentiation and expansion (>95% CD56, >95% CAR, >95% CD16). In vitro functional studies including antigen-specific cytokine release assays, mixed lymphocyte reaction, and NK cell cytotoxicity assays in the presence of peripheral blood mononuclear cells demonstrated functional persistence from allo-reactive immune cells. In vivo studies with xenograft mouse model are ongoing. Collectively, the data provide evidence of the robust capability of our proprietary iPSC product platform to support multi-loci engineering of iPSCs at the single-cell level, the creation of master multiplexed-engineered iPSC lines, and the production of next-generation, off-the-shelf CAR-iNK cell therapies for use in minimally-conditioned patients.
Long-Term Stability of iPSC-Derived CD34+ Cell Banks Supports the Sustainable Manufacture of Off-the-Shelf Immunotherapies
Human induced pluripotent stem cells (hiPSC) have the unique dual properties of unlimited self-renewal and differentiation capacity into all three somatic cell lineages. To further leverage these attributes, we have established a versatile iPSC product platform that enables multiplexed engineering of hiPSCs at the single-cell level and have developed a proprietary differentiation protocol to support definitive hematopoiesis for the derivation of CD34+ hematopoietic progenitor (iCD34) cells. We have shown that these iCD34 cells exhibit multilineage differentiation to diverse subsets of immune cells, including Natural Killer (NK) and aß T cells. The highly efficient and scalable differentiation platform is chemically-defined and cGMP-compatible, and iCD34 cells can be cryopreserved, banked and stored. Here we show the successful cryopreservation and long-term cryogenic storage (2, 4, and 6 years) of iCD34 cells, as well as the ability of these long-term stored iCD34 cells to serve as an intermediate feedstock for mass production of iPSC-derived NK and T (iNK and iT, respectively) cell therapies.
A diverse set of iCD34 cells, including those engineered with a high-affinity, non-cleavable CD16 (hnCD16) and/or with an anti-CD19 chimeric antigen receptor (CAR19), were manufactured, cryopreserved, and stored in the vapor phase of liquid nitrogen at ≤ -150°C for up to 6 years. To determine the impact of long-term cryopreservation on cell quality, long-term stored iCD34 lots were thawed and assessed for viability, recovery, phenotype, differentiation potential and iNK / iT cell functional potency. Post-thaw viability and enumeration was determined by trypan blue dye exclusion or acridine orange/propidium iodide staining in conjunction with Annexin V staining by flow cytometry, which was found to be similar to recently cryopreserved batches. Phenotypic flow analysis showed consistent identity and purity with pre-cryopreservation trends. To evaluate their differentiation potential, iCD34 cells were subsequently differentiated and expanded into iNK and iT cells. Flow cytometry analysis confirmed iNK and iT cell identity and purity, and differentiation yields were comparable with recently cryopreserved batches. Lastly, functionality was assessed via antibody dependent cellular cytotoxicity (ADCC) and antigen-specific cytotoxicity. In combination with monoclonal antibodies, hnCD16-iNK cells continued to show enhanced ADCC and production of the pro-inflammatory cytokines against antigen-bearing tumor cell lines. Similarly, CAR19-expressing iNK and iT cells maintained antigen-specific activity CD19+ tumor cell lines in various cytotoxicity assays.
This study demonstrates that manufactured, cryopreserved, and stored iCD34 cells are stable for a minimum of 6 years in the vapor phase of liquid nitrogen and shows that cryopreserved iCD34 cells can serve as a robust starting material for mass production of iPSC-derived cell-based immunotherapies. We observed no changes in vitality, phenotype, differentiation potential or iNK and iT cell potency. Taken together, our studies show that long-term stored iCD34 cells can serve as an intermediate feedstock for rapid mass production of multiplexed engineered iNK and iT cell therapies.
Generation of Human Myeloid Derived Suppressor Cells from Induced Pluripotent Stem Cells (IPSC) for Graft versus Host Disease Therapy
Allogeneic hematopoietic stem cells transplantation (aHSCT) is a widely used treatment for hematological disorders. Graft versus host disease (GVHD), a donor anti-host cell tissue destructive response, is a life-threatening aHSCT complication. Front line pharmaceutical treatment is not uniformly effective and has toxic side-effects. Myeloid derived suppressor cells (MDSC) are a heterogenous population of immature myeloid cells which are immunosuppressive. Their potential in GVHD therapy has been proven in different rodent models. However, a high MDSC to T cell ratio and multiple doses of MDSC are needed for GVHD therapy. But in vitro generation of MDSC limits its clinical usage by low yield and the need for personalized patient products. To remove the barriers that limit its application, we developed a method to generate MDSC in vitro from human iPSC derived CD34+ cells. We achieved a large-scale of cell expansion using OP9-DLL4 mouse stromal cells, cytokines to support CD34 differentiation followed those for myeloid lineage. In 19 days, 205-1085 iMDSC were derived from one CD34 of which 98% of iPSC derived MDSC (iMDSC) were CD45+CD33+, typical for a myeloid lineage phenotype. As with peripheral blood (PB) MDSC human iMDSC generated in our culture system are composed of two major populations: monocytic MDSC (51% CD45+CD33+ CD14+) and granulocytic MDSC (11% CD45+CD33+CD66b+). In contrast, PB MDSC expansion was < 5-fold. MDSC possess immunosuppressive function, key for a successful GVHD therapy. To investigate this property of iMDSC, we applied our iMDSC in a T cell proliferation assay and compared the potency of the immune suppressive effect to PB MDSC. iMDSC inhibited anti-CD3/28 bead driven proliferation of CD4 and CD8 T cells by 56% and 57%, respectively, at 1:2 (iMDSC:PBMC) while a correspondently lower % suppression was seen for CD4 (39%) and CD8 (30%) cells . Suppression was contact-dependent and associated with increased inhibitory receptor, resulting in decreased Teffector cell proinflammatory cytokine expression. We next investigated the suppressive capability of monocytic CD14+ vs granulocytic CD14- iMDSC subsets. We found that the %proliferation of CD4 (57%) and CD8 (55%) T cells suppressed by CD14+ iMDSC compared favorably to CD14- iMDSC for CD4 (19%) and CD8 (9%) at the same ratio. Previous studies have shown that LPS and ATP which are released due to tissue injury in the GVHD environment will induce inflammasome activation that we reported subverted in vivo suppression of murine GVHD lethality by bone marrow derived MDSC. To our surprise, iMDSC still retained 95% of their suppressive function after the LPS and ATP induced inflammasome activation while PB MDSC lost 77% of their suppressive function. Since MDSC have been reported to be sensitive to freeze-thaw that will be important for off-the-shelf clinical application, we investigated the function of iMDSC from frozen stock and found that iMDSC remained 86% of their function after the freeze thaw cycle. In summary, we developed a method for generating MDSC from human iPSC on a large scale. These iMDSC had potent immunosuppressive function and were resistant to inflammasome and freeze-thaw induced function loss facilitating the use of iMDSC as a potential alternative to or adjunct for GVHD therapy.
Long-Term Stability Assessment of IPSC-Derived T and NK Cells Support the Feasibility of Off-the-Shelf Therapeutic Applications
Cell-based immunotherapies have shown remarkable promise in the fight against various cancers. Induced pluripotent stem cell (iPSC)-derived natural killer (NK) and T (iNK and iT, respectively) cells can be mass produced and administered off-the-shelf to patients, and several iPSC-derived cell-based cancer immunotherapies are now undergoing human clinical testing. Our iPSC product platform leverages the use of clonal master iPSC lines that serve as the starting material for the manufacture of multiplexed-engineered, cell-based cancer immunotherapies that can be fully characterized, stored, and administered on-demand to patients. Here we demonstrate long-term stability (1, 3 and 6 years) of our engineered iNK and iT cell drug product candidates following long-term cryogenic storage. Multiple iNK and iT cell product candidates, including those engineered with a high-affinity, non-cleavable CD16 (hnCD16) and/or with an anti-CD19 chimeric antigen receptor (CAR19), were manufactured, cryopreserved and stored in the vapor phase of liquid nitrogen for up to 6 years. To assess the impact of cryogenic storage on these product candidates, cryopreserved lots were thawed and assessed for cell health, recovery, identity and functionality. Post-thaw recovery and viability was evaluated using acridine orange/propidium iodide staining and Annexin V staining by flow cytometry, and we found cell health was consistent with newly frozen cells suggesting that stability was maintained over years of cryogenic storage. Phenotypic analysis via flow cytometry was used to monitor product identity and purity and shown to be similar to pre-cryopreservation cells. Further, functionality was evaluated via pro-inflammatory cytokine production, antigen-specific cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC). Tumor necrosis factor-alpha and interferon-gamma production and secretion were examined in response to PMA/ionomycin or tumor cell lines via intracellular flow cytometry and electrochemiluminescence immunoassays and demonstrated consistent pro-inflammatory cytokine production. Notably, cryopreserved CAR-expressing iNK and iT cells maintained antigen-specific cytotoxicity against leukemia and lymphoma tumor cell lines in cytotoxicity assays, and hnCD16-expressing cells continued to show enhanced ADCC toward tumor cell lines when combined with tumor-targeting monoclonal antibodies. As there is no accelerated method established to model the shelf-life of cryopreserved cell therapies, periodic examination of stored drug product against release criteria provides the most thorough and accurate assessment of the feasibility of long-term storage. This study demonstrated that iNK and iT cells, including those that are genetically engineered, have a minimum shelf-life of 6 years when stored in the vapor phase of liquid nitrogen. No significant changes in viability, cell recovery, phenotype and tumor-killing potency were observed. Collectively, the data illustrate that engineered iNK and iT cell products can be mass produced, cryopreserved, and long-term stored for use as off-the-shelf cell-based cancer immunotherapy.
iPSC-Derived CD38-Null NK Cells in Combination with CD38-Targeted Antibody Represent a Novel Therapeutic Strategy to Avoid Host Immune Cell Rejection for Off-the-Shelf Cell-Based Cancer Immunotherapy
Conditioning chemotherapies that temporarily suppress a patient’s immune system are commonly used in both autologous and allogeneic cell-based cancer immunotherapy. However, the intensity and frequency of lympho-conditioning can impact immune reconstitution and increase a patient’s susceptibility to adverse outcomes, such as severe infections. Genetic deletion of cell-surface human leukocyte antigen (HLA) molecule expression has long been known to abrogate T-cell alloreactivity. Loss of class I HLA elicits NK cell-mediated recognition and attack though, and therefore additional immune-modulating strategies must be applied. To this end, mouse models using immune cells expressing certain inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell alloreactivity. However, in the human system, HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells. Likewise, the expression of signal regulatory protein alpha (SIRPa), the major interactor for CD47, is mostly restricted to human macrophages and dendritic cells and not human NK cells. In this study, we provide details of a novel therapeutic strategy that combines engineered iPSC-derived NK (iNK) cells that uniformly lack CD38 surface expression with anti-CD38 antibodies to avoid host immune cell alloreactivity for off-the-shelf cell therapy. When testing iNK cells engineered with knockout of beta-2-microglobulin (B2M KO) to ablate HLA class I expression, B2M KO iNK cells were depleted over time in mixed lymphocyte reaction (MLR) assays containing peripheral blood mononuclear cells (PBMCs), suggesting activation of a “missing self” response by PBMC-containing NK cells. To overcome this mechanism of NK cell alloreactivity, we also genetically knocked-out CD38 (CD38 KO) to derive CD38 KO / B2M KO iNK cells having the potential to avoid anti-CD38 antibody-mediated fratricide. When combined with CD38-targeted antibody, the depletion of B2M KO CD38 KO iNK cells was abrogated as expected, and B2M KO CD38 KO iNK cell numbers were increased by 3.5-fold, comparable to the iNK cell numbers cultured without PBMCs. In contrast to these observations seen with B2M KO CD38 KO iNK cells, the combination of anti-CD38 antibody with B2M KO iNK cells resulted in fratricide and reduction of iNK cell counts. B2M KO iNK cells impaired expansion of PBMC-containing T cells over the duration of co-culture, resulting in 50% lower fold T-cell expansion at the peak of the control response, while B2M WT iNK cells stimulated T-cell activation and depleted iNK cells over time. However, parallel co-cultures maintained in the presence of anti-CD38 antibodies showed complete iNK cell resistance (both B2M KO and B2M WT iNK cells) to allogeneic PBMC attack, suggesting that anti-CD38 antibodies affords some control of allogeneic T-cell responses as well. Ongoing in vivo studies suggest that co-administration of anti-CD38 antibodies can significantly enhance the persistence of iNK cells in the presence of allogeneic PBMCs as seen in the blood, spleen and bone marrow. These data demonstrate the potential advantages of combining iPSC-derived CD38-null NK cells with anti-CD38 antibodies as a novel therapeutic strategy for reducing conditioning chemotherapy, depleting alloreactive lymphocytes, and promoting off-the-shelf cell therapy.
