Master Cell Line Research Articles

Abstract 5240: Novel CD3-Fusion Receptor enables combination of T-cell engagers and allogeneic CAR T cells to promote enhanced antitumor activity and overcome antigen escape

Abstract Chimeric antigen receptor (CAR) T cells have shown remarkable clinical success in the treatment of hematologic malignancies. However, the durability of response of CAR T therapy can be impaired by heterogenous expression of the CAR-specific target antigen (1° Ag) on tumors. In combination with a CAR, T-cell engagers (TCEs) that target additional tumor antigen (2° Ag) can be a unique approach to address tumor heterogeneity and overcome antigen escape. Engineered induced pluripotent stem cell (iPSC) master cell lines are a renewable source material from which iPSC-derived CAR T (CAR-iT) cells can be manufactured and administered in an off-the-shelf manner. In developing allogeneic CAR-iT cells, the T-cell receptor (TCR) must be eliminated to prevent graft-versus-host disease. However, the absence of TCR expression leads to loss of surface CD3 and a corresponding loss of TCE compatibility. Here we discuss a novel CD3ε fusion receptor (CD3-FR) with a modified transmembrane and endodomain, which allows for CD3 surface expression on TCR-less T cells and effective combination of off-the-shelf allogeneic CAR-T cells and TCEs. CAR+ CD3-FR+ iPSCs were differentiated into CAR-iT cells, uniformly expressing both modalities (>95% CAR+, >90% CD3ε+) and absent of TCR. To demonstrate that CD3-FR+ CAR-iT cells can elicit TCE-dependent activity, 1° Ag- (CD19-)/2° Ag+ (EpCAM+) target cells were used in cytotoxicity assays. As shown, CD3-FR- CAR-iT cells failed to kill 1° Ag- target cells. In contrast, CD3-FR+ CAR-iT cells demonstrated potent activity toward the target cells (>80% cytolysis) but only in the presence of the TCE targeting 2° Ag. To test the ability of CD3-FR+ CAR-iT cells to mitigate antigen escape, we used a tumor cell population heterogenous for the CAR-specific target (50% 1° Ag-) but homogenous for TCE-specific target (100% 2° Ag+). In co-culture assays, CD3-FR- CAR-iT cells failed to prevent tumor cell growth due to 1° Ag- tumor escape. However, CD3-FR+ CAR-iT cells in the presence of TCE exhibited robust control (80% cytolysis) of the heterogenous tumor, mitigating 1° Ag- tumor escape via targeting of 2° Ag. Next, we tested the ability of CD3-FR+ CAR-iT cells to control heterogeneous tumor in vivo in a disseminated leukemia model. As expected, CD3-FR+ CAR-iT cells exhibited significantly better tumor growth inhibition in the presence of TCE (p-value <0.005) compared to controls lacking the CD3-FR modality. Ex vivo analysis of tumor in the bone marrow confirmed 1° Ag- tumor escape in the control treated mice, but not in mice treated with CD3-FR+ CAR-iT and TCE targeting 2° Ag. Taken together, these studies present a novel opportunity to leverage a novel CD3 fusion receptor and enable combination of TCEs with engineered off-the-shelf CAR T-cell products, uniquely harnessing two potent therapeutic modalities for improving cancer therapy. Citation Format: Dan Lu, Eigen Peralta, Hui-Yi Chu, Soo Park, Masanao Tsuda, Earl Avramis, Matthew Denholtz, Alec Witty, Tom Lee, Bahram Valamehr. Novel CD3-Fusion Receptor enables combination of T-cell engagers and allogeneic CAR T cells to promote enhanced antitumor activity and overcome antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5240.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.MethodsA clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.ResultsWe successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.ConclusionsiPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.

Abstract 2919: A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy

Abstract Natural killer (NK) cells are innate immune cells that play a key role in in tumor immune surveillance. NK cells are self-tolerant to healthy cells and can kill tumor cells in both hematological and solid malignancies. Recent advances allow for the derivation of immune cells such as NK cells, from human induced pluripotent stem cells, that can be utilized for cancer immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-5 x 109Jaya cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable next generation, off-the-shelf, affordable, cancer immunotherapies. We have developed a novel method to produce high purity, clinical scale iNK cells and improved the process for the expansion of NK cells efficiently and consistently. This method does not require cell sorting step and the usage of murine derived stromal cells. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hematopoietic progenitor cells were subsequently differentiated into mature iNK cells in the absence of cell sorting and independent of stroma cells support. Using this novel method, differentiated NK cells could be expanded more than 8,000-fold to enable us to potentially produce 1 x 1012 iNK cells starting from 1 x 106 undifferentiated iPSC. This cell production scale can supply hundreds of doses in clinic from one cGMP manufacturing campaign. iNK cells produced using this method display the typical phenotype of NK cell activating receptor including NKG2D, NKp44, NKp46, DNAM-1 etc. Importantly, these iNK cells demonstrate better anti-tumor activities and higher levels of IFNγ and TNFα, compared to primary peripheral blood-derived NK cells isolated from healthy donors. Moreover, this protocol has been adapted and optimized for clinical scale manufacturing of iNK cells from genetically engineered, clonal master iPSC cell line with knock out of CISH (CISH KO iNK), a key intracellular checkpoint of NK cell anti-tumor immunity. CISH KO iNK cells produced using this optimized method show significantly better anti-tumor activities against multiple liquid and solid tumor cell lines including K562, Raji, Daudi, SUP-B15, MOLT-4, SKOV-3, OVCAR-4, HCC-827, ZR-75, and BT-474 etc. in vitro and in vivo in an AML xenograft mouse model, compared with unmodified iNK cells. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of iPSC-derived NK cells with increased potency, creating next-generation NK cell-based immunotherapies. Citation Format: Davide Bernareggi, Caryn Gonsalves, Max Schabla, Alejandra Gárate-Carrillo, Duygu Ozmadenci, Jaya Thangaraj, Qin Li, Leah Mitchell, Dan S. Kaufman, Robert Hollingsworth, Huang Zhu. A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy [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 2919.

An Optimized Protocol for piggyBac-Induced iPSC Generation from hPBMCs by Automatic Electroporation.

Thanks to the Yamanaka transcription factors, pluripotency is recovered in the cell culture dish during in vitro cell reprogramming. Induced pluripotent stem cells (iPSCs) have been introduced to the scientific world as a source of disease models, which are predominantly used in drug discovery and monitoring disease pathophysiology, and as a source of master cell lines for developing cellular therapies. Successfully attaining iPSCs requires careful optimization of many factors, including selection of the transfection method and the appropriate transfection agents; culturing conditions before and after transfection; recovery conditions after transfection, freezing, and thawing; storage conditions; and the choice of cost-effective cell and colony characterization and molecular verification steps. In our optimized procedure, we describe the isolation of peripheral blood mononuclear cells (PBMCs) after blood harvest and their efficient reprogramming into iPSCs using automated electroporator, with piggyBac.

A CD3 Fusion Receptor (CD3-FR) Uniquely Enables Compatibility of Allogeneic CAR-T and -NK Cells with T Cell Engagers to Enhance Antitumor Function and Limit Antigen Escape

Chimeric antigen receptor (CAR) T cells have shown remarkable clinical success in the treatment of many malignancies. However, certain challenges remain, including broad patient access and durability of response. We have shown that a scalable manufacturing platform, where genetically edited induced pluripotent stem cell (iPSC) master cell lines are created to serve as a renewable starting material for the derivation of uniformly engineered CAR T or NK cells, can facilitate broad patient access in an off-the-shelf manner. To improve durability of response, often plagued by antigen heterogeneity found in cancer, we are pursuing several combination strategies, merging cell therapy with therapeutic agents such as checkpoint blockade therapy and monoclonal antibodies to increase anti-tumor activity and multi-antigen targeting of cancer. T cell engagers have also shown remarkable advancements in the treatment of various cancer types, but they are also afflicted by their own challenges and would equally benefit from combination strategies. Unfortunately, T cell engagers are not compatible with allogeneic adoptive cell therapy, as the cells used in the allogenic setting do not express the CD3 molecule used for interaction with the engagers, as the T cell receptor (TCR) that supports CD3 surface expression, is either not expressed in NK cells or has been ablated in T cells to prevent graft-versus-host disease. To investigate the anti-tumor synergy between off-the-shelf cell therapy and T cell engagers, we developed a novel CD3ε fusion receptor (CD3-FR) to uniquely support the expression of a functional CD3 on TCR-less allogeneic T and NK cells and to enable compatibility between allogeneic cell therapy and T cell engagers. Initially, CD3-FR constructs with various co-stimulating endodomains were confirmed to elicit T cell engager-dependent NFAT activity in TCR alpha chain (TRAC) knockout (T-KO) Jurkat cells. Next, iPSCs already containing a CAR inserted into the TRAC locus were engineered to contain CD3-FR. CAR+ CD3-FR+ iPSCs were differentiated into CAR-iT cells, uniformly expressing both modalities (>95% CAR+, >90% CD3ε+, TCR not detected). To demonstrate that CD3-FR+ CAR-iT cells can elicit T cell engager-dependent antitumor activity, CAR antigen (1° Ag) negative but T cell engager (2° Ag) positive target cells were used in various cytotoxicity assays. As expected, control CAR-iT cells failed to kill the 1° Ag negative tumors despite addition of T cell engager targeting 2° Ag. On the other hand, CD3-FR+ CAR-iT cells showed potent activity toward target cells but only in the presence of the T cell engager (>80% cytolysis). Furthermore, when comparing the expression of activation markers in CD3-FR+ CAR-iT cells in co-cultures with or without the T cell engager, we observed an increased proportion of CD69+ (61% vs. 28%) and CD25+ (37% vs. 2%) cells in the CD3-FR+ CAR-iT cell co-cultures containing the T cell engager. Cytokine production was also tested, with increased TNF (5-fold) and IFNγ (7-fold) measured in supernatants from CD3-FR+ co-cultures upon the addition of T cell engager, further demonstrating T cell engager-dependent functionality and specificity of the CD3-FR construct. To test the ability of CD3-FR CAR-iT cells to mitigate 1° Ag escape, we used a target cell population heterogenous for the CAR-specific target (1° Ag, 50% positive) but homogenous for EpCAM (2° Ag, 100% positive). After three days, the standard CAR-iT cells failed to control tumor cell growth due to antigen escape. In contrast, CD3-FR+ CAR-iT cells exhibited robust control (80% cytolysis) of the 1° Ag heterogenous targets in the presence of αEpCAM, indicating mitigation of 1° Ag escape. Lastly, CD3-FR was edited into iPSC-derived NK (iNK) cells to demonstrate compatibility of CD3-FR+ NK cells with T cell engagers. An xCelligence-based killing assay was performed in the presence of a T cell engager, where CD3-FR+ iNK cells showed superior target cell killing (~80% lysis) after 96 hours compared to parental iNK cells (~30% lysis), demonstrating the potential for synergy between T cell engagers and NK cells (Figure 1). Taken together, these studies present a novel opportunity to combine T cell engager technology with off-the-shelf CAR-iT or iNK cell products, uniquely merging two powerful therapeutics whose combination was previously underappreciated in improving cancer therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD82 Is Sufficient to Uniquely Identify Pluripotent Stem Cell-Derived Hemogenic Endothelium with the Hematopoietic Lineage Potency to Give Rise to Bona Fide Lymphocytes

Chimeric antigen receptor (CAR)-T and natural killer (NK) cells represent compelling opportunities for the treatment of hematological malignancies and solid tumors. However, autologous and donor-derived allogeneic CAR-T and NK cell therapies are limited based on the quantity and quality of the starting engineered cells. We previously described our off-the-shelf induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) and CAR-NK (CAR-iNK) cell platforms that eliminate some of the hurdles associated with patient-derived cells by creating a homogenous cell product that is derived from a renewable bank of iPSC master cell lines. A major challenge in the manufacturing of iPSC derived immune cells is the derivation of hemogenic endothelium (HE), which is the rare and transient direct precursor of hematopoietic cells, making its in vitro specification from iPSCs inefficient and challenging. Furthermore, there is not a reliable marker or set of markers for the identification of HE in culture. HE identification has traditionally relied on detection of the transcription factor RUNX1 and the establishment of reporter cell lines, neither of which are suitable for the manufacture of HE cells destined to become therapeutic immune cells. We have established a cytokine driven protocol for the differentiation of iPSCs that improves the efficiency of the derivation of RUNX1+ HE cells. To identify candidate HE surface markers, we compared iPSC-derived cells containing a high percentage of HE, based on RUNX1 expression, to control differentiated cells containing a low percentage of HE, via a cell surface antibody array. Through the array we identified several surface markers that were expressed by a higher percentage of CD34+ endothelial cells compared to control CD34+ cells. Several of the candidates identified, including CD44, CD61, CD226 and CD143 have been previously identified by other groups as candidate HE markers, thereby validating our screening method. We identified CD82 as a novel candidate HE marker, with expression uniquely restricted to the CD34+ endothelial population and positively correlating with RUNX1 expression (Fig. 1A). Furthermore, we demonstrated via limiting dilution assays that HE is enriched in the CD34+CD43-CD82+ population (HE frequency, 1/38) compared to the CD34+CD43-CD82- population(HE frequency, 1/736) (Fig. 1B). Even more strikingly, isolation of cells based on CD82 alone was sufficient to enrich for HE cells (HE frequency, 1/44) and outcompeted CD34 enrichment alone, suggestive of its unique ability to identify the HE population (Fig. 1B). To determine if CD82+ HE cells could give rise to lymphocytes we carried them forward into T cell differentiation and showed thatthey efficiently gave rise to CAR-iT cells (i.e. >95% CD7+ T lymphocytes). The identification of CD82 for the enrichment of iPSC derived HE is a critical step to the development of definitive hematopoiesis with ability to give rise to all lineages of the hematopoietic compartment and facilitates the scaled and consistent manufacture of high-quality T and NK cells for off-the-shelf therapeutic applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FT555: Off-the-Shelf CAR-NK Cell Therapy Co-Targeting GPRC5D and CD38 for the Treatment of Multiple Myeloma

Recent improvement in patient outcomes with the use of novel cellular immunotherapies for multiple myeloma (MM) has raised the prospect for the emergence of a curative treatment. While BCMA-targeted chimeric antigen receptor (CAR)-T cell therapies have been successful in treating MM, CAR-T cell manufacturing challenges preventing broad patient access and treatment relapse drive the need for additional targeted therapies with emphasis on multi-antigen targeting and off-the-shelf availability. GPRC5D, a tumor-associated orphan G-protein-coupled receptor found to be highly expressed in MM, is a potentially attractive target that has demonstrated promising clinical benefit when targeted via immunotherapy modalities. Here, we describe the development of FT555, an induced pluripotent stem cell (iPSC)-derived CAR-NK (CAR-iNK) cell product with the unique and effective ability to simultaneously co-target GPRC5D and CD38 (an additional tumor-associated antigen of MM) via combination with daratumumab, and which can be mass produced and is available off-the-shelf to support broad patient access. FT555 is a CAR-NK cell derived from an iPSC master cell line that has been multiplexed-engineered at the clonal level to contain four unique modalities; a novel GPRC5D-specific CAR fine-tuned for NK cell biology; a high-affinity, non-cleavable CD16 (hnCD16) to maximize antibody-dependent cellular cytotoxicity (ADCC) when combined with a monoclonal antibody (mAb); a unique IL-15/IL-15 receptor fusion protein (IL15RF) to promote cytokine-independent function; and CD38 knockout to promote NK cell fitness and uniquely prevent anti-CD38 mAb-mediated fratricide. Sourced from a renewable engineered iPSC master cell line, FT555 is a pure population of engineered NK cells (>95% CD56+) exhibiting uniform expression of CAR-GPRC5D (>90%), hnCD16 (>90%) and IL15RF (>90%), with complete elimination of CD38 expression (not detected). In cytotoxicity assays, FT555 shows antigen specificity, dose-dependent potency and importantly, when combined with the anti-CD38 mAb daratumumab, exhibits resistance to fratricide. In a serial restimulation killing assay, FT555 demonstrates persistent tumor-specific activity against GPRC5D-positive MM.1S WT target cells when compared to isogenic GPRC5D knock-out (KO) targets (70.9% control of tumor by AUC against WT targets vs. 3.5% control against GPRC5D KO targets in the third round of stimulation). When used in combination with daratumumab, FT555 targets CD38 and eliminates CAR-resistant GPRC5D KO target cells (57.1% target killing when combined with daratumumab vs. 3.5% target killing in the absence of mAb in the third round of stimulation), demonstrating the capacity for FT555 to target GPRC5D+ and CD38+ cells through CAR and hnCD16 engagement, respectively. In a disseminated xenograft in vivo model of MM where the MM.1S cell line shows comprehensive tumor engraftment, FT555 demonstrates robust killing kinetics and tumor clearance as a single dose, resulting in control of MM progression for up to 42 days and increased survival to 80 days vs. 37 days for the untreated control arm (Figure 1A, 1B, FT555; 99.9% tumor growth inhibition (TGI) at D37, 151% increased lifespan (ILS)). Durability of FT555 is further enhanced with the addition of daratumumab, where tumor growth inhibition deepened and survival improved, with 2 of 5 mice showing complete clearance of tumor cells at Day 80, demonstrating the synergy of anti-tumor activity between CAR and hnCD16 (Figure 1A, 1B, FT555+Dara; 100% TGI at D37, >207% ILS). Furthermore, in a distinct xenograft model of MM, treatment with FT555 resulted in significantly improved TGI against OPM2 tumor targets (FT555; 100% TGI at D51, p < 0.05). Together these studies demonstrate that FT555 is a multiplexed-engineered CAR-NK cell derived from a clonal master iPSC line, which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy with daratumumab to simultaneously target both GPRC5D and CD38 in a single, standardized, and scalable off-the-shelf platform. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Using recombinase-mediated cassette exchange to engineer MIN6 insulin-secreting cells based on a newly identified safe harbor locus.

Aims/IntroductionRecent studies have identified genomic and transcript level changes along with alterations in insulin secretion in patients with diabetes and in rodent models of diabetes. It is important to establish an efficient system for testing functional consequences of these changes. We aimed to generate such a system using insulin‐secreting MIN6 cells.Materials and MethodsMIN6 cells were first engineered to have a tetracycline‐regulated expression system. Then, we used the recombination‐mediated cassette exchange strategy to explore the silencing‐resistant site in the genome and generated a master cell line based on this site.ResultsWe identified a site 10.5 kbps upstream from the Zxdb gene as a locus that allows homogenous transgene expression from a tetracycline responsible promoter. Placing the Flip/Frt‐based platform on this locus using CRISPR/Cas9 technology generated modified MIN6 cells applicable to achieving cassette exchange on the genome. Using this cell line, we generated MIN6 subclones with over‐ or underexpression of glucokinase. By analyzing a mixed population of these cells, we obtained an initial estimate of effects on insulin secretion within 6 weeks. Furthermore, we generated six MIN6 cell sublines simultaneously harboring genes of inducible overexpression with unknown functions in insulin secretion, and found that Cited4 and Arhgef3 overexpressions increased and decreased insulin secretion, respectively.ConclusionsWe engineered MIN6 cells, which can serve as a powerful tool for testing genetic alterations associated with diabetes, and studied the molecular mechanisms of insulin secretion.

Multi-Functional Genetic Engineering of Pluripotent Cell Lines for Universal Off-the-Shelf Natural Killer Cell Cancer Immunotherapy

Since the initial identification of natural killer (NK) cells, considerable effort has been made to harness their inherent anti-tumor capacities for the treatment of cancer. Despite recent clinical advancements utilizing the adoptive transfer of allogeneic NK cells, particularly with haplo-identical NK cells for acute myeloid leukemia, significant opportunities remain to further augment the anti-tumor activity of NK cells. Clinical trials have shown the persistence of adoptively transferred NK cells to be generally correlated with improved patient outcome. Natural killer cell persistence can be enhanced through several strategies, including by the administration of long-lived sub-populations such as adaptive-memory NK cells or of NK cells that are genetically modified to prolong survival or attenuate rejection by the host immune system. However, conventional sources of primary allogeneic NK cells have proven to be highly variable and difficult to genetically modify.Human induced pluripotent stem cells (hiPSCs) are unique in their capacity to self-renew and differentiate into any specialized cell type of the body. In addition, hiPSCs can be readily genetically-edited, and single hiPSCs can be clonally selected for the creation of engineered master pluripotent cell lines. As such, hiPSCs represent a renewable and reliable source for generating off-the-shelf engineered cell therapy products that are homogenous and display augmented anti-tumor properties.To enhance the persistence of hiPSC-derived NK cells, we employed a multi-dimensional strategy for hiPSC modification. First, hiPSCs were derived from fibroblast cells through a proprietary cellular reprogramming process that promotes telomere elongation. Second, hiPSCs were genetically-engineered to express cell surface-bound IL-15 to promote NK cell expansion during hiPSC differentiation and survival upon adoptive transfer. Third, additional hiPSC modifications were introduced to disrupt cell surface HLA Class I presentation and to drive cell surface expression of the non-classical HLA molecule HLA-G, to protect against host-mediated rejection by CD8+ T cells and NK cells, respectively. Lastly, we explored the influence of NKG2C to promote the adaptive-memory NK cell phenotype.We observed that hiPSC-derived NK cells displayed telomere length that was on average ~2-fold longer as compared to peripheral blood NK cells. The introduction of surface-bound IL-15 supported enhanced proliferation during differentiation with minimal requirements for cytokine support. Disruption of HLA-Class I expression decreased the proliferation of allogeneic CD8+ T cells upon challenge with modified hiPSCs as compared to wild-type hiPSCs (64% for wild-type vs. 29% for modified). This modification increased the cytotoxic activity of NK cells against modified hiPSCs (50% killing at 66 hours for modified as compared to 94 hours for wild-type). To this end, we further modified the hiPSCs to express the non-classical HLA molecule HLA-G. HLA-G protected against NK cell cytotoxicity and extended median survival of modified hiPSCs from 35 hours to 58 hours in an in vitro co-culture system. Finally, engineered hiPSC-derived NK cells can be effectively expanded (>1000x in 14 days) to support an efficient central manufacturing and banking method that enables multi-dose strategies to further support product persistence. Combined, the data suggest that our multi-engineered strategy can support the generation of a universal hiPSC backbone for the derivation of NK cells with improved persistence and anti-tumor efficacy. We are currently adding targeting modalities such as enhanced CD16 or chimeric antigen receptor expression to the discussed universal hiPSC backbone to generate master pluripotent cell lines to enable the manufacture of off-the-shelf NK cell cancer immunotherapy products. DisclosuresBjordahl:Fate Therapeutics: Employment, Equity Ownership. Clarke:Fate Therapeutics Inc.: Employment, Equity Ownership. Gaidarova:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics Inc.: Employment, Equity Ownership. Groff:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment, Equity Ownership. Lan:Fate Therapeutics Inc.: Employment, Equity Ownership. Abujarour:Fate Therapeutics Inc.: Employment. Bonello:Fate Therapeutics Inc.: Employment. Robinson:Fate Therapeutics Inc.: Employment. Hardy:Fate Therapeutics: Employment. Valamehr:Fate Therapeutics: Employment, Equity Ownership.

KPMW135, a Biosuperior CD3 Bispecific Version of Rituximab Created By a Novel Chemical Conjugation Technology Demonstrates Increased Anti-Tumor Activity By Adding T Cell-Mediated Cytotoxicity Activity to the Existing Mechanisms of Rituximab

Background We describe KPMW135, a novel CD20 x CD3 bispecific molecule, in which a CD3-directed ScFv (SP34) is conjugated to rituximab (RTX). RTX is a chimeric monoclonal antibody (mAb) specific for CD20, used to treat B-cell lymphomas and lymphocytic leukemias. Monoclonal Ab therapy enhancer (MATE™) enables efficient site-directed chemical conjugation to "off-the-shelf" therapeutic mAbs and allows the development of bispecific therapeutic candidates. Our MATE platform allows for the chemical engineering of existing Abs without the need to create new DNA vectors or genetic engineering of master cell lines. This approach is superior to previous conjugation methods that 1) lack site-directed conjugation specificity by indiscriminately binding to available amino acid residues or 2) require genetic engineering to create conjugate tags. Methods SP34 scFv was constructed using published CDRs. The expression was in FreeStyle 293 cells, followed by affinity purification and SEC to obtain monomeric scFv. The binding of CD20 to KPMW135 was measured by Bio-Layer Interferometry using Octet (Fortebio). Equilibrium dissociation constant (Kd) was calculated using the Octet HT software. The binding of CD3εδ, CD16a, and FcRn were determined by ELISA using neutravidin-coated plates. Biotinylated Human CD3ε &amp; CD3δ heterodimer protein (Avi tag), CD16a (Avi Tag) and human CD16a were used. Readout was determined with anti-human F(ab) HRP. For in vitro T cell-mediated cytotoxicity assays, unfractionated and NK-cell depleted PBMCs were prepared from freshly-thawed and PHA + IL-2 prestimulated PBMCs. Daudi (CD20+) B lymphoblast cells were engineered to stably express a beta-gal reporter fragment using KILR retroparticles (Eurofins). Target cells were treated with KPMW135, RTX, and controls. PBMCs were introduced at an effector:target ratio of 15:1 and incubated for 18h. Luminescence signal was obtained with luminometer to reflect target cell death. Cynomolgus monkeys were intravenously injected with RTX or KPMW135 (30 μg/kg). Endpoints included clinical observations, cytokine profile and flow cytometric immunophenotyping of T cells, monocytes, granulocytes, NK cells, and B cells (CD45, CD3, CD16, CD14, NKG2A, HLA DR) and cellular activation (CD44 and CD69). Results Binding affinity of CD20 to KPMW135 was 0.75nM. CD20 binding to RTX and KPMW135 was similar, thus conjugation of the CD3-binding ScFv did not negatively affect affinity to CD20. ELISA results show KPMW135 binds to CD3εδ, FcRn, and CD16a, indicating that the conjugation does not interfere with binding to their various moieties. In vitro T cell-mediated cytotoxicity results show the activity of Fc/FcR and CD20 binding by the RTX moiety versus CD3 binding to effector T cells. T cell enrichment by functionally prestimulated PBMCs increased killing of target cells by 2-fold. Mechanically depleted NK cells verified that target cell death was induced by T cells, rather than by NK cells via ADCC. KPMW135 elicited target cell killing with an EC50 of 0.03-0.07nM or 0.09-021nM for prestimulated or freshly thawed PBMCs, respectively. Studies in Cynomolgus monkeys showed that KPMW135 can activate T cells in vivo as shown by increased CD69 and CD44 expression (3- and 2-fold, respectively) with a peak at 4h post-dose compared to equivalent dose RTX. A pronounced depletion of B cells was observed in KPMW135 treated animals as early as half hour post-dose, with a partial recovery by days 7-14, while an equivalent dose of RTX induced a transient B-cell depletion followed by a quicker return to baseline levels. Conclusions KPMW135 is a CD20 x CD3 bispecific created by chemical conjugation of CD3-specific ScFv to "off-the-shelf" RTX using our proprietary MATE™ technology, which maintains RTX native binding to FcRs via the Fc domain and its native binding to CD20 on target cells. KPMW135 retains RTX native ability to target B lymphoid malignancies via Fc effector mechanisms like ADCC and ADCP, but now adds T cell-mediated cytotoxicity. Our in vitro data shows increased target cell killing by KPMW135 compared to RTX, and our in vivo study confirms KPMW135 ability to induce target dependent T cell activation and recruitment, making it superior to unconjugated RTX. CD3 MATEs generated with other Abs show similar increased T cell-mediated cytotoxicity, confirming that this technology can be applied to generate "off-the-shelf" biosuperior antibodies. Disclosures Vidal: Kleo Pharmaceuticals: Current Employment, Current equity holder in private company. Cukan:Kleo Pharmaceuticals: Current Employment. Vaill:Kleo Pharmaceuticals: Current Employment. Bunin:Kleo Pharmaceuticals: Current Employment. Rossi:Kleo Pharmaceuticals: Current Employment. Iben:Kleo Pharmaceuticals: Current Employment. Trinh:Kleo Pharmaceuticals: Current Employment. McGrath:Kleo Pharmaceuticals: Current Employment. Berbasova:Kleo Pharmaceuticals: Current Employment. Alvarez:Kleo Pharmaceuticals: Current Employment. Rastelli:Kleo Pharmaceuticals: Current Employment. Welsch:Kleo Pharmaceuticals: Ended employment in the past 24 months.

Comprehensive Analysis of Genomic Safe Harbors as Target Sites for Stable Expression of the Heterologous Gene in HEK293 Cells.

Human cell lines are being increasingly used as host cells to produce therapeutic glycoproteins, due to their human glycosylation machinery. In an attempt to develop a platform for generating isogenic human cell lines producing therapeutic proteins based on targeted integration, three well-known human genomic safe harbors (GSHs)-AAVS1, CCR5, and human ROSA26 loci-were evaluated with respect to the transgene expression level and stability in human embryonic kidney (HEK293) cells. Among the three GSHs, the AAVS1 locus showed the highest eGFP expression with the highest homogeneity. Transgene expression at the AAVS1 locus was sustained without selection for approximately 3 months. Furthermore, the CMV promoter showed the highest expression, followed by the EF1α, SV40, and TK promoters at the AAVS1 locus. Master cell lines were created using CRISPR/Cas9-mediated integration of the landing pad into the AAVS1 locus and were used for faster generation of recombinant cell lines that produce therapeutic proteins with recombinase-mediated cassette exchange.

A system for site-specific integration of transgenes in mammalian cells.

Mammalian cell expression systems are the most commonly used platforms for producing biotherapeutic proteins. However, development of recombinant mammalian cell lines is often hindered by the unstable and variable transgene expression associated with random integration. We have developed an efficient strategy for site-specific integration of genes of interest (GOIs). This method enables rapid and precise insertion of a gene expression cassette at defined loci in mammalian cells, resulting in homogeneous transgene expression. We identified the Hipp11 (H11) gene as a "safe harbor" locus for gene knock-in in CHO-S cells. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated homologous recombination, we knocked in a DNA cassette (the landing pad) that includes a pair of PhiC31 bacteriophage attP sites and genes facilitating integrase-based GOI integration. A master cell line, with the landing pad inserted correctly in the H11 locus, was established. This master cell line was used for site-specific, irreversible recombination, catalyzed by PhiC31 integrase. Using this system, an integration efficiency of 97.7% was achieved with green fluorescent protein (GFP) after selection. The system was then further validated in HEK293T cells, using an analogous protocol to insert the GFP gene at the ROSA26 locus, resulting in 90.7% GFP-positive cells after selection. In comparison, random insertion yielded 0.68% and 1.32% GFP-positive cells in the CHO-S and HEK293T cells, respectively. Taken together, these findings demonstrated an accurate and effective protocol for generating recombinant cell lines to provide consistent protein production. Its likely broad applicability was illustrated here in two cell lines, CHO-S and HEK293T, using two different genomic loci as integration sites. Thus, the system is potentially valuable for biomanufacturing therapeutic proteins.

Abstract LB-073: Generation of novel single cell-derived engineered master pluripotent cell line as a renewable source for off-the-shelf TCR-less CAR T cells in support of first-of-kind clinical trial

Abstract Adoptive transfer of autologous T cells expressing chimeric antigen receptor (CAR) has shown great promise in the treatment of blood malignancies. Challenges for the application of current CAR T cell therapies to broader and more diverse patient populations include inherent variability, cost of manufacture, and the requirement for precise genetic engineering to generate a highly homogenous and consistent CAR T cell product. We have previously reported pre-clinical data supporting the development of FT819, a first-of-kind off-the-shelf CAR T cell product candidate. FT819 is generated from a renewable clonal master human induced pluripotent stem cell (hiPSC) line derived from a single cell engineered to contain bi-allelic disruption of the T cell receptor (TCR) and a novel CD19 CAR targeted into the T cell receptor α constant (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease. For the manufacture of a clinical-grade FT819 clonal master hiPSC line, we sourced peripheral blood mononuclear cells from a fully consented and eligible donor with protocol overseen by an independent Institutional Review Board. Sourced T cells were enriched (&amp;gt;98%) through positive selection for TCRαβ, and cryopreserved cells were confirmed to have stable genome by karyotyping. Using our proprietary non-integrating cellular reprogramming platform, αβ T cells were reprogrammed into hiPSCs. Concurrently with the reprogramming process, reprogrammed cells received nuclease and donor template to mediate targeting of CD19 CAR into the TRAC locus with bi-allelic knockout of the TCR. To generate clonal lines, engineered cells were sorted by flow cytometry for various markers and single cells were seeded into individual wells of feeder-free 96-well plates. hiPSC clones were screened for bi-allelic integration of CAR into the TRAC locus by amplifying the genomic DNA flanking the homologous recombination site and confirmed by a SNP phasing assay. Clones were further screened for random integration of donor template by quantitative PCR (qPCR), and the CAR copy number was confirmed by droplet digital PCR. Out of 545 hiPSC clones screened, 27 clones (5%) had bi-allelic TRAC targeting with no detectable random integration. Maintenance of pluripotency was confirmed in 19 out of the 27 engineered hiPSC clones (70%). Seventeen clones were further tested and were confirmed to be footprint-free of transgenic reprogramming factors. Of the 18 clones tested for genomic stability, 12 clones had normal karyotypes (67%). Validated, TRAC-targeted hiPSC clones were cryopreserved (~150 vials per clone) and are currently being assessed for off-target editing, differentiation propensity into highly-functional T cells, genomic stability, clone identity, sterility and lack of mycoplasma detection. In summary, using our novel iPSC technology platform for reprogramming, single cell engineering and multiplex high-throughput screening of hiPSCs, we have generated clinical-grade clonal master hiPSC lines in support of our first-of-kind clinical trials evaluating FT819 allogenic off-the-shelf hiPSC-derived TCR-less TRAC-CAR19 T cells for the treatment of blood malignancies. Citation Format: Ramzey Abujarour, Yi-Shin Lai, Mochtar Pribadi, Tom Lee, Megan Robinson, Chelsea Ruller, Sjoukje Van der Stegen, Xiuyan Wang, Jolanta Stefanski, Juan Zhen, Jason Dinella, Greg Bonello, Janel Huffman, Helen Chu, Raedun Clarke, Alec Witty, Amanda Medcalf, Jaeger Davis, Stacey Moreno, Pieter Lindenbergh, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. Generation of novel single cell-derived engineered master pluripotent cell line as a renewable source for off-the-shelf TCR-less CAR T cells in support of first-of-kind clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-073.

Functional Assessment for Clinical Use of Serum-Free Adapted NK-92 Cells.

Natural killer (NK) cells stand out as promising candidates for cellular immunotherapy due to their capacity to kill malignant cells. However, the therapeutic use of NK cells is often dependent on cell expansion and activation with considerable amounts of serum and exogenous cytokines. We aimed to develop an expansion protocol for NK-92 cells in an effort to generate a cost-efficient, xeno-free, clinical grade manufactured master cell line for therapeutic applications. By making functional assays with NK-92 cells cultured under serum-free conditions (NK-92SF) and comparing to serum-supplemented NK-92 cells (NK-92S) we did not observe significant alterations in the viability, proliferation, receptor expression levels, or in perforin and granzyme levels. Interestingly, even though NK-92SF cells displayed decreased degranulation and cytotoxicity against tumor cells in vitro, the degranulation capacity was recovered after overnight incubation with 20% serum in the medium. Moreover, lentiviral vector-based genetic modification efficiency of NK-92SF cells was comparable with NK-92S cells. The application of similar strategies can be useful in reducing the costs of manufacturing cells for clinical use and can help us understand and implement strategies towards chemically defined expansion and genetic modification protocols.

Pluripotent Cell-Derived Off-the-Shelf TCR-Less CAR-Targeted Cytotoxic T Cell Therapeutic for the Allogeneic Treatment of B Cell Malignancies

▪The advent of off-the-shelf chimeric antigen receptor (CAR) T cell therapeutics is widely recognized to be a major potential advancement for the treatment of cancer. Several obstacles currently hamper the broad use of CAR T cells, including the inherent variability and cost of manufacturing of autologous cellular populations, the absolute requirement for precise genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity. Here we present pre-clinical data for FT819, a first-of-kind off-the-shelf human induced pluripotent stem cell (hiPSC)-derived CAR T cell product. FT819 is defined by the precise genetic engineering of multiple targeting events at the single cell level to create a clonal master iPSC line. The engineered features include the targeted integration of a novel, modified CD19 CAR into the T cell receptor α (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease (GvHD), and the expression of a high-affinity, non-cleavable form of CD16 (hnCD16) to deliver an adjustable system to address tumor antigen escape.Through a proprietary cellular reprogramming platform, peripheral blood derived T cells are converted to hiPSCs, engineered to contain the modified CD19 CAR targeted into the TRAC locus and hnCD16, and clonally selected to create a master hiPSC line (TRAC-TiPSC, FT819). Molecular characterization of the TRAC-TiPSC master cell line by 5‘ junction, 3‘ junction and internal sequence PCR confirmed homology directed repair and bi-allelic targeting of the CD19 CAR into the TRAC locus. The origin of the clonal master cell bank was confirmed to be a TCRαβ T cell by PCR-mediated detection of TCRδ locus deletion and methyl-seq analysis of the TCRα locus. Flow cytometric analysis demonstrated the maintenance of a uniform population of hiPSCs (>95% SSEA4/TRA-1-81/OCT4/NANOG) and expression of hnCD16 transgene (>95% CD16).Utilizing our stage-specific T cell differentiation protocol, we demonstrate that the TRAC-TiPSCs yield TRAC-iT cells with uniform expression of the CAR (>95%), complete elimination of TCR surface expression and clinically enabling expansion through the manufacturing process (>50,000 fold). To confirm the lack of alloreactivity conferred by the deletion of endogenous TCR expression, mixed lymphocyte reactions were performed using TRAC-iT, primary TCR+ T cells and primary TCR+CAR+ T cells as responders and HLA-mismatched peripheral blood mononuclear cells (PBMCs) as targets. In comparison to primary T cells and primary CAR-T cells, TRAC-iT did not respond and proliferate in response to TCR stimulation or HLA-mismatched PBMCs indicating that the risk of GvHD was alleviated.In vitro functional studies established that TRAC-iT possess a potent cytotoxic T lymphocyte response to CD19 antigen challenge in a similar manner to peripheral blood CAR T cells as demonstrated by expression of markers indicative of degranulation (CD107a/b, Granzyme B), T cell activation (CD69, CD25), and production of INFγ, TNFα and IL2. Importantly, TRAC-iT targeted tumor in an antigen specific manner as verified by lysis of CD19+, but not CD19-, tumor cell lines as seen by in vitro cytolytic assays (50% killing E:T; TRAC-iT = 1:8, primary CAR-T = 1:4).In vivo studies demonstrated that TRAC-iT cells effectively control tumor progression in a mouse model of acute lymphoblastic leukemia Nalm6 (TRAC-iT versus no treatment, p<0.0001). To validate the capability of TRAC-iT to simultaneously target multiple antigens, TRAC-iT was co-cultured with mixtures of CD19+CD20+ and CD19-CD20+ tumor cells in the presence of anti-CD20 monoclonal antibody, Rituxan. In vitro cytolytic assays demonstrate that only TRAC-iT cells can effectively identify and eliminate CD19-CD20+ tumor cells when combined with Rituxan. Importantly, the antibody-dependent cellular-cytotoxicity did not appear to interfere with CAR function as TRAC-iT maintained its directed cytotoxic capacity. Collectively, these preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf product than can be effectively and safely used in the treatment of B cell malignancies in the allogeneic setting. DisclosuresClarke:Fate Therapeutics Inc.: Employment. Chang:Fate Therapeutics Inc.: Employment. Husain:Fate Therapeutics Inc.: Employment. Lai:Fate Therapeutics Inc.: Employment. Peralta:Fate Therapeutics Inc.: Employment. Stokely:Fate Therapeutics Inc.: Employment. Abujarour:Fate Therapeutics Inc.: Employment. Dinella:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Pribadi:Fate Therapeutics Inc.: Employment. Chu:Fate Therapeutics Inc.: Employment. Truong:Fate Therapeutics Inc.: Employment. Sabouri-Ghomi:Fate Therapeutics Inc.: Employment. Meza:Fate Therapeutics Inc.: Employment. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Sadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Screen and Verification for Transgene Integration Sites in Pigs

Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs. After CRISPR/Cas9-mediated site-specific integration on Pifs501, we detected high EGFP expression in different pig cell types and ubiquitous EGFP expression in diverse tissues of transgenic pigs without adversely affecting 600 kb neighboring gene expression. Promoters integrated on Pifs501 exhibit hypomethylated modification, which suggest a permissive epigenetic status of this locus. We establish a versatile master cell line on Pifs501, which allows us to achieve site-specific exchange of EGFP to Follistatin with Cre/loxP system conveniently. Through in vitro and in vivo functional assays, we demonstrate the effectiveness of this screening method, and take Pifs501 as a potential site for transgene insertion in pigs. We anticipate that Pifs501 will have useful applications in pig genome engineering, though the identification of genomic safe harbor should over long-term various functional studies.

Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.

Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease

Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.

Abstract 609: Overcoming host histocompatibility barrier to create a renewable source of off-the-shelf effector lymphocytes for adoptive immunotherapy

Abstract 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 (&amp;gt;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 (&amp;gt;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

Abstract 3755: Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy

Abstract 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 (&amp;gt;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 (&amp;gt;95% CD16+ hnCD16-NK vs. &amp;lt;10% CD16+ PB-derived NK cell, upon target cell-mediated activation), and demonstrated enhanced antibody binding compared to PB-derived NK cells expressing the low-affinity variant. In addition to increased expression of the cytolytic molecules perforin and granzyme B and enhanced direct cytotoxicity against tumor targets, hnCD16-NK cells displayed superior ADCC capacity and cytokine production in response to CD16 stimulation. Importantly, manufacture of hnCD16-NK cells was proven to be highly scalable, delivering up to 107 fold expansion over a 35 day period. The maintained proliferative capacity can be in part associated with longer telomere length seen in hnCD16-NK cells. Furthermore, deletion of classical human leukocyte antigen molecules and ectopic expression of immunosuppressive proteins engineered at the iPSC level provided the ability of hnCD16-NK cells to potentially overcome the host histocompatibility barrier and to improve persistence in the allogeneic setting. In conclusion, the preclinical data presented herein highlight the therapeutic value of hnCD16-iNK cells as an ideal ADCC-mediated “off-the-shelf” NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity, manufacturing reliability and preclinical efficacy. 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