Abstract Immune cell therapy has proven highly effective for the treatment of multiple myeloma (MM). However, key challenges remain that include disease relapse, limited patient access, and inability to effectively combine with existing standard-of-care therapies. Rapid progress in the development of off-the-shelf, multiplexed-engineered, induced pluripotent stem cell (iPSC)-derived cell therapies enables large-scale manufacture of immune cells incorporating multiple novel synthetic controls of cell function to improve cell fitness, enhance cell function, and enable synergistic combination with existing effective therapies such as CD38-targeted antibody (mAb) therapy. We have developed an iPSC-derived chimeric antigen receptor T (CAR-iT) cell therapy that uniquely leverages elements of both adaptive and innate immunity by incorporating a BCMA-targeted CAR (BCMA-CAR) derived from a scFv domain exhibiting high-binding affinity in the low nanomolar range, and a high-affinity, non-cleavable CD16 (hnCD16) Fc receptor to enable antibody-dependent cellular cytotoxicity with mAb therapy. The genetic deletion of CD38 gene was also incorporated to eliminate the possibility for CD38-mediated fratricide, and genetic deletion of TRAC gene was introduced to remove the potential risk of graft-versus-host disease in an allogeneic setting. These CAR iT cells, which were generated from a clonally-derived iPSC line, demonstrated homogenous expression of each genetic edit (>95% BCMA-CAR and hnCD16; <1% CD38 and TCR surface expression). Using a stringent, disseminated xenograft mouse model of multiple myeloma, MM.1S, which was allowed to achieve complete systemic engraftment during the initial 4 days, treatment with BCMA-CAR iT cells demonstrated comparable tumor cell clearance (p=0.0024 vs. vehicle at Day 17) to primary BCMA-CAR T cells (p=0.002 vs. vehicle). In combination with a CD38-targeted mAb to exploit hnCD16 and biallelic CD38 KO, BCMA-CAR iT cells are capable of dual-antigen targeting to address antigen escape and promote durable tumor control. To this end, a single dose of BCMA-CAR iT cells combined with daratumumab exhibited near complete TGI for the duration of the study (p<0.0001 vs. vehicle at Day 37). Together, these studies demonstrate CAR iT cells incorporating a high-avidity BCMA CAR and high-affinity, non-cleavable CD16 Fc receptor can uniquely leverage elements of both adaptive and innate immunity and can be combined with CD38-targeted mAb to potentially outcompete primary BCMA CAR T cells. As these CAR iT cells can be administered off-the-shelf, key challenges associated with current immune cell therapy, such as patient access and inability to synergize with standard-of-care therapies, can be addressed for the treatment of relapsed/refractory MM. Citation Format: John Reiser, Alison O'Connor, Bryan Hancock, Spas Markov, Brian Groff, Alma Gutierrez, Miguel Meza, Mark Jelcik, Yijia Pan, Alex Garcia, Bobby Goulding, Matthew Denholtz, Tom Lee, Ramzey Abujarour, Ryan Bjordahl, Armin Rehm, Raedun Clarke, Jode Goodridge, Bahram Valamehr. High-avidity BCMA CAR and high-affinity, non-cleavable CD16 Fc receptor incorporated in off-the-shelf CAR T cells promote multi-antigen targeting and durable anti-tumor cytotoxicity in the treatment of multiple myeloma [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 3618.

Recent clinical data have shown that autologous anti-CD19 CAR T cells can achieve durable disease remission across several autoimmune diseases (AID) through the elimination of aberrant CD19 expressing (CD19+) B cells, akin to their therapeutic profile in B cell malignancies (BCM). Similar to BCM, the application of CAR T cell therapy across the spectrum of AID faces significant challenges, including the need to target multiple disease-causing cell types and the requirement for patients to receive intense conditioning chemotherapy (CCT), which induces cytopenia, that increases the risk of severe infection and the development of secondary malignancies, and, notably, necessitates patient hospitalization and treatment at specialized centers. To reduce CCT-related toxicities, enable broad outpatient access, and promote durable elimination of AID-associated pathogenic cells, we have developed a multiplexed-engineered, next-generation (NxG) CAR T cell therapy derived from a clonal induced pluripotent stem cell line (CAR iT cell). These NxG CAR iT cells incorporate a suite of novel synthetic edits which are designed to specifically target and eliminate multiple subsets of aberrant immune cells through enhanced homing and bio-distribution to secondary and tertiary tissues, and to mitigate the need for intense CCT administration. Anti-CD19 NxG CAR iT cells consist of a clonal population of genetically edited cells that are deficient in endogenous TCRα chain expression, exhibit a CD8αβ T cell phenotype comparable to that of a potent cytotoxic T cell poised for activation, and lack expression of canonical exhaustion markers following CAR-mediated activation. Moreover, anti-CD19 NxG CAR iT cells demonstrate robust and specific elimination of CD19+ B cells in multiple in vitro cytotoxicity assays, including those containing patient peripheral blood mononuclear cells (PBMCs) as well as those designed to test NxG CAR iT-cell cytotoxic durability over multiple rounds of target cell challenge. In xenograft murine models, where disseminated disease resides in primary, secondary and tertiary tissues, anti-CD19 NxG CAR iT cells displayed tissue-wide functional persistence, including durable elimination of CD19+ B cells in the bone marrow. The addition of a second CAR, targeting B cell maturation antigen (BCMA), to anti-CD19 NxG CAR iT cells showed robust elimination of both CD19 and BCMA expressing B and plasma cells, respectively (target cell depletion >95% p<0.001 vs single CAR containing controls). Similarly, the addition of an anti-CD38 CAR also exhibited enhanced engagement and elimination of aberrant B cells, plasma cells and activated T cells. Both dual-CAR targeting strategies, CD19xBCMA and CD19xCD38, serve as innovative approaches to eliminate many aberrant immune cell types, which can collectively drive disease pathology, and to promote complete remission in AID. These dual-CAR strategies are also applicable in BCM settings, such as lymphoma and multiple myeloma where multiple aberrant immune cells are implicated in disease progression. To overcome the need for administration of intense CCT, NxG CAR iT cells also contain a novel allo-immune defense receptor (ADR), which has been shown to promote CAR T cell expansion, function, and persistence in an allogeneic setting. ADR-armed NxG CAR iT cells displayed enhanced persistence and cytotoxicity in an in vitro allogeneic re-stimulation assay, relative to control T cells without ADR, eliminating target cells through multiple rounds of re-challenge in the presence of alloreactive PBMCs. Similarly, ADR-armed NxG CAR iT cells maintained tumor growth inhibition and persistence in vivo in the presence of unmatched T cells. Importantly, NxG CAR iT cells can be reproducibly manufactured at scale to support off-the-shelf availability and cost-effective utilization. Collectively, NxG CAR iT cells represent a promising off-the-shelf approach to cell therapy for the treatment of AID and BCM, with the unique potential to elicit durable elimination of an array of aberrant immune cells, to avoid toxicities associated with intense CCT, and to maximize patient access and reach.

Aim: We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Methods: Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). Results: iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by in vitro and in vivo. Conclusion: CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine. Combined chemical and CRISPRa-mediated approach leads to efficient generation of human iPSCs.

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

Adoptive T-cell therapy with chimeric antigen receptor (CAR) has shown promising results in cancer treatment, however, antigen escape and tumor heterogeneity are major causes for disease relapse. While CARs are known to trigger an effective immune response through surface antigen recognition many solid tumor cancer antigens are intracellular and presented by MHC molecules recognized by T cell receptors (TCRs). In addition, many therapeutic antibodies have shown clinical efficacy in solid tumor settings. However, antibody-dependent cellular cytotoxicity (ADCC) is mediated by the CD16 Fc receptor naturally expressed on NK cells although its application in T cells is yet not fully appreciated. Utilizing our proprietary induced pluripotent stem cell (iPSC) platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-T cells (CAR-iT cells), we aimed to study the combination of these three targeting modalities, CAR, TCR, and CD16, to determine whether challenges associated with the treatment of solid tumors, which are heterogeneous and challenging to treat, may be overcome.To test the base line activity of CAR-iT cells in the solid tumor setting, we selected our anti-MICA/B CAR, previously shown to effectively target stress ligands found on transformed cells, to demonstrate effective anti-tumor activity against multiple solid tumor cell lines (72 hrs cytotoxicity: A2058 = 99%; 786-O = 98%; versus non-specific CAR-iT cells: A258 = 13%; 786-O = 17%). To test compatibility of TCR in our iT cell platform, we engineered MR1-TCR in iT cells to show increased cytokine release and degranulation upon stimulated with MR1 positive lung carcinoma epithelial cells line A549 (fold change compared to un-stimulated: IFNg = 210, p = 0.0032; TNFa = 76.9, p = 0.0005; CD107ab = 115.0, p=0.0013). Notably, with the engineering of tumor antigen specific TCR in TCR-less CAR-iT cells, CD3 complex can be re-established to provide an opportunity to combine with bispecific T cell engagers. Lastly, combining CAR-iT cells with MR1-TCR and hnCD16 uniquely demonstrated synergistic tumor growth inhibition and validated our approach to target multiple antigens at once for an effective anti-tumor response (A549 cytotoxicity: tumor only = 3.68±2.04%; effector+TCR = 41.31±2.27%; effector+TCR+ADCC = 90.28±1.87%). In summary, using the unique approach to engineer iPSCs at the clonal level to create a distinct population of engineered iT cells, we successfully demonstrated the compatibility between CAR, TCR, and hnCD16 to mitigate tumor heterogeneity. This approach is an ideal strategy to create off-the-shelf cellular immunotherapy for a promising therapeutic approach to combat heterogeneous and difficult to treat solid tumors, including those that are resistant due to antigen escape. Citation Format: Chia-Wei Chang, Bi-Huei Yang, Eason Lin, Soheila Shirinbak, Wen-I Yeh, Mochtar Pribadi, Helen Chu, Alma Gutierrez, Earl Avramis, Jason ORourke, Tom Lee, Alec Witty, Eigen Peralta, Martin Hosking, Bahram Valamehr. Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2756.

Background Antigen escape and tumor heterogeneity remain significant hurdles to the development of curative treatments in many cancers. To address tumor heterogeneity, the intro-duction of pairs of chimeric antigen receptors (CARs) in donor T cells has been demonstrated, however, this adds com-plexity to a manufacturing process already challenged by cellular product consistency. To tackle tumor heterogeneity while maintaining product purity, we applied our induced pluripo-tent stem cell (iPSC)-derived T cell (iT) platform to design an off-the-shelf cell therapy capable of targeting multiple tumor antigens through complementary activation pathways, including targeting of both cell-surface antigens as well as intracellular/ neoantigens. cell disseminated in vivo model was used to mimic cancer heterogeneity and to evaluate the in vivo potency of tri-modal iT cells at mitigating tumor heterogeneity and antigen escape. Results Assessment of individual edits in tri-modal iT cells demonstrated independent functionality by exhibiting increased antigen-mediated IFN g and TNF a production, and degranula-tion compared to the control group (p<0.0001). Using 9-day daily restimulation assay, each edit produced significant tumor reduction compared to tumor only control (p<0.0001). By stimulating tri-modal iT cells with multiple antigens simultane-ously using various solid tumor lines (A549, Caski and MDA-MB-231), we found that co-activation by two or three targeting edits significantly enhanced tumor killing (p<0.0001). Fur-thermore, when challenged with in vivo heterogenous tumor models, we found that the co-activation of all three targeting moieties in tri-modal iT cells achieved nearly complete tumor clearance (p<0.0001). Ex vivo bone marrow analysis further confirmed antigen-specific target elimination, reenforcing the specificity and potency of the tri-modal iT cells. Conclusions Our data highlight the potency and broad applic-ability of tri-modal iT cell expressing CAR, TCR, and hnCD16. This consistent and scalable approach to multiplex-engineered T-cell therapy is an ideal strategy to mitigate antigen escape and combat difficult to treat heterogeneous solid tumors.

FT819 is a first-of-kind, allogeneic, off-the-shelf CAR T-cell therapy derived from a clonal master induced pluripotent stem cell (iPSC) line precisely engineered to insert a novel 1XX anti-CD19 chimeric antigen receptor (CAR) under the regulation of the T-cell receptor alpha constant (TRAC) locus for optimized control of anti-tumor activity and to completely delete T-cell receptor (TCR) expression to eliminate the potential of graft-versus-host disease (GvHD). Unlike conventional allogeneic CAR T-cell therapies which require repeatedly sourcing of T cells from various donors as the starting material, the use of a clonal master engineered iPSC line serves as a renewable starting cell source and ensures routine mass production of a uniformly engineered, homogenous CAR T-cell product for broad patient access.

Chimeric antigen receptor (CAR) is known to trigger an effective immune response through surface antigen recognition enhanced by T-cell activation signal one (ex. CD3) and signal two (ex. CD28); however, targeting neoantigens and intracellular antigens remains a challenge. On the other hand, the T-cell receptor (TCR) can target neo/intracellular antigens presented by MHC molecules, but often the response is not as potent. The CD16 Fc receptor, which is naturally expressed on NK cells, mediates antibody-dependent cellular cytotoxicity (ADCC), but its application in T cells is yet not fully appreciated.

Abstract Genetic engineering of T cells using a chimeric antigen receptor targeting CD19 antigen (CAR19) is now a well-established treatment of B cell malignancies. While cellular immunotherapies are entering front line treatment, substantial limitations currently hamper the broad application of adoptive T cell therapies in diverse patient population including dysfunctional starting material, lack of product consistency and purity post genetic engineering and inefficient quantity produced for true on-demand availability. FT819 is a first-of-kind off-the-shelf CAR19-T cell product generated from a renewable pluripotent stem cells for large-scale clinical manufacturing. We previously reported the engineering and characterization of the FT819 clonal master cell bank (MCB) derived from a single cell comprising targeted integration of a novel CD19 1XX CAR into the T-cell receptor (TCR) α constant locus to provide optimally regulated CAR expression and elimination of graft versus host (GvH) response. Here we preview the nonclinical study for the original investigational new drug application of FT819. Derived in a manufacturing process analogous to pharmaceutical drug product development, pilot runs from the MCB demonstrated FT819 can be consistently and uniformly manufactured in cGMP compliance, cryopreserved at clinical scale to support off-the-shelf clinical application with greater than 1e5 fold increase in cellular yield from the starting MCB and can be thawed and directly used for facilitated treatment. Repeatedly, FT819 displayed a uniform product profile of ≥95% CAR+, TCR-, CD45+, CD7+ and CD3+ [intracellular] with majority of CD8 T cells expressing CD8β. FT819 global gene expression profile displayed high similarity to primary CAR19-T cells confirming its identity as a T lymphocyte. Functional assessment demonstrated that FT819 possesses potent antigen specific cytolytic activity against leukemia and lymphoma cell lines (p=0.0004). Additional specificity studies demonstrated on-target, off-tumor cytolysis of CD19+ B cells in mixed lymphocyte reaction assay (85% lysis of CD19+ B cells versus < 2% lysis of T cells). Inability of FT819 to produce a GvH response was confirmed in a co-culture assay with anti-TCR crosslinking antibodies. Disseminated leukemia xenograft mouse studies demonstrated the ability of directly thawed and infused FT819 to control tumor growth (p=0.0003 at day 21). In a systemic administered leukemia model FT819 also showed sustained localization in the bone marrow up to 45 days post injection. Ongoing in vivo studies will assess long-term survival and avoidance of GvH disease. Collectively, these studies demonstrate that FT819 is a potent, consistent and uniform CAR19 T cell product and can be effectively and safely used off-the-shelf in the treatment of B cell malignancies with an original Phase 1 clinical trial planned in 2020. Citation Format: Mili Mandal, Raedun Clarke, Sjoukje van der Stegen, Chia-Wei Chang, Yi-Shin Lai, Alec Witty, Mushtaq Husain, Cheng-Jang Wu, Bi-Huei Yang, Chad Dufaud, Gloria Hsia, Helena Shaked, Laurel Stokely, Helen Chu, Mochtar Pribadi, Gilberto Hernandez, Jason ORourke, Alma Gutierrez, Ramzey Abujarour, Tom Lee, Jolanta Stefanski, Juan Zhen, Meilan Wu, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. FT819 path to IND: First-of-kind off-the-shelf CAR19 T-cell for B cell malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3245.

The development of chimeric antigen receptor (CAR) T cell therapeutics is widely recognized as a significant advancement for the treatment of cancer. However, several obstacles currently impede the broad use of CAR T cells, including the inherent process variability, cost of manufacturing, the absolute requirement for precise and uniform genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity and tumor growth. Utilizing our previously described induced pluripotent stem cell (iPSC)-derived T (iT) cell platform, we illustrate here the unique ability to address these challenges by creating a consistent CAR iT cell product that can be repeatedly manufactured in large quantities from a renewable iPSC master cell bank that has been engineered to mitigate the occurrence of graft versus host disease (GvHD), antigen escape and tumor relapse. Utilizing our proprietary cellular reprogramming and engineering platform and stage-specific T cell differentiation protocol, we demonstrate that iPSC can be engineered at the single cell level to generate a fully characterized clonal iPSC line, which can then be accessed routinely to yield CAR iT cells in a highly scalable manufacturing process (>100,000 fold expansion). Through bi-allelic targeting of a CAR into the T cell receptor alpha constant (TRAC) region, we generated CAR iT cells with uniform CAR expression (99.0 ± 0.5% CAR+) and complete elimination of T cell receptor (TCR) expression to avoid GvHD in the allogeneic setting. We elected to utilize the 1XX-CAR configuration, which has demonstrated superior anti-tumor performance relative to other CAR designs and when introduced into iT cells displayed enhanced antigen specificity (% specific cytotoxicity at E:T=10:1, antigen positive group: 86.4 ± 7.8; antigen null group: 8.9 ± 3.5). To enhance persistence without reliance on exogenous cytokine support, we engineered signaling-fusion complexes, including IL-7 receptor fusion (RF), into iPSC and studied its impact on iT phenotype, persistence, and efficacy. In vitro, IL-7RF clones demonstrated improved anti-tumor activity in a serial antigen dependent tumor challenge assay (Day 10, relative tumor counts, IL-7RF group: 1.95 ± 0.01; control group: 57.56 ± 4.55, P<0.000001). In a preclinical in vivo model of disseminated leukemia, IL-7RF clones demonstrate enhanced tumor growth inhibition (Day 34, Log [BLI], IL-7RF group: 6.68 ± 1.93; control group: 9.99 ± 0.23, P=0.0143). We next investigated a unique strategy to incorporate multi-antigen targeting potential into anti-CD19 1XX CAR iT cells with the addition of a high-affinity non-cleavable CD16 (hnCD16) Fc receptor. The combination of hnCD16 with anti-CD19 1XX CAR culminated in iT cells capable of multi-antigen specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. Utilizing CD19 negative leukemia cells as targets, superior antibody-dependent cellular cytotoxicity (ADCC) was demonstrated by the combination of hnCD16 CAR iT and Rituximab (% specific cytotoxicity at E:T=1:1, hnCD16 group + Rituximab: 75.64 ± 2.12; control group + Rituximab: 16.98 ± 3.87, P<0.001). To address T cell fitness, the role of CD38 knockout (KO) in T cells was investigated, which we have previously shown to mediate NK cell resistance to oxidative stress induced apoptosis. CD38 gene was disrupted at the iPSC stage to generate 1XX-CAR T cells that lack CD38 expression (% CD38+ population, CD38WT group: 69.67 ± 24.34; CD38KO group: 0.12 ± 0.11) and upon antigen mediated stimulation, CD38KO CAR iT cells showed higher percentages of degranulation (2.3-fold increase in CD107a/b), and IFNγ (4.1-fold increase) and TNFα (2.5-fold increase) production. Antigen specific in vitro tumor killing also was enhanced in CD38KO CAR iT cells (EC50, 3.2-fold decrease). Lastly, to avoid the potential host-mediated rejection, the inclusion of allogeneic defense receptor (ADR) which has been shown to significantly reduce host-mediated rejection will be discussed. Collectively, the described studies demonstrate that iPSCs are an ideal cellular source to generate large-quantities of uniformly multi-edited off-the-shelf CAR T cell products that include a best-in-class CAR design, enhanced product modalities, and complete elimination of TCR expression to avoid the potential of GvHD while maintaining high anti-tumor efficacy in allogeneic setting. Disclosures Hsia: Fate Therapeutics Inc.: Current Employment. Clarke:Fate Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Lee:Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Long-term follow-up of adoptive transfer of autologous T cells expressing a chimeric antigen receptor (CAR) directed to CD19 antigen has demonstrated encouraging, durable clinical outcome in various B cell malignancies. However, to make such CAR-T cells available to a broader base and to reach a more diverse patient population, challenges associated with product consistency, cost of manufacture, precision genetic engineering and on-demand availability still need to be addressed. FT819 is a first-of-kind off-the-shelf CAR-T cell product candidate derived from a renewable master pluripotent cell line. FT819 comprises precise genetic engineering of multiple targeting events at the single cell level and is produced using a clonally-derived master cell bank (MCB) that serves as the starting material to support consistent and reproducible clinical manufacturing. The engineered features of FT819 include the targeted integration of a novel CD19 1XX-CAR into the T-cell receptor α constant (TRAC) locus to provide antigen specificity, enhanced efficacy and temporally-regulated CAR expression driven by an endogenous (TCR) promoter. Such features are designed to also eliminate the possibility of graft versus host disease (GvHD) by nullifying the TCR. To develop the MCB for FT819, αβ T cells were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently engineered to direct CD19 1XX-CAR into the TRAC locus with knockout of the TCR. To generate clonal lines, engineered iPSCs were sorted by flow cytometry for various markers and single cells were seeded into individual wells of feeder-free 96-well plates. Engineered iPSC clones were screened for 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 and the CAR copy number was confirmed by droplet digital PCR. Genome stability of each clone was also confirmed by karyotype analysis. Overall, the described screening initiative surveyed 774 clones to select the ideal MCB for FT819. Utilizing our stage-specific T cell differentiation and expansion protocol, we demonstrated that T cells derived from the FT819 (FT819-iTs) expanded greater than 100,000-fold during the clinical manufacturing process and the cells expressed greater than 95% T lymphocyte markers such as CD45, CD7, intracellular CD3, and TRAC-regulated CAR. Further modifications to the T cell differentiation protocol resulted in enhanced expression of CD8 αβ from less than 25% to greater than 70% of the total population. In addition, expression of CD2, CD5, and CD27 was increased by approximately 5- to 20-fold. In vitro functional studies showed that FT819-iTs possess antigen specificity as confirmed by cytokine release and cytotoxic T lymphocytes (CTL) assays. Upon stimulation with a wild type acute lymphoblastic leukemia line, Nalm-6, FT819-iTs expressed 30% CD107a/b compared to 2% when stimulated by Nalm-6 CD19KO. In an in vitro CTL assay, greater than 80% of Nalm-6 WT cells were lysed with effector to target (E:T) ratio at 10:1 as compared to Nalm-6-CD19KO, which showed less than 10% lysis at the same E:T ratio. Finally, in an in vivo tumor model, FT819-iTs generated from our original and modified T cell differentiation protocols showed similar tumor burden control and prolonged survival rate when compared to primary CAR-T cells (days of survival >80days, p>0.1). In a more stringent in vivo model, FT819-iTs generated from the modified differentiation protocol demonstrated higher anti-tumor response and better animal survival rate compared to iTs from the original T cell differentiation protocol (Day 30 p<0.005). Small molecules are known to modulate cell functions and when treated with compound A, FT819-iTs further delayed tumor growth and increased anti-tumor potency when compared to DMSO treated group (Day 17, P<0.05). Collectively, the preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf CAR T cell product candidate with the first-of-kind Phase 1 clinical trial for the treatment of B cell malignancies in an allogeneic setting study planned for 2020. Disclosures Chang: Fate Therapeutics: Employment. Van Der Stegen:Memorial Sloan Kettering Cancer Center: Employment. Mili:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Lai:Fate Therapeutics: Employment. Witty:Fate Therapeutics: Employment. Lindenbergh:Memorial Sloan Kettering Cancer Center: Employment. Yang:Fate Therapeutics: Employment. Husain:Fate Therapeutics: Employment. Shaked:Fate Therapeutics: Employment. Groff:FATE THERAPEUTICS: Employment. Stokely:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Chu:Fate Therapeutics: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. ORourke:Fate Therapeutics: Employment. Gutierrez:Fate Therapeutics: Employment. Riviere:Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Fate Therapeutics: Consultancy; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents & Royalties; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Cigarette smoke (CS) exposure is well known to lead to progressive lung destruction that is characterized by enlarged airspaces and a decrease in lung elastic recoil. We hypothesized that VEGF expression by airway cells may protect against the early onset of excessive lung damage. Adult VEGF LoxP mice (5 months of age) were exposed to daily periods (5 days/week) of “nose‐only” CS or remained in room air (RA) for 4 months. Half of the mice in each exposure group were instilled one month earlier with AAV‐Cre to delete the VEGF gene in airway cells or a control virus (AAV‐LacZ). An additional group of VEGF promoter/luciferase reporter mice were exposed to CS to monitor VEGF transcriptional activation by in vivo optical imaging. Static lung mechanics were characterized from pressure‐volume curves and oxygen saturation levels were measured in awake mice breathing 21% O 2 or 10% O 2 by a pulse oximeter. VEGF transcriptional activity in the lungs peaked at 7 days and returned towards baseline level by 14 days (Day 0, 1.14e7 ± 2.4e6 relative light units (RLU); Day 1, 3.46e7 ± 9.5e6 RLU, Day 7, 7.40e7 ± 1.9e7 RLU, Day 14, 3.10e7 ± 5.4e6 RLU, p=0.055). There was a progressive left shift and increased hysteresis of the P‐V relationship in mice from the RA‐LacZ < CS‐LacZ < RA‐Cre < CS‐Cre groups. At an average airway pressure of 20 cmH 2 O, lung volumes were 0.9 ml RA‐LacZ group, 1.0 ml CS‐LacZ group, 1.1 ml RA‐Cre group, and 1.2 ml CS‐Cre group. O 2 saturation levels were not different among groups when breathing RA. In mice challenged with 10% O 2, the oxygen saturation levels were lowest in the CS‐Cre group (RA‐LacZ, 71.0 ± 1.8%; CS‐LacZ, 75.2 ± 4.9%; RA‐Cre, 70.4 ± 3.9%; CS‐Cre, 62.6 ± 3.5%, p<0.05). These data suggest that airway epithelial VEGF is vital for maintaining lung function and when deleted from the airway, obstructive physiology develops. This pattern is further exacerbated by exposure to chronic cigarette smoke. Support or Funding Information Department of Veterans Affairs

Locomotor skeletal muscle function is impaired in patients with chronic obstructive pulmonary disease (COPD). The mechanisms responsible for these skeletal muscle abnormalities are not well understood. We hypothesized that the severity of lung damage following chronic exposure to cigarette smoke (CS) would determine the mitochondrial oxidative capacity of locomotor and respiratory skeletal muscle. Adult VEGF LoxP mice with or without lung‐targeted VEGF gene deletion (5 months of age) were used as a model of progressively compromised static lung mechanics following exposure to 2 × 30 min of daily, nose‐only CS for an additional 4 months. Deletion of the VEGF gene was accomplished by instilling half the mice with AAV‐Cre one month before initiating CS exposure. The remaining mice received a control AAV (LacZ). Further, both groups (Cre and LacZ) were assigned either to CS exposure or room air control (RA). Mitochondrial respiration in permeabilized fiber bundles from the diaphragm and medial gastrocnemius was measured using high‐resolution respirometry. Gastrocnemius ADP‐stimulated O 2 consumption with malate and glutamate as substrates was 78±6%, 66±7% and 60±11% lower in CS‐LacZ, RA‐Cre, and CS‐Cre, respectively, than RA‐LacZ ( p <0.05). Gastrocnemius electron transport system capacity was 68±5% ( p <0.05), 53±11% ( p <0.05) and 48±21% (NS) lower in the CS‐LacZ, RA‐Cre, and CS‐Cre, respectively, than RA‐LacZ. Diaphragm ADP‐stimulated O −2 consumption was 56±9% lower in CS‐LacZ fibers than RA‐LacZ ( p <0.05). Diaphragm fiber electron transport system capacity was not different between the groups. Mice with a COPD‐like phenotype exhibited impaired locomotor muscle oxidative capacity irrespective of whether the lung damage was caused by cigarette smoke exposure or VEGF gene deletion. In contrast, diaphragm mitochondrial respiration only showed a deficit in ADP‐stimulated O 2 consumption with CS exposure. Thus, mitochondrial respiration in locomotor skeletal muscle appears to be less well‐preserved than diaphragm in this model of COPD. Support or Funding Information Department of Veterans Affairs

Pulmonary arterial hypertension (PAH) is a progressive, incurable disease of the pulmonary vasculature, leading to high pulmonary vascular resistance and right ventricular failure. In addition, PAH patients show marked skeletal muscle abnormalities that may develop before right heart pathology. Thus, skeletal muscle dysfunction may be more closely related to the primary pathology of PAH than originally thought. We hypothesized that IL‐33, recently implicated in the cardiopulmonary pathogenesis of PAH, is also requisite for skeletal muscle maladaptation.Using adult C57BL/6J mice, we induced PAH with 3 wks of chronic hypoxia (FiO2 0.10) and weekly subcutaneous injections (20 mg.kg−1) of the VEGF receptor kinase inhibitor Su5416 (SuHx). SuHx was applied in both wild‐type (WT) and IL‐33 receptor gene ablated (ST2−/−) mice. Further, vehicle control (DMSO) WT mice were generated using the same injection volume and schedule and were housed in normoxia (CON). Mitochondrial function was measured in permeabilized diaphragm and soleus using a titration protocol that included maximal ADP‐stimulated respiration (JO2max) and non‐phosphorylating electron transport system capacity (ETS).Diaphragm JO2max was reduced in WT‐SuHx vs. WT‐CON (85±26 vs 162±62 pmol.s.mg‐−1; p<0.05), but not in ST2−/−‐SuHx (148±102 pmol.s.mg‐−1; p>0.72 vs WT‐CON). Diaphragm ETS followed a similar pattern in WT‐SuHx vs WT‐CON (90±29 vs 167±63 pmol.s.mg‐−1; p<0.05), and was not different in ST2−/−‐SuHx vs WT‐CON (152±104 pmol.s.mg‐−1; p>0.73). Flux control ratios (JO2max/ETS) were not different between groups (p>0.75). Soleus JO2max was reduced in both WT‐SuHx and ST2−/−‐SuHx vs WT‐CON (70±33 and 86±38 vs 140±40 pmol.s.mg‐−1; p<0.05). Soleus ETS was also reduced in WT‐SuHx and ST2−/−‐SuHx vs WT‐CON (68±24 and 86±38 vs 140±30 pmol.s.mg‐−1; p<0.05). Similar to diaphragm, soleus flux control ratios were not different between groups (p>0.73).The SuHx model showed a profound reduction in skeletal muscle mitochondrial oxidative capacity, suggestive of similar abnormalities to those reported in patients with PAH. As the flux control ratios were not different in SuHx, reduced mitochondrial volume‐density was a more likely mechanism, rather than a deficit in the quality of integrated function between electron transport and phosphorylation. Global gene deletion of the IL‐33 receptor (ST2) prevented diaphragm mitochondrial deficits in SuHx, but soleus muscle was less well‐protected. Our data strengthen the hypothesis that inflammatory cytokines, such as IL‐33, may play a critical role in skeletal muscle maladaptation to PAH.Support or Funding InformationThis research was supported by the US Department of Veterans Affairs.