Search

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer. acceptedVersion

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti-PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an "off-the-shelf" source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a "cold" tumor "hot" by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.

Background Gliomas represent the most common brain tumors within the central nervous system, with glioblastoma being the most aggressive type.1 Conventional treatment combines several approaches including surgery, chemotherapy, and radiation.2 However, the prognosis for glioblastoma remains unfavorable, with only 5% of patients surviving more than 5 years post-diagnosis.3 Thus, new treatment approaches are urgently needed. Natural killer (NK) cells directly lyse malignantly transformed or virally infected cells and secrete inflammatory cytokines that polarize cytotoxic immunity. Allogeneic NK cell adoptive transfer has shown clinical benefit in patients with advanced cancer.4–7 However, limitations of this approach include relatively low numbers of donor NK cells that can be isolated during an apheresis and variability in the quality of NK cells between donors. To overcome these limitations, we have developed a GMP manufacturing strategy to mass produce NK cells from induced pluripotent stem cells (iPSCs) as an approach to off-the-shelf cancer immunotherapy. We refer to these cells as ‘iNK’ (iPSC-derived NK) cells. Here, we provide preclinical data demonstrating the efficacy of iNK cells for immunotherapy against glioblastoma. Methods We generated iNK cells using previously published methods.8–10 iNK cells were used as effectors against an array of patient-derived glioblastoma lines in 2-dimensional live imaging IncuCyte assays where iNK cell-mediated killing was observed over the course of 48 hours. To investigate iNK cell infiltration and cytotoxicity in a more physiological context that accounts for the 3-dimensional architecture of the tumor, we also performed live imaging IncuCyte assays using iNK cells as effectors against glioblastoma spheroids. To test the anti-tumor function of iNK cells in vivo, we implanted patient-derived glioblastoma cells into mice via intracranial injection. Seven days later, 5 mice received intratumoral injections of iNK cells, and 5 mice received vehicle alone (as a control; figure 1A). All mice were monitored for weight and survival over 100 days. Results iNK cells exhibited strong and sustained cytotoxicity against 6 primary patient-derived mesenchymal glioblastoma lines in 2-dimensional IncuCyte assays and complete infiltration and destruction of glioblastoma spheroids in 3-dimensional IncuCyte assays. In xenogeneic adoptive transfer experiments, all mice receiving intratumoral injections of iNK cells survived out to day 100, while all mice in the vehicle group became moribund and had to be sacrificed by day 60 (figure 1B). Conclusions iNK cells are highly cytotoxic against glioblastoma cells, and our preclinical in vivo data provides proof-of-concept for future clinical trials. Ethics Approval This project has been approved by the University of Minnesota IACUC. Approval ID: 1812-36595A References Louis D N, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee W K, Ohgaki H, Wiestler O D, Kleihues P, Ellison D W. The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 2016;131:803–820. Stupp R, Mason W P, van den Bent M J, Weller M, Fisher B, Taphoorn M J B, Belanger K, Brandes A A, Marosi C, Bogdahn U, Curschmann J, Janzer R C, Ludwin S K, Gorlia T, Allgeier A, Lacombe D, Cairncross J G, Eisenhauer E, Mirimanoff R O, European Organization for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987–996. Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholz-Sloan JS, Villano JL. Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Biomarkers Prev 2017;23:1985–1996. Miller J S, Soignier Y, Panoskaltsis-Mortari A, McNearney S A, Yun G H, Fautsch S K, McKenna D, Le C, Defor T E, Burns L J, Orchard P J, Blazar B R, Wagner J E, Slungaard A, Weisdorf D J, Okazaki J, McGlave P B. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005;105:3051–3057. Bachanova V, Cooley S, Defor T E, Verneris M R, Zhang B, McKenna D H, Curtsinger J, Panoskaltsis-Mortari A, Lewis D, Hippen K, McGlave P, Weisdorf D J, Blazar B R, Miller J S. Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood 2014;123:3855. Ciurea S O, Schafer J R, Bassett R, Denman C J, Cao K, Willis D, Rondon G, Chen J, Soebbing D, Kaur I, Gulbis A, Ahmed S, Rezvani K, Scpall E J, Lee D A, Champlin R E. Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplant. Blood 2017;130:1857–1868. Romee R, Rosario M, Berrien-Elliott M M, Wagner J A, Jewell B A, Schappe T, Leong J W, Abdel-Latif S, Schneider S E, Willey S, Neal C C, Yu L, Oh T, Lee S, Mulder A, Cooper M A, Fehniger T A. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukiemia. Sci Transl Med2016:8;375ra123. Valamehr B, Abujarour R, Robinson M, Le T, Robbins D, Shoemaker D, Flynn P. A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci Rep2012:2;213. Valamehr B, Robinson M, Abujarour R, Rezner B, Vranceanu F, Le T, Medcalf A, Lee T T, Fitch M, Robbins D, Flynn P. Platform for induction and maintenance of transgene-free hiPSCs resembling ground state pluripotent stem cells. Stem Cell Reports 2014;2:366–381. Zhu H, Blum R H, Bjordahl R, Gaidarova S, Rogers P, Lee T T, Abujarour R, Bonello G B, Wu J, Tsai P-F, Miller J S, Walcheck B, Valamehr B, Kaufman D S. Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor immunity. Blood 2020;135:399–410.

Approximately 19,940 new cases of acute myeloid leukemia (AML) will be diagnosed in 2020 with an estimated 11,180 deaths, which has remained steady for 3 decades. We established that allogeneic natural killer (NK) cell infusions have an antileukemic effect and induce remission in 25-40% of relapsed/refractory AML patients. After hundreds of individual donor product infusions, we noted therapeutic limitations to include effector potency, cell numbers,persistence, specificity and exportability. To overcome these barriers, we developed a robust genetic editing and manufacturing platform for the uniform engineering and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). This manufacturing system allows efficient production of iPSC-derived NK (iNK) cells engineered to enhance persistence and potency, and enables distribution of highly homogenous iNK cells to multiple clinical sites and multi-dosing to treat patients on-demand with advanced cancer. We hypothesized that iNK cells could be combined with the anti-CD38 monoclonal antibody daratumumab (dara) to directly target various tumors and harness their antibody-dependent cellular cytotoxicity (ADCC) potential. Previously, we reported on our ability to effectively target multiple myeloma with our FT538 program (the iNK defined below), now FDA approved for clinical trials. Here we discuss applying these same engineered iNK cells to specifically target and kill AML blasts induced to upregulate CD38 by retinoic acid exposure. We utilized iNK cells expressing a high-affinity, non-cleavable version of CD16 (the Fc receptor that mediates ADCC) termed hnCD16 alone or combined with CD38 knockout (KO) to prevent dara-mediated NK cell fratricide (Fig. 1A). CD38 KO iNK cells had intact calcium flux in response to ionomycin or CD16 crosslinking (Fig. 1B). Adding dara to peripheral blood NK cells or hnCD16 iNK cells triggered ADCC-mediated fratricide, while hnCD16/CD38 KO iNK cells that cannot be targeted by dara were unaffected (Fig 1C). CD38, expressed intracellularly and on the plasma membrane, functions as an NADase, degrading nicotinamide adenine dinucleotide (NAD+) into ADP-ribose and nicotinamide. Because NAD+ levels influence several key metabolic pathways, we assessed the impact of CD38 KO on iNK cell metabolism. We found that, relative to hnCD16 iNK cells, hnCD16/CD38 KO iNK cells had significantly higher intracellular NAD+, NADH, and ATP levels (Fig. 1D). Additionally, hnCD16/CD38 KO iNK cells exhibited elevated mitochondrial oxidative phosphorylation (Fig. 1E) and marked resistance to oxidative stress to hydrogen peroxide exposure (Fig. 1F). Together, these results demonstrate a significant enhancement of NK cell mitochondrial oxidative phosphorylation and redox homeostasis in iNK CD38 KO cells. To enhance hnCD16/CD38 KO iNK cells further, we incorporated a third modification consisting of an IL-15 receptor signaling complex (IL-15RF). This receptor complex is expressed on the cell surface and provides IL-15 signals required for NK cell survival and proliferation independent of exogenous cytokine. To determine the efficacy of hnCD16/CD38 KO/IL-15RF iNK cells combined with dara to target AML, we first assessed CD38 expression on THP-1 cells (an AML cell line) and primary AML blasts incubated with or without retinoic acid. We observed high CD38 expression on both THP-1 cells and primary AML cells further elevated with retinoic acid treatment (Fig. 1G). THP-1 and primary AML cells were labeled with CellTrace dye and used as targets for killing assays using hnCD16/CD38 KO/IL-15RF iNK cells. Compared to hnCD16/CD38 KO/IL-15RF iNK cells cultured with targets alone, adding dara led to higher target cell killing, especially after retinoic acid exposure (Fig. 1H). In 12-hour live imaging experiments testing iNK cell cytotoxicity against THP-1 cells, similar results were observed (Fig. 1I). Collectively, our results show that utilizing the iNK cell platform to uniformly express hnCD16 and IL15RF combined with complete CD38 KO is an effective strategy to promote effective ADCC against CD38+ cells in the absence of fratricide, and that CD38 KO reprograms NK cells for higher oxidative metabolic fitness for improved persistence and anti-tumor function. Furthermore, we have generated proof-of-concept data supporting triple gene-modified iNK cells combined with dara as a novel AML immunotherapy. Disclosures Cichocki: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Bjordahl:Fate Therapeutics: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Ge:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Felices:GT Biopharma: Consultancy. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Blazar:Fate Therapeutics Inc.: Research Funding; KidsFirst Fund: Research Funding; Magenta Therapeutics: Consultancy; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Tmunity: Other: Co-founder. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Miller:GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy. OffLabel Disclosure: FT538 is a genetically modified induced pluripotent stem cell derived NK cell product that can be combined with daratumumab to target CD38 on AML after retinoid acid induction.

Inhaled glucocorticoids form the mainstay of asthma treatment because of their anti-inflammatory effects in the lung. Exposure to the air pollutant ozone (O3) exacerbates chronic airways disease. We and others showed that presence of the epithelial-derived surfactant protein-D (SP-D) is important in immunoprotection against inflammatory changes including those induced by O3 inhalation in the airways. SP-D synthesis requires glucocorticoids. We hypothesized here that O3 exposure impairs glucocorticoid responsiveness (including SP-D production) in allergic airway inflammation. The effects of O3 inhalation and glucocorticoid treatment were studied in a mouse model of allergic asthma induced by sensitization and challenge with Aspergillus fumigatus (Af) in vivo. The role of O3 and glucocorticoids in regulation of SP-D expression was investigated in A549 and primary human type II alveolar epithelial cells in vitro. Budesonide inhibited airway hyperreactivity, eosinophil counts in the lung and bronchoalveolar lavage (BAL) and CCL11, IL-13, and IL-23p19 release in the BAL of mice sensitized and challenged with Af (p < 0.05). The inhibitory effects of budesonide were attenuated on inflammatory changes and were completely abolished on airway hyperreactivity after O3 exposure of mice sensitized and challenged with Af. O3 stimulated release of pro-neutrophilic mediators including CCL20 and IL-6 into the airways and impaired the inhibitory effects of budesonide on CCL11, IL-13 and IL-23. O3 also prevented budesonide-induced release of the immunoprotective lung collectin SP-D into the airways of allergen-challenged mice. O3 had a bi-phasic direct effect with early (48 h) activation of SP-D mRNA (sftpd) in vitro. Dexamethasone and budesonide induced sftpd transcription and translation in human type II alveolar epithelial cells in a glucocorticoid receptor and STAT3 (an IL-6 responsive transcription factor) dependent manner. Our study indicates that O3 exposure counteracts the effects of budesonide on airway inflammation, airway hyperreactivity, and SP-D production. We speculate that impairment of SP-D expression may contribute to the acute O3-induced airway inflammation. Asthmatics exposed to high ambient O3 levels may become less responsive to glucocorticoid treatment during acute exacerbations.

Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, which leads to airway hyperresponsiveness (AHR) to contractile stimuli and airway obstruction. Blocking T helper 2 (T(H)2) differentiation represents a viable therapeutic strategy for allergic asthma, and strong TCR-mediated ERK activation blocks T(H)2 differentiation. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protected against allergic asthma by simultaneously reducing airway inflammation and AHR though independent mechanisms. Targeted deletion of DGKζ in T cells decreased type 2 inflammation without reducing AHR. In contrast, loss of DGKζ in airway smooth muscle cells decreased AHR but not airway inflammation. T cell–specific enhancement of ERK signaling was only sufficient to limit type 2 airway inflammation, not AHR. Pharmacological inhibition of DGK diminished both airway inflammation and AHR in mice and also reduced bronchoconstriction of human airway samples in vitro. These data suggest that DGK is a previously unrecognized therapeutic target for asthma and reveal that the inflammatory and AHR components of asthma are not as interdependent as generally believed.

Cancer immunotherapies have revolutionized cancer treatment by showing clinical efficacy across multiple cancer indications. However, tumor heterogeneity and evasion from host immune cell surveillance often limit the durability and efficacy of these strategies as monotherapies. Consequently, it is becoming common practice to combine existing anticancer treatments and novel immunotherapies to maximize clinical efficacy. The pan tumor-associated antigens MICA and MICB (MICA/B) are surface proteins induced by cellular stress, often associated with tumorigenesis, and are recognized by the NK cell activating receptor NKG2D. To evade immune recognition, cancer cells often proteolytically shed the membrane distal domains of MICA/B, leading to reduced NKG2D recognition. To combat this pervasive tumor escape mechanism and create a ubiquitous cancer targeting platform, we have developed a novel CAR-iPSC-derived NK (iNK) cell that targets the conserved α3 domain of MICA/B, rendering it resistant to inhibition by shed MICA/B. To enhance effector cell function, persistence and multi-antigen capacity, further genetic editing at the iPSC stage was conducted to equip the CAR-iNK cells with a unique IL-15/IL-15 receptor fusion, the knockout of CD38 and a novel high-affinity, non-cleavable CD16 (hnCD16) to enhance antibody-dependent cellular cytotoxicity (ADCC). In this study, we evaluated the function of multiplexed engineered MICA/B CAR iNK cells (termed FT536) in combination with monoclonal antibodies (mAbs), to elicit multi-antigen targeting, and radiation therapy, to augment surface MICA/B expression. FT536 showed superior in vitro cytotoxicity and in vivo tumor control against an array of MICA/B expressing tumor lines. Furthermore, ADCC, induced in combination with cetuximab or trastuzumab, enhanced the potency of FT536 against various solid tumor lines (p Citation Format: John Goulding, Robert Blum, Bryan Hancock, Moyar Ge, Brian Groff, Soheila Shirinbak, Joy Grant, Martin Hosking, Mochtar Pribadi, Yijia Pan, Hui-Yi Chui, Shohreh Sikaroodi, Lauren Fong, Janel Huffman, Wen-I Yeh, Chia-Wei Chang, Thomas Dailey, Miguel Meza, Cokey Nguyen, Lucas Ferrari de Andrade, Tom Lee, Ryan Bjordahl, Kai W. Wucherpfennig, Bahram Valamehr. FT536: Preclinical development of a novel off-the-shelf CAR-MICA/B NK cell immunotherapy combined with radiation and antibody treatments as a first-of-kind pan-cancer targeting strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1591.

The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O3), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein-D (SP-D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP-D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate-dependent manner. Combined allergic airway sensitization and O3 exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S-nitrosylation related de-oligomerisation of SP-D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP-D. Importantly, S-nitrosylation abolished the ability of SP-D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP-D and eosinophils is destroyed by the NO-rich oxidative lung tissue environment in asthma exacerbations. Demonstration that specific carbohydrate-dependent inhibition of murine and human eosinophil extracellular DNA trap release is abolished by s-nitrosylation of the SP-D molecule.

The mammalian Target of Rapamycin (mTOR) protein controls the machinery necessary for T-cell activation, differentiation, and memory formation, as a component of mTOR complex 1 (mTORC1) and mTORC2, which function both downstream and upstream of AKT. Invariant natural killer T (iNKT) cells are a unique T-cell subset that exists in a primed state, capable of rapid activation, and produces large quantities of cytokines. iNKT-cell effector differentiation is dependent on the mTORC1 complex; however, the requirements for mTORC2 in iNKT cells have been controversial. In this issue, Sklarz et al. [Eur. J. Immunol. 2017. 47: XXX-XXX] provide a careful analysis of the requirements for the mTORC2 component Rictor in iNKT cells, providing a new twist in this unfolding tale. The authors demonstrate that Rictor is required for iNKT-cell proliferation and survival during the key stage of intrathymic expansion and that Rictor supports the development of NKT17 cells, an effector subset which depends on the transcription factor RORγt and produces interleukin (IL)-17, in both the thymus and the lung. IL-4-producing NKT2 cells develop in the absence of Rictor but the cytotoxic potential of iNKT cells is Rictor-dependent.

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, with significant opportunity to optimize natural killer (NK) cell and mAb interaction to improve antibody-dependent cellular cytotoxicity (ADCC). NK cells are critical mediators of ADCC, where they recognize and kill malignant cells coated with antibody through the Fc receptor CD16. However, NK cell function is often impaired in cancer patients, which limits the induction of ADCC in mAb therapy. To enhance ADCC in combination with commercialized mAb therapies, we have developed FT516; a novel, off-the-shelf NK cell immunotherapeutic engineered to uniformly express a high-affinity, non-cleavable version of CD16 (hnCD16). FT516 is manufactured from a renewable master induced pluripotent stem cell (iPSC) line with the potential to generate hundreds to thousands of doses of allogeneic NK cells uniformly expressing hnCD16 (hnCD16 iNK cells) per manufacturing run. In an in vivo xenograft model of disseminated lymphoma, FT516 reduced tumor burden below the limit of detection at day 28 after transplant when delivered in combination with rituximab, which was significantly more potent than peripheral blood NK cells (p = 0.03). This was attributed to enhanced CD16-mediated activation of FT516, as observed through improved calcium flux and enhanced activation of signaling pathways such as ERK (p = 0.016), LAT (p = 0.0007), and ZAP70 (p = 0.0003), leading to enhanced ADCC and cytokine production. FT516 also maintained tumor cell specificity, with preferential targeting of K562 leukemia cells when presented with a mixture of K562 and normal PBMC targets (p < 0.0001). We also explored strategies to further engineer therapeutic function to enhance FT516 efficacy. Combined expression of hnCD16 with an IL-15/IL-15ra fusion construct enhanced the persistence of iNK cells and allowed survival of up to 8 weeks in vivo without exogenous cytokine (p < 0.0001), with correlation to improved efficacy. In vitro modeling of FT516 with daratumumab demonstrated ADCC against multiple myeloma (MM) targets. However, as reported, daratumumab induced NK cell fratricide through binding of CD38 on NK cells. To rescue daratumumab-mediated fratricide, we specifically deleted CD38 at the iPSC level and demonstrated that fratricide was undetectable in hnCD16 CD38-/- iNK cells ( Citation Format: Ryan Bjordahl, Huang Zhu, Paul Rogers, Svetlana Gaidarova, Moyar Q. Ge, Robert Blum, Frank Cichocki, Jode Goodridge, Helen Chu, Greg Bonello, Tom Lee, Brian Groff, Ramzey Abujarour, Bruce Walcheck, Jeffrey Miller, Dan Kaufman, Bahram Valamehr. FT516, an off-the-shelf engineered NK cell therapeutic product for universal anti-tumor targeting strategy in combination with monoclonal antibodies [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 3191.

Epicutaneous allergen‐specific immunotherapy (EPIT) is proposed as an alternative route for allergen‐specific immunotherapy (AIT). The induction of allergen‐specific blocking IgG antibodies represents an important mechanism underlying AIT, but has not been investigated for EPIT. Here, we compared the induction of allergen‐specific blocking IgG in outbred guinea pigs which had been immunized with recombinant birch pollen allergen Bet v 1 using patch delivery system (PDS) with or without heat‐labile toxin (LT) from Escherichia coli or subcutaneously with aluminum hydroxide (Alum)‐adsorbed rBet v 1. Only subcutaneous immunization with Alum‐adsorbed rBet v 1 and epicutaneous administration of rBet v 1 with PDS in combination with LT from E. coli induced allergen‐specific IgG antibodies blocking allergic patients' IgE, but not immunization with rBet v 1 via PDS alone. Our results suggest that patch vaccination with rBet v 1 in combination with LT may be a promising strategy for allergen‐specific immunotherapy against birch pollen allergy.

Adoptive transfer of haplo-donor NK cells can achieve remission in patients with relapsed or refractory acute myeloid leukemia (AML). This strategy is limited by NK cell heterogeneity, lack of specificity, and single dosed strategies. To overcome these barriers, we genetically modified induced pluripotent stem cells (iPSCs) to stably over-express NKG2C alone or along with DAP12. CD94/NKG2C is a heterodimeric receptor that binds to HLA-E and associates with DAP12, a protein containing an immunoreceptor tyrosine-based activating motif. Efficient expression of CD94/NKG2C on the cell surface requires the presence of DAP12, and charged amino acids in the transmembrane domains of DAP12 and NKG2C mediate this interaction. Physiologic NKG2C expression is limited to subsets of NK and CD8+ T cells that expand in response to cytomegalovirus (CMV) infection. Engineered iPSCs were differentiated into CD34+ hematopoietic progenitor cells and subsequently differentiated into CD56+ NK cells (iNK cells) using a stepwise differentiation and expansion strategy. Surface NKG2C expression was low on unmodified iNK cells (10.7%) with higher levels of NKG2C on the surface of iNK cells derived from the NKG2C only line (65.1%). These results demonstrate that transgenic NKG2C can pair with endogenous CD94 and DAP12 to form stable structures on the cell surface. Importantly, surface NKG2C expression was highest on iNK cells derived from the NKG2C/DAP12 line (84.1%) with higher MFI, demonstrating the importance of engineering a line with both components (Figure 1A). To validate function via engagement of NKG2C on iNK cells, we used a redirected antibody dependent cellular cytotoxicity (ADCC) assay where P815 cells (which bind the Fc portion of antibodies) were coated with a monoclonal anti-NKG2C agonist antibody at a range of concentrations (0.01-10 mg/ml) and co-cultured with unmodified iNK cells, NKG2C iNK cells or NKG2C/DAP12 iNK cells. We found that at all concentrations tested, NKG2C/DAP12 iNK cells demonstrated the strongest functional response as measured by the frequencies of surface CD107a expression, interferon (IFN)-g production and tumor necrosis factor (TNF) production. Intermediate functional responses were observed for NKG2C iNK cells (Figure 1B). Having demonstrated stable expression and intact signaling of NKG2C/DAP12 in iNK cells, we next created a Tri-specific Killer Engager (TriKE) molecule with single chain Fv fragments specific for NKG2C (on iNK cells) and CD33 (on AML cells), as well as IL-15 (to support iNK cell survival and proliferation). This NKG2C/CD33/IL-15 TriKE was tested in functional assays where NKG2C iNK cells or NKG2C/DAP12 iNK cells were used as effectors against CD33+ THP1 cells (an AML cell line shown to be refractory to NK cell cytotoxicity). Without the addition of NKG2C/CD33/IL-15 TriKE, the frequencies of both NKG2C iNK cells and NKG2C/DAP12 iNK cells producing IFN-g was relatively low (< 10%). Addition of NKG2C/IL-15/CD33 TriKE in the assay markedly increased the frequency of IFN-g production by both NKG2C iNK cells (19.1%) and NKG2C/DAP12 iNK cells (25.8%) (Figure 1C). Finally, we directly tested the ability of the NKG2C/IL-15/CD33 TriKE molecule to trigger target killing by engineered iNK cells monitored over a 24-hour period. THP1 target cells were dye-labeled and plated in wells with either NKG2C iNK cells or NKG2C/DAP12 iNK cells at a 5:1 effector-to-target ratio with or without NKG2C/CD33/IL-15 TriKE. We found that in the absence of TriKE, there was no killing of THP1 targets. In contrast, both NKG2C iNK cells and NKG2C/DAP12 iNK cells mediated robust THP1 killing with the addition of NKG2C/CD33/IL-15 TriKE, with NKG2C/DAP12 iNK cells exhibiting the strongest response (Figure 1D). Our results provide a proof-of-concept that iNK cells engineered to express NKG2C/DAP12 can be used in combination with a novel NKG2C/IL-15/CD33 TriKE molecule to effectively target cancer cells. Engaging NK cells through NKG2C, restricted to adaptive NK cells or genetically modified iPSC will be more specific than CD16, which will bind to CD16A on NK cells but also have off-target binding to CD16B on neutrophils. Because of the enormous expansion and engineering capacity of the iNK cell platform, we are in a unique position to create an "off-the-shelf" NK cellular therapy that is targeted, specific and efficacious. Disclosures Cichocki: Fate Therapeutics Inc.: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding. Chu:Fate Therapeutics Inc.: Employment. Ge:Fate Therapeutics Inc.: Employment. Bjordahl:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

The development of PD1/PDL1 targeting checkpoint inhibitors (CI) has transformed the oncology landscape, providing long term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. Novel therapeutic approaches with the ability to overcome CI resistance are needed, and there is significant opportunity for therapies capable of additively or synergistically enhancing T-cell activation and recruitment when combined with CI. Adoptive transfer of NK cells from healthy donors has the potential to recruit T cells to the tumor microenvironment and augment T-cell activation at the tumor site. NK cells have both direct anti-tumor activity and the capacity to secrete inflammatory cytokines and chemokines upon activation, enabling the cells to play a unique and critical role in regulating anti-tumor T cell activity. We sought to determine whether FT500, an off-the-shelf NK cell product derived from a clonal master pluripotent cell line, could synergize with CI to relieve local immunosuppression and enhance T-cell activation and recruitment to the tumor site. FT500 is universally negative for cell surface PD1, and expression of PDL1 on tumor lines had no discernable effect on FT500 cytotoxicity. Similarly, addition of PDL1 blocking antibody had no effect on FT500 cytotoxicity or degranulation, suggesting that FT500 is inherently resistant to PDL1-PD1 mediated inhibition. Additionally, activation of FT500 induced the secretion of soluble factors capable of enhancing T-cell activation, as evidenced by increased upregulation of CD69. We hypothesized that FT500 might also enhance CI by promoting recruitment of T cells to the tumor site. Using conventional in vitro transwell migration assays, we found that FT500 produced soluble factors that promoted the migration of activated T cells. Additional profiling confirmed FT500 production of a range of chemokines, including CCL3, CCL4, CXCL10 and CCL22. Furthermore, using an in vivo recruitment model, FT500 was able to recruit T cells out of the circulation and into the peritoneal cavity. Similarly, utilizing a three-dimensional tumor spheroid model in vitro, infiltration of T cells into tumor spheroids was significantly enhanced when combined with FT500, suggesting that FT500 can enhance tumor infiltration of T cells. Our data suggest that FT500 is a potent producer of chemokines and can facilitate the recruitment of T cells to the tumor site. In addition to its direct cytotoxic potential, FT500 is also able to enhance T-cell activation, suggesting an ability to synergize with CI to reduce tumor burden. Together, our data provide evidence supporting the combination of FT500, an off-the-shelf NK cell cancer immunotherapy, with CI to overcome checkpoint blockade resistance. Citation Format: Ryan Bjordahl, Sajid Mahmood, Svetlana Gaidarova, Ramzey Abujarour, Raedun Clarke, Laurel Stokely, Paul Rogers, Moyar Ge, Megan Robinson, Betsy Rezner, Tom Tong Lee, Bahram Valamehr. FT500, an off-the-shelf NK cell cancer immunotherapy derived from a master pluripotent cell line, enhances T-cell activation and recruitment to overcome checkpoint blockade resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3576.

Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13–dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma.

The roles of NK cells, surfactant protein D (SP-D), and IFN-γ, as well as the effect of ozone (O3) inhalation, were studied on recirculation of pulmonary dendritic cells (DC) to the mediastinal lymph nodes. O3 exposure and lack of SP-D reduced NK cell IFN-γ and lung tissue CCL21 mRNA expression and impaired DC homing to the mediastinal lymph nodes. Notably, addition of recombinant SP-D to naive mononuclear cells stimulated IFN-γ release in vitro. Because NKp46, a glycosylated membrane receptor, was necessary for dose-dependent SP-D binding to NK cells in vitro and DC migration in vivo, we speculate that SP-D may constitutively stimulate IFN-γ production by NK cells, possibly via NKp46. This mechanism could then initiate the IFN-γ/IL-12 feedback circuit, a key amplifier of DC lymph node homing. Inhibition of this process during an acute inflammatory response causes DC retention in the peripheral lung tissue and contributes to injury.