The low five-year survival rate for patients with acute myeloid leukemia (AML), primarily caused due to disease relapse, emphasizes the need for better therapeutic strategies. Disease relapse is facilitated by leukemic stem cells (LSCs) that are resistant to standard chemotherapy and promote tumor growth. To target AML blasts and LSCs using Natural Killer (NK) cells, we have developed a trispecific killer engager (TriKE™) molecule containing a humanized anti-CD16 heavy chain camelid single domain antibody (sdAb) that activates NK cells, an IL-15 molecule that drives NK cell priming, expansion and survival, and a single-chain variable fragment (scFv) against human CLEC12A (CLEC12A TriKE). CLEC12A is a myeloid lineage antigen that is highly expressed by AML cells and LSCs, but not expressed by normal hematopoietic stem cells (HSCs), thus minimizing off-target toxicity. The CLEC12A TriKE induced robust NK cell specific proliferation, enhanced NK cell activation and killing of both AML cell lines and primary patient derived AML blasts in vitro while sparing healthy HSCs. Additionally, the CLEC12A TriKE was able to reduce tumor burden in pre-clinical mouse models. These findings highlight the clinical potential of the CLEC12A TriKE for the effective treatment of AML.
Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen. The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects. The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. Figure 1. Figure 1. Disclosures Vallera: GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.
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A major limitation of the use of cellular therapies is the loss of donor-derived cells because of immune incompatibility. While induced pluripotent stem (iPS) cells offer the potential for autologous transplant therapies, questions have been raised using a mouse model that specific antigens mediate the rejection of grafts after syngeneic transplants with iPS, but not embryonic stem (ES) cells. In this study, we examined whether the human homologs of these markers, HORMAD1, ZG16, and Cyp3A, are differentially expressed in human iPS versus ES cells, as well as undifferentiated and in vitro-differentiated cells. Both qRT-PCR and flow cytometric analyses demonstrated similar gene and protein expression profiles for iPS and ES cells regardless of differentiation state. Our data are consistent with a recent study in mice that showed no evidence of rejection of differentiated syngeneic iPS cells. Furthermore, our results suggest that expression of these gene products cannot predict differences in clinical outcomes between human iPS and ES-derived cells.
BackgroundNatural Killer (NK) cells can eliminate cancer cells through the release of cytotoxic granules triggered by interactions with natural ligands or through antibody-dependent cellular cytotoxicity (ADCC).1–3 NK cell-based treatments have had therapeutic success for hematological malignancies but strategies to treat solid tumors have been limited due to immunosuppression within the tumor microenvironment (TME).4–6 An important and understudied aspect of NK cell immunosuppression is the low oxygen (hypoxia) environment created by proliferating tumor cells. We used the novel AVATAR incubator system to model oxygen levels of three key tissues that NK cells inhabit in vivo: the peripheral blood (12% O2), the bone marrow (5% O2 ) and the TME (1% O2).MethodsNK cells were incubated in the AVATAR incubators for 24 hours, 72 hours and 7 days. We conducted a mass cytometry (CyTOF) analysis to assess phenotype, flow cytometry-based assays to assess proliferation and an IncuCyte machine and immunofluorescent imaging to measure cytotoxicity of NK cells incubated at different oxygen conditions. We evaluated NK cell metabolism using Seahorse assays, gene expression using RNA-Seq and are in the process of evaluating epigenetic regulation using ATAC-Seq.ResultsNK cells from the 1% O2 condition express fewer activating receptors (CD16, NKG2D, Nkp30, Nkp46, DNAM-1) and less perforin and granzyme than NK cells from the higher oxygen conditions (figure 1). NK cells in the 1% O2 condition also have decreased aggregation of perforin and granzyme granules at the immune synapse. This translates to reduced natural cytotoxicity and ADCC responses against tumor targets (figure 2). We also observe a sharp decrease in proliferation in the NK cells at 1% O2 (figure 3). This is partly due to an increase in CISH gene expression that makes the cells less responsive to cytokine stimulation.7 The RNA-Seq analysis revealed that NK cell metabolism closely resembles cancer cell metabolism under hypoxic conditions, specifically an increased expression of genes related to glycolysis, amino acid synthesis and central carbon metabolism. This change in metabolism was confirmed using Seahorse assays. We also observed changes in genes related to epigenetic regulation specifically, increases in histone demethylases and decreases in DNA methyltransferases (figure 4).Abstract 675 Figure 1Oxygen concentration alters NK cell phenotype300,000 enriched NK cells were incubated for 24 hrs, 72 hrs and 7 days at noted incubator conditions with 1 ng/ml IL-15. At the end of the incubation, cells were barcoded and stained with a custom panel for CyTOF evaluation. Data is show here for CD16 (A), NKG2D (B), Perforin (C) and Granzyme B (D). N=3 (data concatenated).Abstract 675 Figure 2Oxygen concentration effects NK cell cytotoxicity300,000 enriched NK cells were plated per well in 96-well round-bottom plates with 1 ng/ml IL-15. Plates were inserted in noted incubator conditions for 24 hours, 72 hours or 7 days. At the end of the incubation period NK cells were counted and plated at a 2:1 E:T ratio with fluorescently labeled K562 targets or fluorescently labeled labeled Raji targets + Rituximab (10 ug/ml) and cells were imaged every 30 minutes. Data is shown here for K562 targets (A) and raji targets (B) at the 7 day timepoint. Representative of six separate experiments.Abstract 675 Figure 3Oxygen concentration impacts NK cell proliferation300,000 PMBCs were CellTrace labeled and plated per well in 96-well round-bottom plates with noted treatments. The NK cells were incubated under noted incubator conditions for 7 days. At the end of 7 days, LiveDead dye was used to assess viability (A), while proliferation was assessed by evaluating CellTrace dye dilution on gated (CD56+CD3-) NK cells (B). (N=6)Abstract 675 Figure 4RNA-Seq reveals changes in gene expressionAn RNA-Seq analysis was performed on enriched NK cells incubated in noted oxygen and pressure concentrations for 24 hours, 72 hours or 7 days. A heat map of epigenetic regulation genes (A) and glycolysis genes (B) are shown for the day 7 timepoint. (N=4)ConclusionsThese results indicate that NK cells who enter the solid TME are fundamentally different than those in the bone marrow or the blood stream. Overall, the insights gained from these experiments can help overcome hypoxia induced immune suppression in the tumor microenvironment and improve NK cell-based immunotherapy for solid tumors.AcknowledgementsWe thank XCell biosciences for providing us with the AVATAR incubators used for these experimentsTrial RegistrationN/AEthics ApprovalN/AConsentN/AReferencesVivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol 2008;9(5):503–10.Waldhauer I, Steinle A. NK cells and cancer immunosurveillance. Oncogene. 2008;27(45):5932–43.Voskoboinik I, Smyth MJ, Trapani JA. Perforin-mediated target-cell death and immune homeostasis. Nat Rev Immunol 2006;6(12):940–52.Miller JS, Soignier Y, Panoskaltsis-mortari A, Mcnearney SA, Yun GH, Fautsch SK, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005;105(8):3051–8.Romee R, Cooley S, Berrien-Elliott MM, Westervelt P, Verneris MR, Wagner JE, et al. First-in-human phase 1 clinical study of the IL-15 superagonist complex ALT-803 to treat relapse after transplantation. Blood 2018;131(23):2515–2527.Björklund AT, Carlsten M, Sohlberg E, Liu LL, Clancy T, Karimi M, et al. Complete remission with reduction of high-risk clones following haploidentical NK-Cell therapy against MDS and AML. Clin Cancer Res 2018;24(8):1834–1844.Delconte RB, Kolesnik TB, Dagley LF, Rautela J, Shi W, Putz EM, et al. CIS is a potent checkpoint in NK cell–mediated tumor immunity. Nat Immunol 2016;17(7):816–24.
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For Natural Killer (NK) cell-based immunotherapy to succeed against solid tumors, NK cells need to enter a highly immunosuppressive tumor microenvironment (TME) and retain functionality. The objective of this study is to evaluate how hypoxia exerts an immunosuppressive effect on NK cells. We used the novel AVATAR™ system to model oxygen levels of three key tissues that NK cells inhabit in vivo: the peripheral blood (12% O2), the bone marrow (5% O2) and the TME (1% O2). NK cells incubated at 1% O2 have decreased proliferation and cytotoxicity compared to NK cells incubated at higher oxygen conditions. To assess what contributes to these changes, we conducted a mass cytometry (CyTOF) analysis, real time imaging assays, metabolic assays and gene expression (RNA-seq and ATAC-seq) analysis. We observed oxygen dependent changes in expression of activating receptors, Ki-67, perforin and granzyme. Hypoxia impacts aggregation of perforin and granzyme granules at the immune synapse. Under hypoxic conditions, NK cell metabolism resembles cancer cell metabolism with increased glycolysis, amino acid synthesis and central carbon metabolism. These changes are accompanied by mitochondrial defects. Gene expression analysis revealed that changes in NK cell metabolism and function are mediated at the epigenetic level by histone demethylases. Treatment with histone demethylase inhibitors rescued NK cell cytotoxicity as measured using an IncuCyte machine. These results indicate that NK cells who enter the TME are fundamentally different than those in the bone marrow or blood stream. The insights gained from this study will be leveraged to overcome hypoxia induced immune suppression in the TME to enhance NK cell-based immunotherapy for solid tumors.
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