Session: 703. Cellular Immunotherapies: Basic and Translational: Novel Approaches for Next Generation Cellular Immunotherapies
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Biological therapies, Translational Research, Diseases, Therapies, immunology, Myeloid Malignancies, Biological Processes, Natural Killer (NK) Cell Therapies, Human
W-NK characterization was performed by scRNA-seq (10X Genomics) and multidimensional flow and mass cytometry.Functionality was evaluated by in vitro cytotoxicity assays against AML cell lines (THP-1, and HL-60) and bioenergetics assays (Seahorse XF), under different conditions: conventional (N) media (pH 7, glucose 11 mM), or two models for TME (TME-aligned media - pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1, and malignant ascites), and in hypoxia (0.1% O2; AVATAR). In vivo efficacy was evaluated in NSG mice bearing THP-1 AML tumors. Proteomic data was acquired by tandem-mass spectrometry (Sciex Zeno TOF 7600). Cell trafficking was measured by fluorescent-labeled W-NK in NSG mice.
W-NK cells are transcriptionally distinct from cNKs. Sc-RNA-seq analysis revealed important transcriptional and compositional differences compared to cNK cells, including novel NK clusters expressing metabolic (GLUL), proliferation-associated (Ki67) and IL-15-inducible genes (TXNIP) known to enhance NK responses. Additionally, W-NKs express higher levels of activating receptors (i.e., 2B4, DNAM-1, NKG2D, NKp30, etc.), cytotoxic effector proteins (i.e., granzyme B), and lower levels of inhibitory receptors leading to increased potency in vitro against HL-60 cells (mEC50 1.7 and 5.2; p= 0.032; W-NK vs. cNK respectively) and demonstrated a dose dependent reduction of tumor burden in THP-1 xenografts (p=0.015). W-NK also express high levels of CXCR4 (81.6% positive, MFI 5552), contributing to improved bone marrow (BM) homing (16.5 ± 2.4%) compared to data previously reported for IL-2-expanded NK cells (3%; Sato et al. CCR. 2020). Once in the TME, unlike cNK and T-cells, W-NK cytotoxic function is preserved (TME-media vs. N-media-; W-NK: 96.5% vs. 88.2%, p=0.45; cNK: 44.1% vs.13.5%, p=0.009; T-cell: 53.2 % vs. 25.2%, p=0.041, respectively). This is potentially due to W-NK resistance to hypoxia, and improved metabolic fitness/flexibility. W-NK cytotoxic activity was also preserved in hypoxic conditions (90% vs. 89.5%, p=0.2) compared to cNK (24% vs. 6.9%, p=0.02; normoxia vs. hypoxia). Metabolically, W-NK express higher levels of cell surface nutrient transporters, implying better adaptation to adverse TMEs (i.e., upregulation of amino acids, lactate, and pyruvate). This might enable utilization of diverse macronutrients, activation of metabolic pathways, and optimal ATP manufacturing, and is supported by upregulation of REACTOME pathways capturing amino-acid and lipid metabolism, as well as mitochondrial function. Lastly, W-NK did not attack cells from healthy human tissues including PBMCs, indicating that W-NK are not indiscriminate killers. This observation would predict low myelosuppression and a favorable safety profile.
In summary, W-NK are effective against AML cells, robustly home to the BM, overcome hypoxia, immunosuppression, and nutritional scarcity to survive/function within the leukemia TME. Moreover, they are not restricted to a single antigen target thereby mitigating antigen escape. These features, which overcome challenges for ACT, herald the promise of NK cell therapy and augur positively for patients being treated with W-NK in a Phase 1 study in R/R AML (NCT# 05470140).
Disclosures: Rutella: Wugen: Research Funding. Muth: Wugen: Current Employment, Current equity holder in private company. Leedom: Wugen: Current Employment, Current holder of stock options in a privately-held company. Hamil: Wugen: Current Employment, Current holder of stock options in a privately-held company. Arthur: Wugen Inc.: Current Employment, Current holder of stock options in a privately-held company. Schramm: Wugen: Current Employment, Current holder of stock options in a privately-held company. Baughman: MacroGenics, Inc.: Current equity holder in publicly-traded company. Petit: METAFORA biosystems: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Bhatnagar: Metafora Biosystems: Current Employment. Magee: Wugen: Current Employment, Current holder of stock options in a privately-held company. Berrien-Elliott: WUGEN: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties. Cooper: Wugen: Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties. Davidson-Moncada: Wugen: Current Employment, Current equity holder in private company. Fehniger: AI Proteins: Other: Scientific Advisory Board, Research Funding; Smart Immune: Other: Scientific Advisory Board; HCW Biologics: Research Funding; Orca Bio: Current holder of stock options in a privately-held company; Indapta: Current holder of stock options in a privately-held company; Affimed: Other: Scientific Advisory Board; Wugen: Consultancy, Current holder of stock options in a privately-held company, Research Funding.
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