Session: 203. Lymphocytes, Lymphocyte Activation, and Immunodeficiency, including HIV and Other Infections: Poster III
Hematology Disease Topics & Pathways:
Leukemia, ALL, Biological, antibodies, Diseases, CLL, Lymphoma (any), LGLL, Therapies, Biological Processes, immunotherapy, Lymphoid Malignancies, NK cells, hematopoiesis, immune mechanism, inflammation, stem cells
To further define the ability of ADAM17 to regulate NK cell activity, we have generated and characterized ADAM17-deficient (ADAM17-KO) NK cells derived from CRISPR/Cas9-modified human induced pluripotent stem cells (iPSCs). ADAM17-KO iPSCs successfully differentiate into hematopoietic progenitor cells, then to NK cells that uniformly express typical NK cell surface markers including CD56, CD94, NKG2D, NKp44, and NKp46. ADAM17-KO iPSC-NKs are functional and kill K562 erythroleukemia cells comparable to wildtype iPSC-derived NK cells (WT iPSC-NK cells) and healthy donor-derived peripheral blood NK cells (PB-NK cells) in vitro. Surprisingly, upon differentiation, ADAM17-KO iPSC-NK cells express ~20% lower CD16a surface expression compared to WT iPSC-NK cells, but stably retain CD16a expression after enrichment for CD16a+ cells and over 6 weeks of expansion in culture. WT iPSC-NKs and PB-NKs rapidly lose CD16a surface expression upon stimulation with phorbol esters, while ADAM17 KO iPSC-NK cells maintain over 90% CD16a expression after this stimulation. Additionally, a significantly higher proportion of ADAM17-KO iPSCs express TNF-α (71%) and CD62L (L-Selectin) (36%) – two other known ADAM17 substrates, on the cell surface after stimulation with phorbol esters for 4 hours compared to WT iPSC-NK (7% TNF-α+, 2% L-Selectin+) and PB-NK (2% TNF-α+, 1% L-Selectin+). CD16a+ ADAM17-KO iPSC-NK cells mediate increased CD107a (45%) and IFNγ (39%) expression when co-incubated with RAJI B-lymphoma cells in the presence of the anti-CD20 antibody rituximab, compared to CD16a+ WT iPSC-NK (32% CD107a+, 11% IFNγ) and PB-NK (37% CD107a+, 7% IFNγ) cells. Similarly, CD16a+ ADAM17-KO iPSC-NK cells upregulate increased CD107a (29%) and IFNγ (42%) expression when co-incubated with CAL27 squamous cell carcinoma cells in the presence of the anti-EGFR antibody cetuximab, compared to CD16a+ WT iPSC-NK (12% CD107a+, 8% IFNγ) and PB-NK (14% CD107a+, 6% IFNγ). Long-term (24 hour) cytotoxicity assay against RAJI cells in the presence of rituximab demonstrates higher cytotoxicity in CD16a+ ADAM17-KO iPSC-NK cells compared to CD16a+ WT iPSC-NK and CD16a+ PB-NK cells over time (see associated figure). In vivo studies to determine the therapeutic efficacy of ADAM17-KO iPSC-NK cells compared to WT iPSC-NK and PB-NK cells are ongoing. Together, these studies demonstrate ADAM17-KO iPSC-NK cells derived from a renewable source of gene-edited iPSCs possess enhanced ADCC potential, and provide a promising candidate to be used for standardized, off-the-shelf NK cell-based therapies in conjunction with therapeutic antibodies.
Disclosures: Blum: Fate Therapeutics: Current Employment. Kaufman: Fate Therapeutics: Consultancy.
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