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Contact James Trager, PhD [email protected] www.nkartatx.com Introduction NK cells have been expanded on K562 stimulatory cells expressing membrane-bound (mb) IL-15 and 41BBL (NKSTIM) for clinical use and can be genetically modified to express activating chimeric receptors [1,2,3]. Engineered NK cells targeting CD19 show in vitro and in vivo cytotoxicity against relevant tumor targets that can overcome endogenous resistance to NK cells. NK cells activated in the presence of IL-12, IL-15 and IL-18 develop cytokine induced memory-like phenotype and function; these cells have shown clinical promise [4]. Here we describe NK cell function and phenotype achieved by combining robust expansion driven by K562-mbIL15-41BBL with the induction of a cytokine-induced memory phenotype achieved after exposure to IL-12 and IL-18. Methods NK cell expansion, cytokine secretion, cytotoxicity against tumor lines at various time points, and persistence in culture over 5 weeks was compared with or without exposure to IL- 12 and IL-18 (12-18). The expanded NK were transduced with a CD19 CAR construct, and the resulting cells were evaluated for CAR expression, cytotoxicity and in vivo efficacy against relevant cell lines. NK Expansion with IL-12 and IL-18 Healthy donor PBMC NK were expanded on K562-mbIL15- 41BBL stimulatory cells (NKSTIM) with IL-2 alone or with IL-2 plus IL-12 and IL-18. Expanded NK were then measured for expansion, cytotoxicity and memory-like phenotype. Transduction with CAR constructs & in vivo experiments Expanded NK were genetically modified with a retroviral construct consisting of the FMC63 scFv CD19-OX40- CD3zeta CAR and mbIL-15 to drive persistence. Modified NK were then assayed in vitro and in vivo. Results References 1.Lapteva N., et al. Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications. Cytotherapy (2012) 2.Chihaya I., et al. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. (2005) 3.Yang Y., et al. A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells. Cancer Res. (2013) 4.Romee R., et al. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci Trans Med. (2016) Conclusions IL-12 and IL-18 in combination with Nkarta’s NKSTIM feeder NK expansion platform significantly augments NK phenotype and function. • Addition of IL-12 & IL-18 drives significantly greater in vitro expansion. • NK innate and CD19-CAR driven cytotoxicity significantly improves with addition of IL-12 & 18 to Nkarta’s expansion platform • Exposure to IL-12 and IL-18 in Nkarta’s platform drives greater persistence and improved potency in a NALM-6 preclinical model Stimulatory cells plus IL-12 and IL-18 augments NK cell expansion, transduction, memory phenotype, and in vitro and in vivo CAR NK cytotoxicity & persistence Anmol Vohra, MS; Kathryn Jamboretz, MS; Sasha Lazetic; Daofeng Liu, Ph.D.; Denise Gonzalez; Ivan Chan, Ph.D.; James B. Trager, Ph.D. Nkarta Inc., South San Francisco, CA, USA Figure 1: Nkarta Natural Killer (NK) cell platform showing ex vivo (NK) cell expansion, activation and genetic modification procedures Figure 5. Nkarta CD19 based chimera (NKG2D.aCR) CD19 CAR CD19 scFv Hinge & TM OX40 ICD CD8 CD3z ITAM T2A mbIL-15 Figure 2: Expansion 6 healthy PBMC donors were expanded 7 days in a matrix of 4 concentrations each ranging from 20-0.08 ng/ml IL-12 and IL-18 inoculated at day 0. Day7 fold expansion is shown for IL18 @ 4ng/ml (A) and 20ng/ml (B) 0 10 20 30 40 50 0 20 40 60 80 100 days Percent survival PBS NK19-1 NT NK NK19-1_12/18 D-3 D-7 D-11 D-18 D-25 0 1×10 8 2×10 8 3×10 8 4×10 8 5×10 8 1×10 9 1.5×10 9 2×10 9 2.5×10 9 Days Post Tumor Inoculation BLI (phtoton/sec) PBS Untransduced NK CD19-CAR NK CD19-CAR NK_12/18 Figure 7: Preclinical Model Feeder stimulated NK with IL-12 & IL-18 drives significantly greater activity and persistence in a CD19 CAR-NK NALM-6 xenograft tumor model. 1x10 5 NALM-6 were injected at Day 0 and 2x10 7 CD19-CAR NK with or without 12-18 were inoculated 4 days later, ** P=0.003 IL-12:10 ng/ml IL-12:2ng/ml IL-12:0.4 ng/ml IL-12:0.08 ng/ml Feeder Alone 0 50 100 150 Purified NK Expansion Titering IL-12 With IL-18 at 4ng/ml IL-12 Concentrations Fold 7 Day NK Expansion IL-12:10 ng/ml IL-12:2ng/ml IL-12:0.4 ng/ml IL-12:0.08 ng/ml Feeder Alone IL-12:10 ng/ml IL-12:2ng/ml IL-12:0.4 ng/ml IL-12:0.08 ng/ml Feeder Alone 0 50 100 150 Purified NK Expansion Titering IL-12 With IL-18 at 20ng/ml IL-12 Concentrations Fold 7 Day NK Expansion IL-12:10 ng/ml IL-12:2ng/ml IL-12:0.4 ng/ml IL-12:0.08 ng/ml Feeder Alone Figure 3: Flow Phenotype 3 donors were expanded on NKSTIM with or without soluble 12-18 (10 & 20ng/ml respectively) spiked at day 0 or with NKSTIM feeder engineered to express membrane bound IL-12 and IL-18. Flow cytometry was used to characterize the cells over 7-35 days of expansion. 12-18 drives a decrease in CD57 (A) and increase in CD62L (B) and NKG2C (C) over 21 days. Weekly tracking of memory markers over 5 weeks (D) shows an increase, and then constant NKG2C D21-35 expression and an initial spike and subsequent drop in CD62L. Both NKG2C NK percentages and surface level expression (E) increase in an example donor from D14 to D21 with a concurrent loss of CD57 expression by day 21. A. B. Figure 6: Genetic Modification & Cytotoxicity 12-18 plus NKSTIM expansion drives comparable CD19 CAR expression (A) over 3 weeks and significantly more potent CAR-CD19 driven cytotoxicity against NALM-6 (B & C). 3 donors were expanded with or without 12-18 plus NKSTIM and genetically modified with a retroviral CD19-CAR-mbIL-15 construct. Cells were assayed for cytotoxicity 7 days post transduction for 4 days against NALM-6 and both unmodified and CAR expressing cells showed significantly greater in vitro cytotoxicity over 4 days (B) and at the final time point at day4 (C). Figure 4: Cytokine Production IL-12 & 18 titrations on NKSTIM expanded NK drive potent IFNg accumulation over 3 days. Peripheral blood NK were expanded 7 days and plated with varying concentrations of IL-12 or 18 alone or in combination. Activation with NKSTIM plus 12-18 synergizes to drive high levels of IFNg accumulation. A. B. C. D. E. Feeder Expanded NK Feeder Expanded NK plus 12/18 0.0 0.5 1.0 1.5 CAR Positive NK Cells: . Day18 Post Injection % CAR+ out of Total Peripheral Mu Blood P<.001 NKSTIM &12/18 NKSTIM Alone D14 D21 Feeder Expanded NK Feeder Expanded NK with 12/18 0 1 2 3 4 Total Human NK Cells: . Day18 Post Injection % CD56+ out of Total Peripheral Mu Blood p=0.004 A. B. C. Untransd Untransd 12/18 CD19 CAR CD19 CAR 12/18 0 20 40 60 80 100 IL-12 & IL-18 Increases Cytotoxic Potency % Cytotoxicity p=0.01 p=0.04 NALM-6 CD19-CAR NK CD19-CAR NK 12-18 CD57 SP 7% CD57 SP 19% NKG2C SP 8% NKG2C SP 37% CD57 SP 1.6% CD57 SP 0.4% NKG2C SP 12% NKG2C SP 10%
