Treatment of Alzheimer's Disease subjects with expanded non-genetically modified autologous natural killer cells (SNK01): a phase I study

Abstract

Background: The importance of natural killer (NK) cells of the innate immune system in neurodegenerative disease has largely been overlooked despite studies demonstrating their ability to reduce neuroinflammation (thought to be mediated by the elimination of activated T cells, degradation of protein aggregates and secretion of anti-inflammatory cytokines). SNK01 is an autologous non-genetically modified NK cell product showing increased activity in vitro. We hypothesized that SNK01 can be safely infused to reduce neuroinflammation in Alzheimer's Disease (AD) patients.

Methods: SNK01 was produced and characterized for its ability to eliminate activated T cells, degrade protein aggregates and secrete anti-inflammatory cytokines. In this phase 1 study, SNK01 was administered intravenously every three weeks for a total of 4 treatments using a 3 + 3 dose escalation design (1, 2 and 4 × 10⁹ cells) in subjects with either mild, moderate, or severe AD (median CDR-SB 10.0). Cognitive assessments and cerebrospinal fluid biomarkers associated with protein aggregation, neurodegeneration and neuroinflammation including amyloid-β42 and 42/40, α-synuclein, total Tau, pTau217 and pTau181, neurofilament light, GFAP and YKL-40 analyses were performed at baseline, at 1 and 12 weeks after the last dose. The primary endpoint was safety; secondary endpoints included changes in cognitive assessments and biomarker levels.

Results: In preclinical in vitro studies, SNK01 were able to uptake and degrade the protein aggregates of amyloid-β and α-synuclein, produce anti-inflammatory cytokines and eliminate activated T cells. In the phase 1 clinical study, eleven subjects were enrolled (10 evaluable). No drug-related adverse events were observed. Despite 70% of subjects being treated at relatively low doses of SNK01 (1 and 2 × 10⁹ cells), 50-70% of all enrolled subjects had stable/improved CDR-SB, ADAS-Cog and/or MMSE scores and 90% had stable/improved ADCOMS at one-week after the last dose. SNK01 also appeared to have beneficial effects on protein aggregate levels and neuroinflammatory biomarkers in the cerebrospinal fluid, with decreases in pTau181 and GFAP appearing to be dose-dependent.

Conclusions: SNK01 was well tolerated and appeared to have clinical activity in AD while also having beneficial effects on cerebrospinal fluid protein and neuroinflammatory biomarker levels. A larger trial with a higher dosing/duration has been initiated in the USA in 2023.

Trial registration: www.

Clinicaltrials: gov NCT04678453, date of registration: 2020-12-22.

Keywords: Alzheimer’s Disease; Biomarkers; Natural killer cell.

Fig. 1

Uptake and degradation of extracellular amyloid β (Aβ) aggregates by SNK01. A-E Intracellular levels of Aβ aggregates in SNK01 and HMC3 cells were analyzed by Western blot analysis. A SNK01 cells were treated with varying concentrations of Aβ aggregates for 1 h and harvested. B SNK01 cells were treated with 5 µM of Aβ aggregates and harvested at 1, 3, 6, 9, and 24 h after treatment. C SNK01 cells were treated with 5 µM Aβ aggregates for 1 h and washed 3 times with PBS. Then SNK01 cells were incubated in fresh medium for up to 48 h and harvested at 1, 3, 6, 24, and 48 h after wash. D HMC3 cells were treated with 5 µM of Aβ aggregates for 1, 3, and 6 h. N.C: negative control without treatment with Aβ aggregates. E HMC3 cells were treated with 5 µM Aβ aggregates for 1 h and washed 3 times with PBS. Then cells were incubated further in fresh medium for up to 48 h and harvested at 1, 3, 6, 24, and 48 h after wash. F-J SNK01 cells were incubated with 5 µM of Aβ aggregates for 24, 48, and 72 h and analyzed for viability (F), cytotoxicity at 72-h after treatment (G), degranulation (H), intracellular expression of IFN-γ and TNF-α (I), and surface expression of various activating NK receptors at 72-h after treatment (J)

Fig. 2

Uptake and degradation of extracellular α-synuclein (α-syn) aggregates by SNK01. A-E Intracellular levels of α-syn aggregates in SNK01 and HMC3 cells were analyzed by Western blot analysis. A SNK01 cells were treated with varying concentrations of α-syn aggregates for 1 h and harvested. B SNK01 cells were treated with 5 µg/mL of α-syn aggregates and harvested at 0.5, 1, 3, 6, and 24 h after treatment. C SNK01 cells were treated with 5 µg/mL of α-syn aggregates for 1 h and washed 3 times with PBS. Then SNK01 cells were incubated in fresh medium for up to 24 h and harvested at 0.5, 1, 3, 6, 12, and 24 h after wash. Left panel: soluble fraction with lysis buffer with 1% triton X-100, Right panel: insoluble fraction with lysis buffer with 1% triton X-100. D HMC3 cells were treated with 5 µg/ml of α-syn aggregates for 1 and 3 h. N.C: negative control without treatment with α-syn aggregates. E HMC3 cells were treated with 5 µg/mL of α-syn aggregates for 1 h and washed 3 times with PBS. Then cells were incubated further in fresh medium for up to 48 h and harvested at 1, 3, 6, 24, and 48 h after wash. F-J SNK01 cells were incubated with 5 µg/mL of α-syn aggregates for 24, 48, and 72 h and analyzed for viability (F), cytotoxicity at 72-h after treatment (G), degranulation (H), intracellular expression of IFN-γ and TNF-α (I), and surface expression of various activating NK receptors at 72-h after treatment (J)

Fig. 3

Level of interleukin 10 (IL-10) and TGF-β1 in conditioned media (CM) of SNK01 cells. The production of IL-10 (A) and TGF-β1 (B) by SNK01 cells for 2 days cultivation after stimulation with or without K562 target cells for 3 h before harvest at a 1:1 E:T ratio was investigated by ELISA. CM and Activated CM indicate culture supernatant of SNK01 cells treated without (CM) or with target cells (Activated CM)

Fig. 4

Cytotoxic activity and degranulation activity of SNK01 against stimulated T cell. A Human T lymphocytes were cultured with (P/I) or without (Media) stimulation by PMA (5 ng/mL)/ionomycin (250 ng) (P/I) for 48 h. The cells were stained with PE-CD3, PerCP/Cy5.5-CD69, and APC-CD25 antibodies and then analyzed by flow cytometry. Contour plots represent percentages of the CD25⁺ CD69⁺ cells on CD3⁺ gated T cells. B Cytotoxic activity of SNK01 against T cells was assessed by flow cytometry. T cells labeled with CTV were activated either with (P/I) or without (Media) PMA and ionomycin, and then co-incubated with SNK01 cells at an E:T ratio of 1:1 for 4 h. C-D Degranulation activity and IFN-γ expression of SNK01 cells against T cells. T cells were activated either with (P/I) or without (Media) PMA and ionomycin and then mixed with SNK01 cells at an E:T ratio of 1:1. The expression of CD107a (C) or IFN-γ (D) in CD56 + population was assessed by flow cytometry after 5- or 3- hour incubation, respectively

Fig. 5

Characteristics of SNK01 cells. A The percentages of CD3⁻CD56⁺ NK cells, CD3 + T cells, CD20⁺ B cells, and CD14⁺ monocytes were analyzed flow cytometrically on freshly isolated cells from PBMC using CliniMACS CD56 microbeads (D0; before expansion) and expanded NK cells for 17–18 days of culture (D17-18). B The fold expansion of the total cell population after 17–18 days of culture (D17-18). C The cytotoxic activity of expanded NK cells against the K562 was measured via calcein-release assay at E:T ratios of 10:1 to 0.5:1 in triplicate. Dots and horizontal bars represent the mean value of each patient from 4 cultures for clinical trial and mean ± SD from all patients, respectively

Fig. 6

Cognitive Assessment Changes from Baseline Score. Cognitive assessments measured at Baseline, at Week 11 (1 week after the last treatment infusion), and at the end of the study (Week 22, or early termination), expressed as a change from baseline scores for each assessment scale. Top panels: individual subject plots. Dotted lines are subjects in Cohort 1 (1 × 10⁹ cells), dashed lines are subjects in Cohort 2 (2 × 10⁹ cells) and solid lines are subjects in Cohort 3 (4 × 10⁹ cells). Subject 6 did not complete the Week 22 assessment. Bottom panels: medians with interquartile ranges

Fig. 7

CSF Biomarkers Percent Changes from Baseline. Biomarkers in the CSF, measured at Baseline, at Week 11 (1 week after the last treatment infusion), and at the end of the study (Week 22, or early termination), expressed as a percentage of baseline values. Dotted lines are subjects in Cohort 1 (1 × 10⁹ cells), dashed lines are subjects in Cohort 2 (2 × 10⁹ cells) and solid lines are subjects in Cohort 3 (4 × 10⁹ cells). Subject 12 did not complete the Week 22 assessment