Off-the-shelf CAR natural killer cells secreting IL-15 target spike in treating COVID-19

Engineered natural killer (NK) cells represent a promising option for immune therapy option due to their immediate availability in allogeneic settings. Severe acute diseases, such as COVID-19, require targeted and immediate intervention. Here we show engineering of NK cells to express (1) soluble interleukin-15 (sIL15) for enhancing their survival and (2) a chimeric antigen receptor (CAR) consisting of an extracellular domain of ACE2, targeting the spike protein of SARS-CoV-2. These CAR NK cells (mACE2-CAR_sIL15 NK cells) bind to VSV-SARS-CoV-2 chimeric viral particles as well as the recombinant SARS-CoV-2 spike protein subunit S1 leading to enhanced NK cell production of TNF-α and IFN-γ and increased in vitro and in vivo cytotoxicity against cells expressing the spike protein. Administration of mACE2-CAR_sIL15 NK cells maintains body weight, reduces viral load, and prolongs survival of transgenic mice expressing human ACE2 upon infection with live SARS-CoV-2. These experiments, and the capacity of mACE2-CAR_sIL15 NK cells to retain their activity following cryopreservation, demonstrate their potential as an allogeneic off-the-shelf therapy for COVID-19 patients who are faced with limited treatment options.


Statistics
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Software and code
Policy information about availability of computer code Data collection Aura Version 3.2 was used to collected Bioluminescent imagings; BD FACSDiva version 6 was used to collect data for flow cytometry; RTCA eSight Software 1.0.3 was used to collect data for real-time cell analysis (RTCA).
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The experiments of NSG mice were approved by the City of Hope Animal Care and the experiments of K18-hACE2 mice were approved by the institutional animal care and committee of Northern Arizona University.
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Umbilical cord blood units were obtained from StemCyte Inc., and the covariate-relevant population characteristics were not revealed.

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All participates who provided informed consent had samples collected under a protocol approved by the Institution Review Board at City of Hope National Medical Center.
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Flow Cytometry
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Methodology Sample preparation
For the isolation of lymphocytes from murine spleen, liver and lung, a single cell suspension was generated by mashing the organ through a cell strainer. Cells were washed in FACS buffer (PBS + 1% FBS) before labeling with flurorochrome-conjugated antibodies. Labeling of single-cell suspensions was performed at room temperature for 20 min avoiding the light. For intracellular cytokine stainings were carried out upon permeabilization and fixation with BD fix/perm Kit.

Instrument
Fortessa X20 (BD Biosciences) was used to collect data of flow cytometry.

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Software BD FACSDiva™ Software was used to collect data and FlowJo_v10 was used to analyzed the data for flow cytometry assay.

Cell population abundance
The purity of sorted cells was detected via BD FACSAriaTM Fusion and samples with purity > 95% were used.
Gating strategy Doublets (based on FSC and SSC values) and dead cells (based on live/dead staining) were gated out from the starting cell population to select only live, single cells.
In human NK cells, LNGFR expression, surface markers (e.g. CD16, CD94 et al.) expression, CD107a expression, IFN-γ expression, TNF-α expression were gated from NK cells which were defined as CD56+ population; In mice samples, human NK cells were defined as CD56+CD45+ population, and the expression of LNGFR was gated in human NK cells. The boundary between positive and negative cell population was defined using isotype controls and unstained controls.
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