Efficacy of Targeting SARS-CoV-2 by CAR-NK Cells

  1. Minh Ma
  2. Saiaditya Badeti
  3. Ke Geng
  4. Dongfang Liu

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Abstract

SARS-CoV-2, which causes COVID-19 disease, is one of greatest global pandemics in history. No effective treatment is currently available for severe COVID-19 disease. One strategy for implementing cell-based immunity involves the use of chimeric antigen receptor (CAR) technology. Unlike CAR T cells, which need to be developed using primary T cells derived from COVID-19 patients with lymphopenia, clinical success of CAR NK cell immunotherapy is possible through the development of allogeneic, universal, and ‘off-the-shelf’ CAR-NK cells from a third party, which will significantly broaden the application and reduce costs. Here, we develop a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2. CAR-NK cells were generated using the scFv domain of CR3022 (henceforth, CR3022-CAR-NK), a broadly neutralizing antibody for SARS-CoV-1 and SARS-CoV-2. CR3022-CAR-NK cells can specifically bind to RBD of SARS-CoV-2 and pseudotyped SARS-CoV-2 S protein, and can be activated by pseudotyped SARS-CoV-2-S viral particles in vitro. Further, CR3022-CAR-NK cells can specifically kill pseudo-SARS-CoV-2 infected target cells. Thus, ‘off-the-shelf’ CR3022-CAR-NK cells may have the potential to treat patients with severe COVID-19 disease.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    NKG2D
    suggested: (US Biological Cat# K1893-28, RRID:AB_2265490)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD314
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS.
    anti-human IgG(H+L
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    Anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To establish transient 293T-hACE2-RBD, 293T-hACE2 cells were transfected with 0.5 µg of SARS-CoV-2-RBD plasmid (a gift from Dr. Abraham Pinter) each well in a 24-well plate (Eppendorf) for 48 hours at 37℃ under 5% (v/v) CO2.
    293T-hACE2
    suggested: None
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    293T cells were transfected with SFG-CR3022CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells.
    293T
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierResolved on clinicaltrials.govStatusTitle
    NCT04375176YesRecruitingMonocytes and NK Cells Activit...
    NCT04280224YesRecruitingNK Cells Treatment for COVID-1...
    NCT04365101YesRecruitingNatural Killer Cell (CYNK-001)...
    NCT04324996YesRecruitingA Phase I/II Study of Universa...
    NCT04416139YesRecruitingMesenchymal Stem Cell for Acut...

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and Reagents: PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56,
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    BioLegend), PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    NKG2D
    suggested: (US Biological Cat# K1893-28, RRID:AB_2265490)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD314
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS.
    anti-human IgG(H+L
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: 293T cell line was purchased from the American Type Culture Collection (ATCC).
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    293T-hACE2 cell line is a gift from Dr. Abraham Pinter (Rutgers-New Jersey Medical School, PHRI).
    293T-hACE2
    suggested: None
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 μg of SARS-CoV-2-RBD plasmid and 0.25 μg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis: NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK-92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production: A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, intracellular domain CD28 and 4-1BB, and the ζ chain of the human TCR/CD3 complex.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04375176RecruitingMonocytes and NK Cells Activity in Covid-19 Patients
    NCT04280224RecruitingNK Cells Treatment for COVID-19
    NCT04365101RecruitingNatural Killer Cell (CYNK-001) Infusions in Adults With COVI…
    NCT04324996RecruitingA Phase I/II Study of Universal Off-the-shelf NKG2D-ACE2 CAR…
    NCT04416139RecruitingMesenchymal Stem Cell for Acute Respiratory Distress Syndrom…

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    NKG2D
    suggested: (US Biological Cat# K1893-28, RRID:AB_2265490)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD314
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    anti-human IgG(H+L
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours.
    293T
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    293T-hACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierResolved on clinicaltrials.govStatusTitle
    NCT04375176YesRecruitingMonocytes and NK Cells Activit...
    NCT04280224YesRecruitingNK Cells Treatment for COVID-1...
    NCT04365101YesRecruitingNatural Killer Cell (CYNK-001)...
    NCT04324996YesRecruitingA Phase I/II Study of Universa...
    NCT04416139YesRecruitingMesenchymal Stem Cell for Acut...

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  4. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD314
    suggested: None
    NKG2D
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS.
    anti-human IgG(H+L
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    Anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours.
    293T
    suggested: None
    Then, the following day, the wells were aspirated and 293T-hACE2FFLuc-GFP-RBD and 293T-hACE2 cells were pre-seeded at 1 × 104 target cells/well in 100 µL/well of DMEM supplemented with 10% FBS.
    293T-hACE2
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierResolved on clinicaltrials.govStatusTitle
    NCT04375176YesRecruitingMonocytes and NK Cells Activit...
    NCT04280224YesRecruitingNK Cells Treatment for COVID-1...
    NCT04365101YesRecruitingNatural Killer Cell (CYNK-001)...
    NCT04324996YesRecruitingA Phase I/II Study of Universa...
    NCT04416139YesRecruitingMesenchymal Stem Cell for Acut...

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  5. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    NKG2D
    suggested: (US Biological Cat# K1893-28, RRID:AB_2265490)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD314
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS.
    anti-human IgG(H+L
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    Anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293T cells were transfected with a combination of plasmids containing CR3022-CAR in the SFG backbone, RDF, and PegPam3, as previously described17,18.
    293T
    suggested: None
    To maintain the stable expression of hACE2, 293T-hACE2 cells were cultured in DMEM (Corning) supplemented with 10% (v/v)
    293T-hACE2
    suggested: None
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierResolved on clinicaltrials.govTitleStatus
    NCT04375176YesRecruitingMonocytes and NK Cells Activit...
    NCT04280224YesRecruitingNK Cells Treatment for COVID-1...
    NCT04365101YesRecruitingNatural Killer Cell (CYNK-001)...
    NCT04324996YesRecruitingA Phase I/II Study of Universa...
    NCT04416139YesRecruitingMesenchymal Stem Cell for Acut...

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  6. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    anti-human CD314
    suggested: None
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    NKG2D
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS.
    anti-human IgG(H+L
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours.
    293T
    suggested: None
    Then, the following day, the wells were aspirated and 293T-hACE2FFLuc-GFP-RBD and 293T-hACE2 cells were pre-seeded at 1 × 104 target cells/well in 100 µL/well of DMEM supplemented with 10% FBS.
    293T-hACE2
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3022-CAR-NK-92MI cells, test the efficacy of CR3022-CAR-NK-92MI using in vitro assays, and finally, define the efficacy of eliminating SARS-CoV-2 infected target cells by CR3022-CAR-NK-92MI cells. This work pioneers the use of CR3022-CAR-NK cells to treat SARS-CoV-2 infected patients and will lead to the development of novel immunotherapeutic strategies for patients presenting with severe COVID-19, and combined with other broadly neutralizing antibodies, will support the development of a universal, “off-the-shelf ” CAR-NK based-immunotherapy for COVID-19. Online content Any methods, additional references, source data, and statements of code and data availability are available online. METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend), PE anti-human CD69 antibody (clone FN50, BioLegend), PE anti-human CD8a antibody (clone RPA-T8, BioLegend), APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend), APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend), BV421 anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend), PE/Cy7 anti-human CD244 (2B4) antibody (clone C1.7, BioLegend), PE anti-human CD152 (CTLA-4) antibody (clone BNI3), APC anti-human CD366 (Tim-3) antibody (clone F38-2E2), PerCP/Cy5.5 antihuman TIGIT (VSTM3) antibody (clone A15153G), FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend), BV510 anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA). APC anti-human CD16 antibody (clone 3G8, BD Biosciences), BV711 anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA). PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems. AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Anti-SARS-CoV-2 Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA). Anti-SARS-CoV-2 Spike RBD rabbit polyclonal antibody was purchased from SinoBiological (Beijing, China). Anti-His mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG1 (g1) were purchased from Fisher Scientific (Waltham, MA). Cell lines 293T cell line was purchased from the American Type Culture Collection (ATCC). 293T-hACE2 cell line is a gift from Dr. Abraham Pinter (Rutgers-New Jersey Medical School, PHRI). To maintain the stable expression of hACE2, 293T-hACE2 cells were cultured in DMEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL Penicillin-Streptomycin (Corning), and 1µg/mL of puromycin at 37℃ under 5% (v/v) CO2. To establish transient 293T-hACE2-RBD, 293T-hACE2 cells were transfected with 0.5 µg of SARS-CoV-2-RBD plasmid (a gift from Dr. Abraham Pinter) each well in a 24-well plate (Eppendorf) for 48 hours at 37℃ under 5% (v/v) CO2. Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2. Cells were harvested and immediately used for CD107a degranulation, 51Cr release, and FFLuc reporter assays. CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3. CR3022-CAR retrovirus was harvested after 48-72 hours and transduced to NK92MI cells in a 24-well plate coated with 0.5 µg/ml of RetroNectin diluted in PBS (Clontech). Two days later, cells were transferred to 75 cm2 flask (Corning) and maintained in 35 ml complete NK92MI medium (MEM-a with 12.5% (v/v) FBS, 12.5% (v/v) heat inactivated horse serum, 11 µM bME, 2 µM folic acid, and 20 µM inositol) supplemented 200 U/mL IL-2 (PeproTech). To determine the expression of CAR, cells were stained for CD56 and anti-human IgG(H+L) F(ab’)2 fragment and analyzed by flow cytometry. CR3022-CAR and RBD binding assay To evaluate the binding activity of CR3022-CAR to RBD domain of SARS-CoV-2-S, CR3022-CAR and NK92MI (5 × 105) cells were incubated with 5 µg of His-gp70-RBD recombinant protein is a gift from Dr. Abraham Pinter in DPBS buffer (0.5 mM MgCl2 and 0.9 mM CaCl2 in PBS) in for 30 minutes on ice. Cells were washed twice with PBS, stained with anti-His in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed twice with PBS. Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry. CR3022-CAR and pseudotyped SARS-CoV-2-S viral particles binding assay CR3022-CAR, NK92MI, and 293T-hACE2 (5 × 105) cells were first equilibrated with BM (complete RPMI-1640 containing 0.2% BSA and 10 mM HEPES pH 7.4). Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS. Pseudotyped SARS-CoV-2-S (Genscript), full-length recombinant S protein (Acrobio systems), and S1 subunit recombinant protein (a gift from Dr. Abraham Pinter) were diluted with BM to appropriate concentrations. 4 × 106 IFU of pseudotyped SARS-CoV-2-S, or 2 µg of full-length recombinant S protein, or 2 µg of S1 subunit recombinant protein was added to designated wells of a 96-well V bottom plate. Plate was spun at 600 × g for 30 minutes at 32°C, then was incubated at 37°C under 5% (v/v) CO2 for 1 hour. Cells were washed twice with PBS, stained with anti-S1 in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed thrice with PBS. Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry. Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described. Cells were analyzed on a FACS LSRII or an LSR Fortessa flow cytometer (BD). PMT voltages were adjusted and compensation values were calculated before data collection. Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD). CD107a Degranulation assay The CD107a degranulation assay was described previously3. Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours. The cells were harvested, washed, and stained for CD3, CD56, and CD107a with GolgiStop (BD Biosciences) for 30 minutes, and analyzed by flow cytometry. FFLuc reporter assay To quantify the cytotoxicity of CAR-modified immune cells, we also developed the FFLuc reporter system assay. Briefly, an optical 96-well plate (Greiner BioOne™ No: 655098) was precoated with Retronectin (0.5 µg/ml in PBS) and placed at 4°C overnight. Then, the following day, the wells were aspirated and 293T-hACE2FFLuc-GFP-RBD and 293T-hACE2 cells were pre-seeded at 1 × 104 target cells/well in 100 µL/well of DMEM supplemented with 10% FBS. The plate was centrifuged for 5 minutes at 350 ´ g. In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml. Serial dilutions of effector cells were then prepared according to the effector/target ratio using NK-92MI medium. Then, the effector cells were added to each well of the optical plate (100 µL/well) and incubated at 37°C under 5% (v/v) CO2 for 4 hours and then the supernatant was gently discarded. 100 µL of working concentration D-Luciferin was added to each well with the lights turned off. A microplate reader (BioTek, USA) was used to quantify the data. The data was quantified by converting the obtained values to percentage of specific lysis by the following equation: Specific Lysis Percentage (%) = [1-(S-E)/(T-M)]×100, where S is the value of luminescence of the sample well, E is the value of luminescence of the “effector cell only” well compared to the sample well, T is the mean value of luminescence of “Target cell only” wells, and M is the mean value of luminescence of “blank medium only” wells. 51 Cr release assay To evaluate the cytotoxic activity of CAR-NK cell, the standard 4-hour 51Cr release assay was used. Briefly, target cells were labeled with 51Cr at 37°C for 2 hours and then resuspended at 1×105/mL in NK-92MI culture medium with 10% FBS without IL-2. Then, 1×104 target cells were incubated with serially diluted CAR-NK or NK-92MI cells at 37°C for 4 hours. After centrifugation, the supernatants were collected and the released 51Cr was measured with a gamma counter (Wallac, Turku, Finland). The cytotoxicity (as a percentage) was calculated as follows: [(sample − spontaneous release) / (maximum release − spontaneous release)] × 100. Statistical Analysis Data were represented as means ± SEM. The statistical significance was determined using a two-tailed unpaired Student t test, a two-tailed paired Student t test, a two-way ANOVA, where indicated. P < 0.05 was considered statistically significant. Figure and Figure legends: Figure 1: Generation of CR3022-CAR-NK92MI cells. (a) Schematic design of CR3022-CAR in SFG retroviral vector. The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ. (b) Generation of CR3022-CAR-NK cells. 293T cells were transfected with SFG-CR3022CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells. (c) Determination of CAR expression by flow cytometry. CR3022-CAR cells were harvested after 4-5 days then stained with anti-CD56 and CAR F(ab)2 domain [IgG Figure 2: CR3022-CAR-NK92MI cells bind to RBD domain of SARS-CoV-2-S protein. (a) Immunophenotyping of CR3022-CAR. Antibodies against various immunomodulatory receptors including TIGIT, LAG-3, TIM-3, KLRG1, CTLA-4, PD-1, CD69, CD8A, NKG2C, CD94, DNAM-1, 2B4, NKG2D, NKP46, and CD16 were used to stain CR3022-CAR and NK-92MI. The expression of these receptors was determined by flow cytometry. (b) Diagram of CR3022-CAR binding to the RBD domain of SARSCoV-2-S recombinant protein. CR3022-CAR binds to RBD of SARS-CoV-2-S protein which is then recognized by anti-His and its corresponding secondary antibody conjugated to a fluorophore. (c) Representative dot plots of CR3022-CAR binding to RBD of SARS-CoV-2. CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. The binding efficiency was determined by flow cytometry. Figure 3: CR3022-CAR-NK-92MI cells bind to pseudotyped SARS-CoV-2-S viral particles. (a) Diagram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S viral particles. CR3022-CAR binds to pseudotyped SARS-CoV-2-S viral particle which is then recognized by anti-spike and its corresponding secondary antibody conjugated to a fluorophore. (b) Representative histogram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S. CR3022-CAR or NK-92MI or 293T-hACE2 cells were incubated with pseudotyped SARS-CoV-2 or full-length spike or S1 subunit recombinant protein. The binding efficiency was determined by flow cytometry. Experimental sample was performed in triplicate with MFI 6759 ± 440 (a.u.). (c) Graph showing the binding efficiency of CR3022-CAR to pseudotyped SARS-CoV-2-S. The values were converted from Figure 2b. Experimental sample was performed in triplicate with binding efficiency 51.4 ± 3.34 (%). Figure 4: Increased CD107a degranulation and killing activity of CR3022-CAR-NK92MI cells against 293T-hACE2 cells transfected with RBD-SARS-Cov-2 Spike. (a) 293T-hACE2 cells were transfected with a plasmid containing firefly luciferase and GFP as well as SARS-CoV-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody. Cells were then harvested and used as target cells for subsequent CD107a degranulation assay and luciferase killing assays. (c) Representative dot plots of CD107a assay and quantitative data of the percentage and mean fluorescence intensity of CD107a positive CR3022-CAR-NK92MI cells are shown. (d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown. Experimental groups were performed in represent the mean ± SEM from at least two independent experiments. Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Then, cells were harvested, stained for CAR F(ab)2 domain [IgG (H+L)], and CD107a. Representative flow cytometry dot plots, CD107a percent of total CAR cells, and CD107a MFI are shown. Figure 5: Increased killing activity of CR3022-CAR-NK-92MI cells against 293ThACE2 cells transfected with SARS-Cov-2 Spike protein Receptor Binding Domain using the 51Cr release platform. (a) 293T-hACE2 cells were transfected with SARSCov-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody. Cells were then harvested and used as target cells for the subsequent 51Cr release assay. (c) Quantitative data of the 51Cr release assay using CR3022-CAR-NK-92MI cells and wild-type NK-92MI cells. Experimental groups were performed in triplicate. * p < 0.05, ** p < 0.01, *** p = 0.001, **** p<0.0001 ns p > 0.05. Data represent the mean ± SEM from at least two independent experiments.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierResolved on clinicaltrials.govTitleStatus
    NCT04375176YesMonocytes and NK Cells Activity in Covid-19 Patien...Recruiting
    NCT04280224YesNK Cells Treatment for COVID-19Recruiting
    NCT04365101YesNatural Killer Cell (CYNK-001) Infusions in Adults...Recruiting
    NCT04324996YesA Phase I/II Study of Universal Off-the-shelf NKG2...Recruiting
    NCT04416139YesMesenchymal Stem Cell for Acute Respiratory Distre...Recruiting

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  7. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD314
    suggested: None
    NKG2D
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    anti-human IgG(H+L
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    Anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To maintain the stable expression of hACE2, 293T-hACE2 cells were cultured in DMEM (Corning) supplemented with 10% (v/v)
    293T-hACE2
    suggested: None
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours.
    293T
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3022-CAR-NK-92MI cells, test the efficacy of CR3022-CAR-NK-92MI using in vitro assays, and finally, define the efficacy of eliminating SARS-CoV-2 infected target cells by CR3022-CAR-NK-92MI cells. This work pioneers the use of CR3022-CAR-NK cells to treat SARS-CoV-2 infected patients and will lead to the development of novel immunotherapeutic strategies for patients presenting with severe COVID-19, and combined with other broadly neutralizing antibodies, will support the development of a universal, “off-the-shelf ” CAR-NK based-immunotherapy for COVID-19. Online content Any methods, additional references, source data, and statements of code and data availability are available online. METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend), PE anti-human CD69 antibody (clone FN50, BioLegend), PE anti-human CD8a antibody (clone RPA-T8, BioLegend), APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend), APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend), BV421 anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend), PE/Cy7 anti-human CD244 (2B4) antibody (clone C1.7, BioLegend), PE anti-human CD152 (CTLA-4) antibody (clone BNI3), APC anti-human CD366 (Tim-3) antibody (clone F38-2E2), PerCP/Cy5.5 antihuman TIGIT (VSTM3) antibody (clone A15153G), FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend), BV510 anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA). APC anti-human CD16 antibody (clone 3G8, BD Biosciences), BV711 anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA). PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems. AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Anti-SARS-CoV-2 Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA). Anti-SARS-CoV-2 Spike RBD rabbit polyclonal antibody was purchased from SinoBiological (Beijing, China). Anti-His mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG1 (g1) were purchased from Fisher Scientific (Waltham, MA). Cell lines 293T cell line was purchased from the American Type Culture Collection (ATCC). 293T-hACE2 cell line is a gift from Dr. Abraham Pinter (Rutgers-New Jersey Medical School, PHRI). To maintain the stable expression of hACE2, 293T-hACE2 cells were cultured in DMEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL Penicillin-Streptomycin (Corning), and 1µg/mL of puromycin at 37℃ under 5% (v/v) CO2. To establish transient 293T-hACE2-RBD, 293T-hACE2 cells were transfected with 0.5 µg of SARS-CoV-2-RBD plasmid (a gift from Dr. Abraham Pinter) each well in a 24-well plate (Eppendorf) for 48 hours at 37℃ under 5% (v/v) CO2. Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2. Cells were harvested and immediately used for CD107a degranulation, 51Cr release, and FFLuc reporter assays. CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3. CR3022-CAR retrovirus was harvested after 48-72 hours and transduced to NK92MI cells in a 24-well plate coated with 0.5 µg/ml of RetroNectin diluted in PBS (Clontech). Two days later, cells were transferred to 75 cm2 flask (Corning) and maintained in 35 ml complete NK92MI medium (MEM-a with 12.5% (v/v) FBS, 12.5% (v/v) heat inactivated horse serum, 11 µM bME, 2 µM folic acid, and 20 µM inositol) supplemented 200 U/mL IL-2 (PeproTech). To determine the expression of CAR, cells were stained for CD56 and anti-human IgG(H+L) F(ab’)2 fragment and analyzed by flow cytometry. CR3022-CAR and RBD binding assay To evaluate the binding activity of CR3022-CAR to RBD domain of SARS-CoV-2-S, CR3022-CAR and NK92MI (5 × 105) cells were incubated with 5 µg of His-gp70-RBD recombinant protein is a gift from Dr. Abraham Pinter in DPBS buffer (0.5 mM MgCl2 and 0.9 mM CaCl2 in PBS) in for 30 minutes on ice. Cells were washed twice with PBS, stained with anti-His in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed twice with PBS. Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry. CR3022-CAR and pseudotyped SARS-CoV-2-S viral particles binding assay CR3022-CAR, NK92MI, and 293T-hACE2 (5 × 105) cells were first equilibrated with BM (complete RPMI-1640 containing 0.2% BSA and 10 mM HEPES pH 7.4). Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS. Pseudotyped SARS-CoV-2-S (Genscript), full-length recombinant S protein (Acrobio systems), and S1 subunit recombinant protein (a gift from Dr. Abraham Pinter) were diluted with BM to appropriate concentrations. 4 × 106 IFU of pseudotyped SARS-CoV-2-S, or 2 µg of full-length recombinant S protein, or 2 µg of S1 subunit recombinant protein was added to designated wells of a 96-well V bottom plate. Plate was spun at 600 × g for 30 minutes at 32°C, then was incubated at 37°C under 5% (v/v) CO2 for 1 hour. Cells were washed twice with PBS, stained with anti-S1 in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed thrice with PBS. Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry. Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described. Cells were analyzed on a FACS LSRII or an LSR Fortessa flow cytometer (BD). PMT voltages were adjusted and compensation values were calculated before data collection. Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD). CD107a Degranulation assay The CD107a degranulation assay was described previously3. Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours. The cells were harvested, washed, and stained for CD3, CD56, and CD107a with GolgiStop (BD Biosciences) for 30 minutes, and analyzed by flow cytometry. FFLuc reporter assay To quantify the cytotoxicity of CAR-modified immune cells, we also developed the FFLuc reporter system assay. Briefly, an optical 96-well plate (Greiner BioOne™ No: 655098) was precoated with Retronectin (0.5 µg/ml in PBS) and placed at 4°C overnight. Then, the following day, the wells were aspirated and 293T-hACE2FFLuc-GFP-RBD and 293T-hACE2 cells were pre-seeded at 1 × 104 target cells/well in 100 µL/well of DMEM supplemented with 10% FBS. The plate was centrifuged for 5 minutes at 350 ´ g. In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml. Serial dilutions of effector cells were then prepared according to the effector/target ratio using NK-92MI medium. Then, the effector cells were added to each well of the optical plate (100 µL/well) and incubated at 37°C under 5% (v/v) CO2 for 4 hours and then the supernatant was gently discarded. 100 µL of working concentration D-Luciferin was added to each well with the lights turned off. A microplate reader (BioTek, USA) was used to quantify the data. The data was quantified by converting the obtained values to percentage of specific lysis by the following equation: Specific Lysis Percentage (%) = [1-(S-E)/(T-M)]×100, where S is the value of luminescence of the sample well, E is the value of luminescence of the “effector cell only” well compared to the sample well, T is the mean value of luminescence of “Target cell only” wells, and M is the mean value of luminescence of “blank medium only” wells. 51 Cr release assay To evaluate the cytotoxic activity of CAR-NK cell, the standard 4-hour 51Cr release assay was used. Briefly, target cells were labeled with 51Cr at 37°C for 2 hours and then resuspended at 1×105/mL in NK-92MI culture medium with 10% FBS without IL-2. Then, 1×104 target cells were incubated with serially diluted CAR-NK or NK-92MI cells at 37°C for 4 hours. After centrifugation, the supernatants were collected and the released 51Cr was measured with a gamma counter (Wallac, Turku, Finland). The cytotoxicity (as a percentage) was calculated as follows: [(sample − spontaneous release) / (maximum release − spontaneous release)] × 100. Statistical Analysis Data were represented as means ± SEM. The statistical significance was determined using a two-tailed unpaired Student t test, a two-tailed paired Student t test, a two-way ANOVA, where indicated. P < 0.05 was considered statistically significant. Figure and Figure legends: Figure 1: Generation of CR3022-CAR-NK92MI cells. (a) Schematic design of CR3022-CAR in SFG retroviral vector. The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ. (b) Generation of CR3022-CAR-NK cells. 293T cells were transfected with SFG-CR3022CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells. (c) Determination of CAR expression by flow cytometry. CR3022-CAR cells were harvested after 4-5 days then stained with anti-CD56 and CAR F(ab)2 domain [IgG Figure 2: CR3022-CAR-NK92MI cells bind to RBD domain of SARS-CoV-2-S protein. (a) Immunophenotyping of CR3022-CAR. Antibodies against various immunomodulatory receptors including TIGIT, LAG-3, TIM-3, KLRG1, CTLA-4, PD-1, CD69, CD8A, NKG2C, CD94, DNAM-1, 2B4, NKG2D, NKP46, and CD16 were used to stain CR3022-CAR and NK-92MI. The expression of these receptors was determined by flow cytometry. (b) Diagram of CR3022-CAR binding to the RBD domain of SARSCoV-2-S recombinant protein. CR3022-CAR binds to RBD of SARS-CoV-2-S protein which is then recognized by anti-His and its corresponding secondary antibody conjugated to a fluorophore. (c) Representative dot plots of CR3022-CAR binding to RBD of SARS-CoV-2. CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. The binding efficiency was determined by flow cytometry. Figure 3: CR3022-CAR-NK-92MI cells bind to pseudotyped SARS-CoV-2-S viral particles. (a) Diagram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S viral particles. CR3022-CAR binds to pseudotyped SARS-CoV-2-S viral particle which is then recognized by anti-spike and its corresponding secondary antibody conjugated to a fluorophore. (b) Representative histogram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S. CR3022-CAR or NK-92MI or 293T-hACE2 cells were incubated with pseudotyped SARS-CoV-2 or full-length spike or S1 subunit recombinant protein. The binding efficiency was determined by flow cytometry. Experimental sample was performed in triplicate with MFI 6759 ± 440 (a.u.). (c) Graph showing the binding efficiency of CR3022-CAR to pseudotyped SARS-CoV-2-S. The values were converted from Figure 2b. Experimental sample was performed in triplicate with binding efficiency 51.4 ± 3.34 (%). Figure 4: Increased CD107a degranulation and killing activity of CR3022-CAR-NK92MI cells against 293T-hACE2 cells transfected with RBD-SARS-Cov-2 Spike. (a) 293T-hACE2 cells were transfected with a plasmid containing firefly luciferase and GFP as well as SARS-CoV-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody. Cells were then harvested and used as target cells for subsequent CD107a degranulation assay and luciferase killing assays. (c) Representative dot plots of CD107a assay and quantitative data of the percentage and mean fluorescence intensity of CD107a positive CR3022-CAR-NK92MI cells are shown. (d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown. Experimental groups were performed in represent the mean ± SEM from at least two independent experiments. Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Then, cells were harvested, stained for CAR F(ab)2 domain [IgG (H+L)], and CD107a. Representative flow cytometry dot plots, CD107a percent of total CAR cells, and CD107a MFI are shown. Figure 5: Increased killing activity of CR3022-CAR-NK-92MI cells against 293ThACE2 cells transfected with SARS-Cov-2 Spike protein Receptor Binding Domain using the 51Cr release platform. (a) 293T-hACE2 cells were transfected with SARSCov-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody. Cells were then harvested and used as target cells for the subsequent 51Cr release assay. (c) Quantitative data of the 51Cr release assay using CR3022-CAR-NK-92MI cells and wild-type NK-92MI cells. Experimental groups were performed in triplicate. * p < 0.05, ** p < 0.01, *** p = 0.001, **** p<0.0001 ns p > 0.05. Data represent the mean ± SEM from at least two independent experiments.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  8. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    NKG2D
    suggested: (US Biological Cat# K1893-28, RRID:AB_2265490)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD314
    suggested: None
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS.
    anti-human IgG(H+L
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293T cells were transfected with a combination of plasmids containing CR3022-CAR in the SFG backbone, RDF, and PegPam3, as previously described17,18.
    293T
    suggested: None
    293T-hACE2 cell line is a gift from Dr. Abraham Pinter (Rutgers-New Jersey Medical School, PHRI).
    293T-hACE2
    suggested: None
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3022-CAR-NK-92MI cells, test the efficacy of CR3022-CAR-NK-92MI using in vitro assays, and finally, define the efficacy of eliminating SARS-CoV-2 infected target cells by CR3022-CAR-NK-92MI cells. This work pioneers the use of CR3022-CAR-NK cells to treat SARS-CoV-2 infected patients and will lead to the development of novel immunotherapeutic strategies for patients presenting with severe COVID-19, and combined with other broadly neutralizing antibodies, will support the development of a universal, “off-the-shelf ” CAR-NK based-immunotherapy for COVID-19. Online content Any methods, additional references, source data, and statements of code and data availability are available online. METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend), PE anti-human CD69 antibody (clone FN50, BioLegend), PE anti-human CD8a antibody (clone RPA-T8, BioLegend), APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend), APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend), BV421 anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend), PE/Cy7 anti-human CD244 (2B4) antibody (clone C1.7, BioLegend), PE anti-human CD152 (CTLA-4) antibody (clone BNI3), APC anti-human CD366 (Tim-3) antibody (clone F38-2E2), PerCP/Cy5.5 antihuman TIGIT (VSTM3) antibody (clone A15153G), FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend), BV510 anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA). APC anti-human CD16 antibody (clone 3G8, BD Biosciences), BV711 anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA). PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems. AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Anti-SARS-CoV-2 Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA). Anti-SARS-CoV-2 Spike RBD rabbit polyclonal antibody was purchased from SinoBiological (Beijing, China). Anti-His mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG1 (g1) were purchased from Fisher Scientific (Waltham, MA). Cell lines 293T cell line was purchased from the American Type Culture Collection (ATCC). 293T-hACE2 cell line is a gift from Dr. Abraham Pinter (Rutgers-New Jersey Medical School, PHRI). To maintain the stable expression of hACE2, 293T-hACE2 cells were cultured in DMEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL Penicillin-Streptomycin (Corning), and 1µg/mL of puromycin at 37℃ under 5% (v/v) CO2. To establish transient 293T-hACE2-RBD, 293T-hACE2 cells were transfected with 0.5 µg of SARS-CoV-2-RBD plasmid (a gift from Dr. Abraham Pinter) each well in a 24-well plate (Eppendorf) for 48 hours at 37℃ under 5% (v/v) CO2. Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2. Cells were harvested and immediately used for CD107a degranulation, 51Cr release, and FFLuc reporter assays. CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3. CR3022-CAR retrovirus was harvested after 48-72 hours and transduced to NK92MI cells in a 24-well plate coated with 0.5 µg/ml of RetroNectin diluted in PBS (Clontech). Two days later, cells were transferred to 75 cm2 flask (Corning) and maintained in 35 ml complete NK92MI medium (MEM-a with 12.5% (v/v) FBS, 12.5% (v/v) heat inactivated horse serum, 11 µM bME, 2 µM folic acid, and 20 µM inositol) supplemented 200 U/mL IL-2 (PeproTech). To determine the expression of CAR, cells were stained for CD56 and anti-human IgG(H+L) F(ab’)2 fragment and analyzed by flow cytometry. CR3022-CAR and RBD binding assay To evaluate the binding activity of CR3022-CAR to RBD domain of SARS-CoV-2-S, CR3022-CAR and NK92MI (5 × 105) cells were incubated with 5 µg of His-gp70-RBD recombinant protein is a gift from Dr. Abraham Pinter in DPBS buffer (0.5 mM MgCl2 and 0.9 mM CaCl2 in PBS) in for 30 minutes on ice. Cells were washed twice with PBS, stained with anti-His in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed twice with PBS. Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry. CR3022-CAR and pseudotyped SARS-CoV-2-S viral particles binding assay CR3022-CAR, NK92MI, and 293T-hACE2 (5 × 105) cells were first equilibrated with BM (complete RPMI-1640 containing 0.2% BSA and 10 mM HEPES pH 7.4). Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS. Pseudotyped SARS-CoV-2-S (Genscript), full-length recombinant S protein (Acrobio systems), and S1 subunit recombinant protein (a gift from Dr. Abraham Pinter) were diluted with BM to appropriate concentrations. 4 × 106 IFU of pseudotyped SARS-CoV-2-S, or 2 µg of full-length recombinant S protein, or 2 µg of S1 subunit recombinant protein was added to designated wells of a 96-well V bottom plate. Plate was spun at 600 × g for 30 minutes at 32°C, then was incubated at 37°C under 5% (v/v) CO2 for 1 hour. Cells were washed twice with PBS, stained with anti-S1 in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed thrice with PBS. Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry. Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described. Cells were analyzed on a FACS LSRII or an LSR Fortessa flow cytometer (BD). PMT voltages were adjusted and compensation values were calculated before data collection. Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD). CD107a Degranulation assay The CD107a degranulation assay was described previously3. Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours. The cells were harvested, washed, and stained for CD3, CD56, and CD107a with GolgiStop (BD Biosciences) for 30 minutes, and analyzed by flow cytometry. FFLuc reporter assay To quantify the cytotoxicity of CAR-modified immune cells, we also developed the FFLuc reporter system assay. Briefly, an optical 96-well plate (Greiner BioOne™ No: 655098) was precoated with Retronectin (0.5 µg/ml in PBS) and placed at 4°C overnight. Then, the following day, the wells were aspirated and 293T-hACE2FFLuc-GFP-RBD and 293T-hACE2 cells were pre-seeded at 1 × 104 target cells/well in 100 µL/well of DMEM supplemented with 10% FBS. The plate was centrifuged for 5 minutes at 350 ´ g. In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml. Serial dilutions of effector cells were then prepared according to the effector/target ratio using NK-92MI medium. Then, the effector cells were added to each well of the optical plate (100 µL/well) and incubated at 37°C under 5% (v/v) CO2 for 4 hours and then the supernatant was gently discarded. 100 µL of working concentration D-Luciferin was added to each well with the lights turned off. A microplate reader (BioTek, USA) was used to quantify the data. The data was quantified by converting the obtained values to percentage of specific lysis by the following equation: Specific Lysis Percentage (%) = [1-(S-E)/(T-M)]×100, where S is the value of luminescence of the sample well, E is the value of luminescence of the “effector cell only” well compared to the sample well, T is the mean value of luminescence of “Target cell only” wells, and M is the mean value of luminescence of “blank medium only” wells. 51 Cr release assay To evaluate the cytotoxic activity of CAR-NK cell, the standard 4-hour 51Cr release assay was used. Briefly, target cells were labeled with 51Cr at 37°C for 2 hours and then resuspended at 1×105/mL in NK-92MI culture medium with 10% FBS without IL-2. Then, 1×104 target cells were incubated with serially diluted CAR-NK or NK-92MI cells at 37°C for 4 hours. After centrifugation, the supernatants were collected and the released 51Cr was measured with a gamma counter (Wallac, Turku, Finland). The cytotoxicity (as a percentage) was calculated as follows: [(sample − spontaneous release) / (maximum release − spontaneous release)] × 100. Statistical Analysis Data were represented as means ± SEM. The statistical significance was determined using a two-tailed unpaired Student t test, a two-tailed paired Student t test, a two-way ANOVA, where indicated. P < 0.05 was considered statistically significant. Figure and Figure legends: Figure 1: Generation of CR3022-CAR-NK92MI cells. (a) Schematic design of CR3022-CAR in SFG retroviral vector. The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ. (b) Generation of CR3022-CAR-NK cells. 293T cells were transfected with SFG-CR3022CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells. (c) Determination of CAR expression by flow cytometry. CR3022-CAR cells were harvested after 4-5 days then stained with anti-CD56 and CAR F(ab)2 domain [IgG Figure 2: CR3022-CAR-NK92MI cells bind to RBD domain of SARS-CoV-2-S protein. (a) Immunophenotyping of CR3022-CAR. Antibodies against various immunomodulatory receptors including TIGIT, LAG-3, TIM-3, KLRG1, CTLA-4, PD-1, CD69, CD8A, NKG2C, CD94, DNAM-1, 2B4, NKG2D, NKP46, and CD16 were used to stain CR3022-CAR and NK-92MI. The expression of these receptors was determined by flow cytometry. (b) Diagram of CR3022-CAR binding to the RBD domain of SARSCoV-2-S recombinant protein. CR3022-CAR binds to RBD of SARS-CoV-2-S protein which is then recognized by anti-His and its corresponding secondary antibody conjugated to a fluorophore. (c) Representative dot plots of CR3022-CAR binding to RBD of SARS-CoV-2. CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. The binding efficiency was determined by flow cytometry. Figure 3: CR3022-CAR-NK-92MI cells bind to pseudotyped SARS-CoV-2-S viral particles. (a) Diagram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S viral particles. CR3022-CAR binds to pseudotyped SARS-CoV-2-S viral particle which is then recognized by anti-spike and its corresponding secondary antibody conjugated to a fluorophore. (b) Representative histogram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S. CR3022-CAR or NK-92MI or 293T-hACE2 cells were incubated with pseudotyped SARS-CoV-2 or full-length spike or S1 subunit recombinant protein. The binding efficiency was determined by flow cytometry. Experimental sample was performed in triplicate with MFI 6759 ± 440 (a.u.). (c) Graph showing the binding efficiency of CR3022-CAR to pseudotyped SARS-CoV-2-S. The values were converted from Figure 2b. Experimental sample was performed in triplicate with binding efficiency 51.4 ± 3.34 (%). Figure 4: Increased CD107a degranulation and killing activity of CR3022-CAR-NK92MI cells against 293T-hACE2 cells transfected with RBD-SARS-Cov-2 Spike. (a) 293T-hACE2 cells were transfected with a plasmid containing firefly luciferase and GFP as well as SARS-CoV-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody. Cells were then harvested and used as target cells for subsequent CD107a degranulation assay and luciferase killing assays. (c) Representative dot plots of CD107a assay and quantitative data of the percentage and mean fluorescence intensity of CD107a positive CR3022-CAR-NK92MI cells are shown. (d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown. Experimental groups were performed in represent the mean ± SEM from at least two independent experiments. Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Then, cells were harvested, stained for CAR F(ab)2 domain [IgG (H+L)], and CD107a. Representative flow cytometry dot plots, CD107a percent of total CAR cells, and CD107a MFI are shown. Figure 5: Increased killing activity of CR3022-CAR-NK-92MI cells against 293ThACE2 cells transfected with SARS-Cov-2 Spike protein Receptor Binding Domain using the 51Cr release platform. (a) 293T-hACE2 cells were transfected with SARSCov-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody. Cells were then harvested and used as target cells for the subsequent 51Cr release assay. (c) Quantitative data of the 51Cr release assay using CR3022-CAR-NK-92MI cells and wild-type NK-92MI cells. Experimental groups were performed in triplicate. * p < 0.05, ** p < 0.01, *** p = 0.001, **** p<0.0001 ns p > 0.05. Data represent the mean ± SEM from at least two independent experiments.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  9. ScreenIT

    SciScore for 10.1101/2020.08.11.247320: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend)
    anti-human CD3
    suggested: None
    anti-human CD56
    suggested: None
    , PE anti-human CD69 antibody (clone FN50, BioLegend)
    anti-human CD69
    suggested: None
    PE anti-human CD8a antibody (clone RPA-T8, BioLegend),
    anti-human CD8a
    suggested: None
    APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend),
    anti-human CD226
    suggested: None
    APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)
    antihuman KLRG1
    suggested: None
    MAFA
    suggested: None
    anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend)
    anti-human CD335
    suggested: None
    NKp46
    suggested: None
    anti-human CD244 (2B4) antibody (clone C1.7, BioLegend)
    anti-human CD244
    suggested: None
    , PE anti-human CD152 (CTLA-4) antibody (clone BNI3)
    anti-human CD152
    suggested: None
    CTLA-4
    suggested: None
    , APC anti-human CD366 (Tim-3) antibody (clone F38-2E2),
    anti-human CD366
    suggested: None
    Tim-3
    suggested: None
    antihuman TIGIT (VSTM3) antibody (clone A15153G)
    antihuman TIGIT ( VSTM3
    suggested: None
    FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)
    anti-human CD223
    suggested: None
    LAG-3
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA)
    anti-human CD314
    suggested: None
    NKG2D
    suggested: (US Biological Cat# K1893-28, RRID:AB_2265490)
    anti-human CD94
    suggested: None
    APC anti-human CD16 antibody (clone 3G8, BD Biosciences)
    anti-human CD16
    suggested: None
    anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA)
    anti-human CD107a
    suggested: None
    PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems.
    PE anti-human NKG2C/CD159c antibody
    suggested: None
    anti-human NKG2C/CD159c
    suggested: None
    AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
    AF647 Goat anti-human IgG(H+L ) F(ab’)2
    suggested: None
    anti-human IgG(H+L
    suggested: None
    Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA).
    Coronavirus Spike protein ( subunit 1 ) polyclonal antibody
    suggested: None
    Coronavirus Spike protein ( subunit 1
    suggested: None
    mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
    mouse monoclonal antibody IgG1
    suggested: None
    antibody IgG1
    suggested: None
    Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry.
    anti-mouse ( IgG1
    suggested: None
    Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry.
    anti-rabbit
    suggested: None
    The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ.
    human IgG1
    suggested: None
    CD28
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    4-1BB
    suggested: None
    CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To maintain the stable expression of hACE2, 293T-hACE2 cells were cultured in DMEM (Corning) supplemented with 10% (v/v)
    293T-hACE2
    suggested: None
    Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2.
    293T-hACE2-FFLuc-GFP-RBD
    suggested: None
    Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described.
    CR3022-CAR
    suggested: None
    In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml.
    NK-92MI
    suggested: None
    293T cells were transfected with SFG-CR3022CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells.
    293T
    suggested: None
    ( d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown.
    293ThACE2-FFLuc-GFP-RBD
    suggested: None
    Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody.
    CR3022-CAR-NK92MI
    suggested: None
    Software and Algorithms
    SentencesResources
    CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

    We have optimized the NK cell expansion technology to buffer this potential limitation. Thus, in this study, we focused on CR3022-CAR-NK-92MI, a NK-92 cell line expressing IL-2 molecule to sustain the persistence in vivo20. In this study, we provide proof-of-concept for using CR3022-CAR-based cell therapy for treating severe COVID-19 patients. These experiments will expedite preclinical studies and a potential clinical application during the COVID-19 pandemic. Although these findings support the therapeutic potential of CR3022-CAR-NK cells for treating severe COVID-19 patients, there are several limitations presented in the current form of study. First, we use the NK-92 cell line in this study. NK-92-mediated immunotherapy is currently undergoing phase I/II clinical trials21,22. However, NK-92 cells must be irradiated prior to infusion to prevent permanent engraftment because of malignant potential of NK-92 cells. Second, we use pseudotyped SARS-CoV-2-S viral particles, which is different from the natural SARS-CoV-2 virus. Future studies using natural SARS-CoV-2 virus in the ACE2-transgenic mouse model are needed to test the efficacy and toxicity of CR3022-CAR-NK cells. In conclusion, development of this novel CAR-NK cell therapy for the treatment of severe COVID-19 patients with maximal efficacy and minimal toxicity will be required to reduce patient risk and enhance the benefit of these expensive and time-intensive therapies. The studies here characterize the biology of CR3022-CAR-NK-92MI cells, test the efficacy of CR3022-CAR-NK-92MI using in vitro assays, and finally, define the efficacy of eliminating SARS-CoV-2 infected target cells by CR3022-CAR-NK-92MI cells. This work pioneers the use of CR3022-CAR-NK cells to treat SARS-CoV-2 infected patients and will lead to the development of novel immunotherapeutic strategies for patients presenting with severe COVID-19, and combined with other broadly neutralizing antibodies, will support the development of a universal, “off-the-shelf ” CAR-NK based-immunotherapy for COVID-19. Online content Any methods, additional references, source data, and statements of code and data availability are available online. METHODS AND MATERIALS Antibodies and Reagents PE anti-human CD3 antibody (clone OKT3), FITC and PE/Cy7 anti-human CD56 antibody (clone HCD56, BioLegend), PE anti-human CD69 antibody (clone FN50, BioLegend), PE anti-human CD8a antibody (clone RPA-T8, BioLegend), APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend), APC/Fire 750 antihuman KLRG1 (MAFA) antibody (clone SA231A2, BioLegend), BV421 anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend), PE/Cy7 anti-human CD244 (2B4) antibody (clone C1.7, BioLegend), PE anti-human CD152 (CTLA-4) antibody (clone BNI3), APC anti-human CD366 (Tim-3) antibody (clone F38-2E2), PerCP/Cy5.5 antihuman TIGIT (VSTM3) antibody (clone A15153G), FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend), BV510 anti-human CD314 (NKG2D) antibody (clone 1D11), and APC anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA). APC anti-human CD16 antibody (clone 3G8, BD Biosciences), BV711 anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) were purchased from BD Biosciences (San Jose, CA, USA). PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems. AF647 Goat anti-human IgG(H+L) F(ab’)2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Anti-SARS-CoV-2 Coronavirus Spike protein (subunit 1) polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA). Anti-SARS-CoV-2 Spike RBD rabbit polyclonal antibody was purchased from SinoBiological (Beijing, China). Anti-His mouse monoclonal antibody IgG1 (clone H-3) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG1 (g1) were purchased from Fisher Scientific (Waltham, MA). Cell lines 293T cell line was purchased from the American Type Culture Collection (ATCC). 293T-hACE2 cell line is a gift from Dr. Abraham Pinter (Rutgers-New Jersey Medical School, PHRI). To maintain the stable expression of hACE2, 293T-hACE2 cells were cultured in DMEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL Penicillin-Streptomycin (Corning), and 1µg/mL of puromycin at 37℃ under 5% (v/v) CO2. To establish transient 293T-hACE2-RBD, 293T-hACE2 cells were transfected with 0.5 µg of SARS-CoV-2-RBD plasmid (a gift from Dr. Abraham Pinter) each well in a 24-well plate (Eppendorf) for 48 hours at 37℃ under 5% (v/v) CO2. Similarly, 293T-hACE2-FFLuc-GFP-RBD cells were transfected with 0.25 µg of SARSCoV-2-RBD plasmid and 0.25 µg of pHAGE-FFLuc-GFP each well in a 24-well plate (Eppendorf) for 48 hours at 37°C under 5% (v/v) CO2. Cells were harvested and immediately used for CD107a degranulation, 51Cr release, and FFLuc reporter assays. CR3022-CAR construction and retrovirus production A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the CR3022-specific scFv and sub-cloned into the SFG retroviral vector retroviral backbone in-frame with the hinge component of human IgG1, CD28 trans-membrane domain, The method was previously described2, briefly, to produce CR3022-CAR retrovirus, 293T cells were transfected with CR3022-CAR in SFG backbone, RDF, and PegPam3. CR3022-CAR retrovirus was harvested after 48-72 hours and transduced to NK92MI cells in a 24-well plate coated with 0.5 µg/ml of RetroNectin diluted in PBS (Clontech). Two days later, cells were transferred to 75 cm2 flask (Corning) and maintained in 35 ml complete NK92MI medium (MEM-a with 12.5% (v/v) FBS, 12.5% (v/v) heat inactivated horse serum, 11 µM bME, 2 µM folic acid, and 20 µM inositol) supplemented 200 U/mL IL-2 (PeproTech). To determine the expression of CAR, cells were stained for CD56 and anti-human IgG(H+L) F(ab’)2 fragment and analyzed by flow cytometry. CR3022-CAR and RBD binding assay To evaluate the binding activity of CR3022-CAR to RBD domain of SARS-CoV-2-S, CR3022-CAR and NK92MI (5 × 105) cells were incubated with 5 µg of His-gp70-RBD recombinant protein is a gift from Dr. Abraham Pinter in DPBS buffer (0.5 mM MgCl2 and 0.9 mM CaCl2 in PBS) in for 30 minutes on ice. Cells were washed twice with PBS, stained with anti-His in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed twice with PBS. Cells were then stained with anti-mouse (IgG1) secondary antibody in FACS buffer for 30 minutes on ice, washed twice with PBS, and analyzed by Flow Cytometry. CR3022-CAR and pseudotyped SARS-CoV-2-S viral particles binding assay CR3022-CAR, NK92MI, and 293T-hACE2 (5 × 105) cells were first equilibrated with BM (complete RPMI-1640 containing 0.2% BSA and 10 mM HEPES pH 7.4). Due to the non-specific binding to our CR3022-CAR of our secondary antibody, cells were first blocked with anti-human IgG(H+L) F(ab’)2 fragment for 30 minutes on ice in BM and washed thrice with PBS. Pseudotyped SARS-CoV-2-S (Genscript), full-length recombinant S protein (Acrobio systems), and S1 subunit recombinant protein (a gift from Dr. Abraham Pinter) were diluted with BM to appropriate concentrations. 4 × 106 IFU of pseudotyped SARS-CoV-2-S, or 2 µg of full-length recombinant S protein, or 2 µg of S1 subunit recombinant protein was added to designated wells of a 96-well V bottom plate. Plate was spun at 600 × g for 30 minutes at 32°C, then was incubated at 37°C under 5% (v/v) CO2 for 1 hour. Cells were washed twice with PBS, stained with anti-S1 in FACS buffer (0.2% FBS in PBS) for 30 minutes on ice and washed thrice with PBS. Cells were then stained with goat anti-rabbit secondary antibody in FACS buffer for 30 minutes on ice, washed thrice with PBS, and analyzed by Flow Cytometry. Flow Cytometry Analysis NK92MI and CR3022-CAR cells were stained were stained and washed as previously described. Cells were analyzed on a FACS LSRII or an LSR Fortessa flow cytometer (BD). PMT voltages were adjusted and compensation values were calculated before data collection. Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (BD). CD107a Degranulation assay The CD107a degranulation assay was described previously3. Briefly, expanded NK cells (5 × 104) were incubated with 1 × 105 293T or cells in V-bottomed 96-well plates in complete RPMI-1640 media at 37℃ for 2 hours. The cells were harvested, washed, and stained for CD3, CD56, and CD107a with GolgiStop (BD Biosciences) for 30 minutes, and analyzed by flow cytometry. FFLuc reporter assay To quantify the cytotoxicity of CAR-modified immune cells, we also developed the FFLuc reporter system assay. Briefly, an optical 96-well plate (Greiner BioOne™ No: 655098) was precoated with Retronectin (0.5 µg/ml in PBS) and placed at 4°C overnight. Then, the following day, the wells were aspirated and 293T-hACE2FFLuc-GFP-RBD and 293T-hACE2 cells were pre-seeded at 1 × 104 target cells/well in 100 µL/well of DMEM supplemented with 10% FBS. The plate was centrifuged for 5 minutes at 350 ´ g. In a separate 96-well plate, CR3022-CAR-NK92MI and NK-92MI cells were resuspended at a concentration of 1 × 106 cells/ml. Serial dilutions of effector cells were then prepared according to the effector/target ratio using NK-92MI medium. Then, the effector cells were added to each well of the optical plate (100 µL/well) and incubated at 37°C under 5% (v/v) CO2 for 4 hours and then the supernatant was gently discarded. 100 µL of working concentration D-Luciferin was added to each well with the lights turned off. A microplate reader (BioTek, USA) was used to quantify the data. The data was quantified by converting the obtained values to percentage of specific lysis by the following equation: Specific Lysis Percentage (%) = [1-(S-E)/(T-M)]×100, where S is the value of luminescence of the sample well, E is the value of luminescence of the “effector cell only” well compared to the sample well, T is the mean value of luminescence of “Target cell only” wells, and M is the mean value of luminescence of “blank medium only” wells. 51 Cr release assay To evaluate the cytotoxic activity of CAR-NK cell, the standard 4-hour 51Cr release assay was used. Briefly, target cells were labeled with 51Cr at 37°C for 2 hours and then resuspended at 1×105/mL in NK-92MI culture medium with 10% FBS without IL-2. Then, 1×104 target cells were incubated with serially diluted CAR-NK or NK-92MI cells at 37°C for 4 hours. After centrifugation, the supernatants were collected and the released 51Cr was measured with a gamma counter (Wallac, Turku, Finland). The cytotoxicity (as a percentage) was calculated as follows: [(sample − spontaneous release) / (maximum release − spontaneous release)] × 100. Statistical Analysis Data were represented as means ± SEM. The statistical significance was determined using a two-tailed unpaired Student t test, a two-tailed paired Student t test, a two-way ANOVA, where indicated. P < 0.05 was considered statistically significant. Figure and Figure legends: Figure 1: Generation of CR3022-CAR-NK92MI cells. (a) Schematic design of CR3022-CAR in SFG retroviral vector. The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ. (b) Generation of CR3022-CAR-NK cells. 293T cells were transfected with SFG-CR3022CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells. (c) Determination of CAR expression by flow cytometry. CR3022-CAR cells were harvested after 4-5 days then stained with anti-CD56 and CAR F(ab)2 domain [IgG Figure 2: CR3022-CAR-NK92MI cells bind to RBD domain of SARS-CoV-2-S protein. (a) Immunophenotyping of CR3022-CAR. Antibodies against various immunomodulatory receptors including TIGIT, LAG-3, TIM-3, KLRG1, CTLA-4, PD-1, CD69, CD8A, NKG2C, CD94, DNAM-1, 2B4, NKG2D, NKP46, and CD16 were used to stain CR3022-CAR and NK-92MI. The expression of these receptors was determined by flow cytometry. (b) Diagram of CR3022-CAR binding to the RBD domain of SARSCoV-2-S recombinant protein. CR3022-CAR binds to RBD of SARS-CoV-2-S protein which is then recognized by anti-His and its corresponding secondary antibody conjugated to a fluorophore. (c) Representative dot plots of CR3022-CAR binding to RBD of SARS-CoV-2. CR3022-CAR or NK-92MI cells were incubated with SARS- CoV-2-RBD or SARS-CoV-1-RBD recombinant protein. The binding efficiency was determined by flow cytometry. Figure 3: CR3022-CAR-NK-92MI cells bind to pseudotyped SARS-CoV-2-S viral particles. (a) Diagram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S viral particles. CR3022-CAR binds to pseudotyped SARS-CoV-2-S viral particle which is then recognized by anti-spike and its corresponding secondary antibody conjugated to a fluorophore. (b) Representative histogram of CR3022-CAR binding to pseudotyped SARS-CoV-2-S. CR3022-CAR or NK-92MI or 293T-hACE2 cells were incubated with pseudotyped SARS-CoV-2 or full-length spike or S1 subunit recombinant protein. The binding efficiency was determined by flow cytometry. Experimental sample was performed in triplicate with MFI 6759 ± 440 (a.u.). (c) Graph showing the binding efficiency of CR3022-CAR to pseudotyped SARS-CoV-2-S. The values were converted from Figure 2b. Experimental sample was performed in triplicate with binding efficiency 51.4 ± 3.34 (%). Figure 4: Increased CD107a degranulation and killing activity of CR3022-CAR-NK92MI cells against 293T-hACE2 cells transfected with RBD-SARS-Cov-2 Spike. (a) 293T-hACE2 cells were transfected with a plasmid containing firefly luciferase and GFP as well as SARS-CoV-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using anti-RBD antibody. Cells were then harvested and used as target cells for subsequent CD107a degranulation assay and luciferase killing assays. (c) Representative dot plots of CD107a assay and quantitative data of the percentage and mean fluorescence intensity of CD107a positive CR3022-CAR-NK92MI cells are shown. (d) Quantitative data of the luciferase killing assay using CR3022-CAR-NK92MI and wild-type NK-92MI cells against 293ThACE2-FFLuc-GFP-RBD cells is shown. Experimental groups were performed in represent the mean ± SEM from at least two independent experiments. Briefly, 5 x 104 CR3022-CAR-NK92MI cells were cocultured with either 1 x 105 RBD transfected-293ThACE2 cells, 293T-hACE2 cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Then, cells were harvested, stained for CAR F(ab)2 domain [IgG (H+L)], and CD107a. Representative flow cytometry dot plots, CD107a percent of total CAR cells, and CD107a MFI are shown. Figure 5: Increased killing activity of CR3022-CAR-NK-92MI cells against 293ThACE2 cells transfected with SARS-Cov-2 Spike protein Receptor Binding Domain using the 51Cr release platform. (a) 293T-hACE2 cells were transfected with SARSCov-2 Spike protein receptor binding domain for 48 hours. (b) Successful transfection was confirmed by flow cytometry using Anti-RBD antibody. Cells were then harvested and used as target cells for the subsequent 51Cr release assay. (c) Quantitative data of the 51Cr release assay using CR3022-CAR-NK-92MI cells and wild-type NK-92MI cells. Experimental groups were performed in triplicate. * p < 0.05, ** p < 0.01, *** p = 0.001, **** p<0.0001 ns p > 0.05. Data represent the mean ± SEM from at least two independent experiments.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

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  10. Version published to 10.1101/2020.08.11.247320v1 on bioRxiv