CAR-NK Cells Effectively Target SARS-CoV-2-Spike-Expressing Cell Lines In Vitro

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1. Version published to 10.3389/fimmu.2021.652223

   Jul 23, 2021
2. 

   ScreenIT

   Jan 16, 2021

   SciScore for 10.1101/2021.01.14.426742: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Primary NK cell expansion from peripheral blood: Human blood related work was approved by the Rutgers University Institutional Review Board (IRB)
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   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, …

   SciScore for 10.1101/2021.01.14.426742: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Primary NK cell expansion from peripheral blood: Human blood related work was approved by the Rutgers University Institutional Review Board (IRB)
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   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
   750 anti-human KLRG1 (MAFA) antibody (clone SA231A2, BioLegend)	anti-human 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
   anti-human TIGIT (VSTM3) antibody (clone A15153G)	anti-human TIGIT ( VSTM3 suggested: None
   FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend)	anti-human CD223 suggested: None LAG-3 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 SinoBiological (Beijing, China).	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
   Transfected cells were harvested after 48 hours and stained with primary anti-RBD (SinoBiological) followed by a goat anti-rabbit fluorophore-conjugated secondary antibody to determine the expression of RBD by flow cytometry.	anti-rabbit fluorophore-conjugated secondary suggested: None
   The spike protein expression was determined by flow cytometry by staining the transduced cells with anti-RBD antibody (SinoBiological) followed by a goat anti-rabbit fluorophore-conjugated secondary antibody.	anti-RBD suggested: None anti-rabbit fluorophore-conjugated secondary antibody . suggested: None
   Cells were then stained with goat 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
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: 293T, A549, HepG2, and NK-92MI cell lines were purchased from the American Type Culture Collection (ATCC).	HepG2 suggested: None NK-92MI suggested: KCB Cat# KCB 200677YJ, RRID:CVCL_3755)
   Generation of stable A549-Spike and A549-Spike D614G cell lines: pcDNA3.1-SARS-CoV-2 Spike (Addgene plasmid #145302) was used to clone SARS-CoV-2 S gene into the SFG backbone using the In-Fusion Cloning kit (Takara Bio).	D614G suggested: None
   The spike retrovirus supernatant was filtered (0.45 μm) and transduced into A549 cells for an additional 48 – 72 hours at 37°C under 5% (v/v) CO2.	A549 suggested: None
   A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.	A549-Spike suggested: None
   The presence of the SARS-CoV-2 pseudovirus was further confirmed by flow cytometry, transfected 293T-hACE2 cells were stained with primary anti-RBD followed by goat anti-rabbit fluorophore-conjugated secondary antibody.	293T-hACE2 suggested: None
   Transduction of expanded NK cells with S309-CAR: The transduction procedure was previously described, briefly, 293T cells were transfected with a combination of SFG-S309, PegPam3, and RDF.	293T suggested: None
   Briefly, NK-92MI or S309-CAR-NK-92MI or CR3022-CAR-NK-92MI cells (5 × 104) were cocultured with 1 × 105 293T-hACE2, 293T-hACE2-RBD, A549, or A549-Spike cells in the presence of GolgiStop (BD Biosciences) in a V-bottomed 96-well plate in complete RPMI-1640 media at 37°C under 5% CO2 for 2 hours.	293T-hACE2-RBD suggested: None
   Software and Algorithms
   Sentences	Resources
   S309-CAR construction and retrovirus production: A codon-optimized DNA fragment was synthesized by GENEWIZ encoding the S309-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 (36) and analyzed using FlowJo software (36)	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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   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).

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

   However, several limitations in the current study need to be addressed: Firstly, the pseudotyped SARS-CoV-2 S viral particles used in our experiments may not fully recapitulate the authentic SARS-CoV-2 virus. Secondly, our studies provide limited mechanistic insights showing how S309-CAR-NK cells target SARS-CoV-2 virus or SARS-CoV-2 S expressing cells. Currently, we are testing the efficacy of S309-CAR-NK cells on animal studies. Our current study supports the potential efficacy of S309-CAR-NK cells and the safety assessment of S309-CAR-NK cells in vivo. Future studies using wild-type SARS-CoV-2 virus in pre-clinical animal models are needed to test the efficacy and toxicity of S309-CAR-NK cells in vivo. Nevertheless, the 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 clinical benefit of these expensive and time-intensive therapies in the acute care setting. This work pioneers the use of S309-CAR-NK cells to prevent the SARS-CoV-2 infection and treat SARS-CoV-2 infected patients and will lead to the development of novel immunotherapeutic strategies for patients with immunocompromised conditions. These patients include: 1) older adults (particularly over 70-year-old with immune-compromising conditions); 2) those with comorbidities or chronic conditions such as cancer, heart disease, pre-existing lung disease, diabetes, malnutrition, a...

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

   Identifier	Status	Title
   NCT04368728	Active, not recruiting	Study to Describe the Safety, Tolerability, Immunogenicity, …
   NCT04283461	Active, not recruiting	Safety and Immunogenicity Study of 2019-nCoV Vaccine (mRNA-1…
   NCT04375176	Recruiting	Monocytes and NK Cells Activity in Covid-19 Patients
   NCT04280224	Recruiting	NK Cells Treatment for COVID-19
   NCT04365101	Recruiting	Natural Killer Cell (CYNK-001) Infusions in Adults With COVI…
   NCT04324996	Recruiting	A Phase I/II Study of Universal Off-the-shelf NKG2D-ACE2 CAR…

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

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   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.

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   Read the original source
3. 

   Version published to 10.1101/2021.01.14.426742v1 on bioRxiv

   Jan 15, 2021