Targeting SARS-CoV-2 infection through CAR-T like bispecific T cell engagers incorporating ACE2

  1. Mikail Dogan
  2. Lina Kozhaya
  3. Lindsey Placek
  4. Fatih Karabacak
  5. Mesut Yigit
  6. Derya Unutmaz

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Abstract

Despite advances in antibody treatments and vaccines, COVID-19 caused by SARS-CoV-2 infection remains a major health problem resulting in excessive morbidity and mortality and the emergence of new variants has reduced the effectiveness of current vaccines. Here, as a proof-of-concept we engineered primary CD8 T cells to express SARS-CoV-2 Spike protein-specific CARs, using extracellular region of ACE2, and demonstrated their highly specific and potent cytotoxicity towards Spike-expressing target cells. To improve on this concept as a potential therapeutic, we developed a bispecific T cell engager combining ACE2 with an anti-CD3 scFv (ACE2-Bite) to target infected cells and the virus. Similar to CAR-T cell approach, ACE2-Bite endowed cytotoxic cells to selectively kill Spike-expressing targets. Furthermore, ACE2-Bite neutralized the pseudoviruses of SARS-CoV, SARS-CoV-2 wild-type and variants including Delta and Omicron, as a decoy protein. Remarkably, ACE2-Bite molecule showed a higher binding and neutralization affinity to Delta and Omicron variants compared to SARS-CoV-2 wild-type Spike proteins, suggesting the potential of this approach as a variant-proof, therapeutic strategy for future SARS-CoV-2 variants, employing both humoral and cellular arms of the adaptive immune response.

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  1. Version published to 10.1101/2022.01.19.476940v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2022.01.19.476940: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: To generate HEK-293T cells that transiently expressed wild type and mutated spike protein (ATCC; mycoplasma-free low passage stock), the cells were transfected with Spike protein expressing pLP plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol and stained for their spike protein expression 72 hours after the transfection as described in Staining and Flow cytometry Analysis.

    Table 2: Resources

    Antibodies
    SentencesResources
    ACE2 CAR construct: CAR constructs consisting of CD8 alpha signal peptide, extracellular domain of ACE2 molecule or single chain variable fragment (scFv) of anti-CD19 or anti-Spike protein antibodies, CD8 hinge domain, CD8 transmembrane domain, 4-1BB (CD137) intracellular domain and CD3ζ domain were designed with Snapgene and synthesized via Genscript.
    anti-CD19
    suggested: None
    anti-Spike protein
    suggested: None
    CD8 transmembrane domain, 4-1BB (CD137
    suggested: None
    Anti-CD3 antibody single-chain variable fragment, His-Tag, and Hemagglutinin (HA) Tag sequences were obtained from Addgene plasmid #85437.
    Anti-CD3
    suggested: None
    His-Tag
    suggested: None
    Hemagglutinin (HA
    suggested: None
    Activation of CAR CD8 T cells was determined with CD25 staining using CD25-APC antibody (Biolegend).
    CD25-APC
    suggested: None
    CAR expressions of ACE2 CAR and anti-Spike CAR and ACE2 expression of ACE2-293 cells were determined with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression.
    anti-Spike CAR
    suggested: None
    Mouse IgG2a
    suggested: None
    CAR expression of anti-CD19 CAR was determined with Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Acro) followed by a secondary staining with APC conjugated anti-human IgG Fc Antibody (Biolegend) and RFP expression.
    anti-CD19 CAR
    suggested: (Miltenyi Biotec Cat# 130-115-965, RRID:AB_2811310)
    Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity)
    suggested: None
    For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).
    anti-human IgG
    suggested: None
    Detection reagent was prepared using Human CD3 epsilon Protein, Mouse IgG2a Fc Tag (Acro) and Phycoerythrin-conjugated Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody (Fisher) for ACE2-Bite and APC anti-human IgG Fc Antibody clone HP6017 (Biolegend) for ACE2-Fc were added to the wells and incubated for another hour at room temperature.
    Human CD3 epsilon Protein, Mouse IgG2a Fc Tag (Acro)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    VSVG and Spike Protein pseudotyped lentivirus production: The lentiviruses pseudotyped with vesicular stomatitis virus G protein envelope were generated with HEK293T cells.
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    To measure viral titers of VSV-G pseudotyped lentiviruses, virus preparations were serially diluted on Jurkat cells and 3 days post-infection, infected cells were measured using flow cytometry and the number of cells transduced with 1 mL of virus supernatant was calculated as infectious units per milliliter.
    Jurkat
    suggested: KCB Cat# KCB 94018YJ, RRID:CVCL_0065)
    To generate HEK-293T cells that transiently expressed wild type and mutated spike protein (ATCC; mycoplasma-free low passage stock), the cells were transfected with Spike protein expressing pLP plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol and stained for their spike protein expression 72 hours after the transfection as described in Staining and Flow cytometry Analysis.
    HEK-293T
    suggested: None
    All engineered and wild-type HEK-293 and T2 cells were cultured in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FBS; Atlanta Biologicals, Lawrenceville, GA), 8% GlutaMAX (Life Technologies), 8% sodium pyruvate, 8% MEM vitamins, 8% MEM nonessential amino acid, and 1% penicillin/streptomycin (all from Corning Cellgro).
    HEK-293
    suggested: None
    T2
    suggested: None
    To generate T2s and 293s with stable Spike overexpression, wild-type T2 and 293 cells were transduced with 3 MOI of Spike protein overexpressing VSVG lentivirus and proliferated.
    293
    suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)
    CAR expressions of ACE2 CAR and anti-Spike CAR and ACE2 expression of ACE2-293 cells were determined with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression.
    ACE2-293
    suggested: None
    Recombinant DNA
    SentencesResources
    Spike protein constructs: Human codon optimized wild-type full-length SARS-CoV-2 Spike protein sequence was synthesized by MolecularCloud (MC_0101081) and then cloned into pLP/VSVG plasmid from Thermo Fisher under CMV promoter after removing the VSVG sequence via EcoRI-EcoRI restriction digestion. 5’-ACGACGGAATTCATGTTCGTCTTCCTGGTCCTG-3’ and 5’-ACGACGGAATTCTTAACAGCAGGAGCCACAGC-3’ primers were used to generate wild-type SARS-CoV-2 Spike protein sequence without the Endoplasmic Reticulum Retention Signal (ERRS, last 19 amino acids of Spike) (Ou et al., 2020).
    pLP/VSVG
    suggested: None
    E484K and N501Y mutated spike protein sequences without ERRS domain were obtained from VectorBuilder plasmids pRP[Exp]-CMV-human beta globin intron>S(E484K,deltaC19)/3xFLAG and pRP[Exp]-CMV-human beta globin intron>S(N501Y,deltaC19)/3xFLAG, respectively.
    pRP[Exp]-CMV-human
    suggested: None
    Since wild-type Spike protein did not have the FLAG tag and efficiently incorporated into the lentiviruses, FLAG Tags in each construct were removed to have the same amino acid sequences among all Spike constructs with the exception of the necessary mutations, via PCR amplification with 5’-ACGACGGAATTCATGTTCGTTTTCCTTGTTCTGTTGC-3’ and 5’-ACGACGGAATTCTTAGCAACATGATCCGCAAGAGCA-3’ primers and cloned into the same pLP expression plasmid.
    pLP
    suggested: RRID:Addgene_22310)
    Briefly, the lentivector plasmids containing the constructs were co-transfected with vesicular stomatitis virus G protein, pLP1, and pLP2 plasmids into HEK293T cells at 80–90% confluency using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.
    pLP1
    suggested: RRID:Addgene_22614)
    pLP2
    suggested: None
    Software and Algorithms
    SentencesResources
    ACE2 extracellular domain, CD8a signal peptide, CD8 hinge, CD8 transmembrane domain, 4-1BB intracellular domain and CD3ζ domain sequences were obtained from Ensembl Gene Browser and codon optimized with SnapGene by removing the restriction enzyme recognition sites that are necessary for subsequent molecular cloning steps, while preserving the amino acid sequences.
    Ensembl Gene Browser
    suggested: None
    Anti-CD19 and anti-Spike scFv amino acid sequences were obtained from Addgene plasmids #79125 and #155364, respectively, reverse translated to DNA sequences and codon optimized with Snapgene 5.2.4.
    Snapgene
    suggested: (SnapGene, RRID:SCR_015052)
    Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Geometric means of PE fluorescence in different titrations were used to generate the titration curve and the area under the curve was calculated using GraphPad Prism 9.0 software (GraphPad Software).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Statistical Analyses and Reproducibility: All statistical analyses were performed and graphs were prepared using GraphPad Prism V9 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

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

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 37, 38, 39, 41 and 42. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2022.01.19.476940v1 on bioRxiv
  4. Version published to 10.1002/cti2.1421