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

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious and presents a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in treating COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its various mutants. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody (NAbs) that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein and is therefore more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G, N501Y, and E484K mutants. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently reported CR3022-CAR-NK cells. Thus, these results pave the way for generating 'off-the-shelf' S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Keywords: CAR (chimeric antigen receptor); COVID-19; E484K variant; N501Y variant; NK cells; SARS-CoV-2; off-the-shelf.

Figure 1

Generation of S309-CAR-NK-92MI cells. (A) Plasmid construct of S309-CAR. The SFG retroviral vector contains the S309 single chain antibody fragment (accession code 6WS6 on PDB), a human IgG1 CH2CH3 hinge region, a CD28 transmembrane region, followed by the co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ. (B) Determination of S309-CAR-NK expression by flow cytometry. S309-CAR cells were collected and stained with anti-CD56 and CAR F(ab)2 domain [IgG (H+L)] for flow cytometry. The cells were then sorted to achieve a homogenous population of high CAR expression. (C) Representative histogram showing the immunoprofiling of S309-CAR-NK-92MI cells by flow cytometry. S309-CAR-NK-92MI or the wildtype (WT) NK-92MI cells were stained with antibodies against different immunomodulatory receptors including CTLA-4, PD-1, NKG2A, NKG2C, NKG2D, NKp46, CD56, CD16, 2B4, DNAM-1, CD94, KLRG1, TIM-3, LAG-3, and TIGIT.

Figure 2

S309-CAR-NK-92MI cells bind to RBD domain of SARS-CoV-2 S protein and pseudotyped SARS-CoV-2 viral particles. (A) Representative dot plots showing the efficiency of S309-CAR binding to SARS-CoV-2-RBD. S309-CAR-NK-92MI or WT NK-92MI cells were incubated with the RBD recombinant protein of SARS-CoV-1 or SARS-CoV-2. (B) Generation of pseudotyped SARS-CoV-2 viral particles. 293T cells were transfected with various plasmids for 72 hours for the generation of pseudotyped SARS-CoV-2 viral particles. (C) Representative histogram showing S309-CAR-NK-92MI binds to the pseudotyped SARS-CoV-2 viral particles. S309-CAR-NK-92MI, parental NK-92MI, or 293T-hACE2 (positive control) cells were incubated with pseudotyped SARS-CoV-2 viral particles, S1 subunit, or full-length S recombinant protein at 37°C for 1 hour. The experimental sample was performed in triplicates with MFI = 13579 ± 251 (a.u.). (D) Quantitative data of the binding efficiency of S309-CAR-NK-92MI cells to pseudotyped SARS-CoV-2 viral particles. The experimental sample was performed in triplicates with binding efficiency of over 90%. Data represent means ± standard error of the mean (SEM) of three independent experiments represented by each dot. Non-parametric test was employed for panel (D). ns p > 0.05, and ****p < 0.0001.

Figure 3

Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (A) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. (B) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. (C) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab’)2 domain IgG (H+L) and CD107a. Each dot represents an experiment. Data represent means ± SEM from three independent experiments. (D) Representative 4-hour standard Cr⁵¹ release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK-92MI and NK-92MI. Experimental groups were performed in triplicates. Error bars represent means ± SD. The experiment was repeated at least two times. Non-parametric test was used for panels (C). ns p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4

Increased killing activity of expanded S309-CAR-NK^primary against A549 Spike cell line. (A) Schematic representation of human S309-CAR-NK^primary expansion system. Briefly, irradiated (100 Gy) 221-mIL21 feeder cells were cocultured with PBMC supplemented with IL-2 and IL-15 on Day 0. In parallel, 293T cells were transfected with the retrovirus packaging system to produce S309-CAR retrovirus that were then transduced into the expanded PBNK cells in the presence of IL-2 and IL-15. S309-CAR-NK^primary cells were harvested on Day 7 and continued expansion for 21 days. (B) Representative dot plots of expanded primary NK cells and S309-CAR-NK^primary. The purity of NK cells and the expression of CAR were monitored every 3-4 days. (C) Representative histogram of immunophenotyping of S309-CAR-NK^primary cells using flow cytometry. Antibodies against various immunomodulatory receptors including CTLA-4, PD1, NKG2A, CD56, CD16, 2B4, NKG2C, NKG2D, NKp30, NKp46, DNAM-1, CD69, CD94, TIGIT, KLRG1, KIR2DL1, TIM-3, and LAG-3 were used to stain both primary NK cells and S309-CAR-NK^primary. (D) Representative quantitative data of cytotoxic activity of S309-CAR-NK^primary against A549-Spike. Briefly, expanded human S309-CAR-NK^primary cells were blocked with anti-CD16 for 30 minutes and then anti-NKG2D for 30 minutes on ice. The target cells were labeled with Cr⁵¹ for 2 hours prior to coculturing with S309-CAR-NK^primary cells for an additional 4 hours. Representative data are shown in panel (D).

Figure 5

S309-CAR-NK cells are superior to CR3022-CAR-NK cells. (A) Diagram of S309 and CR3022 NAbs binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. (B) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments, each dot representing an experiment. (C) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr⁵¹ release assay. Effector cells were cocultured with Cr⁵¹-labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. Representative data from one experiment is shown. Error bars represent means ± SD. (D) Expanded S309-CAR-NK^primary has increased killing activity against A549-Spike cells compared to primary CR3022-CAR-NK^primary. Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. S309-CAR-NK^primary and CR3022-CAR-NK^primary cells were expanded from three different donors. Representative data from one heathy donor are shown. Error bars represent means ± SD. Non-parametric test was employed for panel (B). ns p > 0.05 and **p < 0.01.

Figure 6

S309-CAR-NK^primary cells can also target SARS-CoV-2 D614G variant. (A) Quantitative data of the binding efficiency of S309-CAR-NK^primary to pseudotyped SARS-CoV-2 and D614G variant viral particles. (B) Intracellular cytokine productions of various effector cells. Briefly, effector cells were cocultured with A549, A549-Spike, or A549-Spike D614G for 4 hours at 37°C. Cells were then stained for surface markers and permeabilized prior to staining for intracellular cytokines. Data were acquired by flow cytometry and the values were normalized with the PMA + ionomycin group (positive control). Data were pooled from at least two independent experiments. (C) Expanded S309-CAR-NK^primary has increased killing activity against A549-Spike and A549-Spike D614G cells compared to CR3022-CAR-NK^primary. Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike or A549-Spike D614G target cells for 4 hours at 37°C. Primary NK cells and CAR-NK^primary cells were expanded from at least four donors. Representative data from one donor is shown. Samples were performed in triplicates. Error bars represent means ± SD. Non-parametric test was employed for panels (A, B). ns p > 0.05 and *p < 0.05.