Specific Activation of T Cells by an ACE2-Based CAR-Like Receptor upon Recognition of SARS-CoV-2 Spike Protein

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the Coronavirus Disease 2019 (COVID-19) pandemic, which is still a health issue worldwide mostly due to a high rate of contagiousness conferred by the high-affinity binding between cell viral receptors, Angiotensin-Converting Enzyme 2 (ACE2) and SARS-CoV-2 Spike protein. Therapies have been developed that rely on the use of antibodies or the induction of their production (vaccination), but despite vaccination being still largely protective, the efficacy of antibody-based therapies wanes with the advent of new viral variants. Chimeric Antigen Receptor (CAR) therapy has shown promise for tumors and has also been proposed for COVID-19 treatment, but as recognition of CARs still relies on antibody-derived sequences, they will still be hampered by the high evasion capacity of the virus. In this manuscript, we show the results from CAR-like constructs with a recognition domain based on the ACE2 viral receptor, whose ability to bind the virus will not wane, as Spike/ACE2 interaction is pivotal for viral entry. Moreover, we have developed a CAR construct based on an affinity-optimized ACE2 and showed that both wild-type and affinity-optimized ACE2 CARs drive activation of a T cell line in response to SARS-CoV-2 Spike protein expressed on a pulmonary cell line. Our work sets the stage for the development of CAR-like constructs against infectious agents that would not be affected by viral escape mutations and could be developed as soon as the receptor is identified.

Keywords: CAR-like receptor; COVID-19; Chimeric Antigen Receptors (CAR); SARS-CoV-2; immunotherapy.

Figure 1

ACE2-CAR design and expression. Generic diagram of the expression vector SFFV/ACE2-CAR Amp(R) (A), which was the same for both WT- (B) and AO- (C) ACE2-CAR molecules, only differing at the ACE2 extracellular domain. Expression of both CAR-like constructs onto Jurkat-TPR cells was detected by staining with anti-IgG, recognizing the hinge region that was unchanged between WT- (D) and AO- (E) ACE2-CAR.

Figure 2

Establishment of ACE2-CAR-expressing Jurkat-TRP cell lines. (A) Schematic representation of the sorting procedure: cells were sorted according to anti-IgG staining, recognizing the IgG-derived hinge domain of the ACE2-CAR molecules. The terms “Low” and “High” of the sorted WT (B) and AO (C) ACE2-CAR-TPR populations refer to lower and higher densities, respectively, of the protein expressed in the cell membrane.

Figure 3

Activation of ACE2-CAR-TPR cells upon stimulation with Spike-coated MACSiBead particles. (A) Schematic illustration of the stimulation of ACE2-CAR-TPR cells with Spike and (B) Histogram overlays showing eGFP (NFAT), CFP (NFκB) or CD69 upregulation 24, 48 and 72 h after stimulation with Spike trimers. A representative experiment out of three performed in triplicate is shown.

Figure 4

Kinetics of the activation of ACE2-CAR-TPR cells upon stimulation with Spike- or anti-CD3+anti-CD28 MACSiBead-coated particles. MFI values for CD69 upregulation (A) as well as NFκB (B) or NFAT promoter activation (C) upon either Spike trimers or CD3+CD28 stimulation. Percentage of the cell that responded to at least one of the analyzed activation markers: NFAT, NFκB and/or CD69 (D). Each symbol represents mean ± SEM of MFI values of three independent experiments performed in triplicate. Statistical analysis is indicated for each condition vs. non-stimulated cells (*** p < 0.001), Spike-stimulated WT-CAR^High vs. WT-CAR^Low (^# p < 0.05, ^### p < 0.001) and Spike-stimulated AO-CAR^High vs. AO-CAR^Low (^xxx p < 0.001).

Figure 5

Expression of SARS-CoV-2 spike protein on A549 cells. (A) Schematics of SARS-CoV-2 Spike-expressing A549 cells and (B) detection, by flow cytometry, of Spike expression of transduced A549 cells.

Figure 6

Jurkat-A549 co-culture assay. Schematic representation of the procedure (A) and illustration of FACS gating strategy (B), which allowed Jurkat (below) to be distinguished from A549 cells (above) based on FSC and SSC as well as on CD69 staining expressed in Jurkat over A549 even in the absence of stimulation. (C–E) Histogram overlays showing eGFP (NFAT), CFP (NFκB) or CD69 upregulation 24, 48 and 72 h after co-culturing with A549-Spike target cells at 1:1 effector:target ratios, at different time points. A representative experiment out of three performed in triplicate is shown.

Figure 7

Kinetics of the activation of ACE2-CAR-TPR cells upon culture with Spike-expressing A549 cells. Kinetics of CD69 upregulation (B) as well as NFκB (C) or NFAT promoter activation (A) upon culture with Spike-expressing A549 cells. In the two first rows, MFI values of the corresponding activation markers for WT-CAR-TPR cells (top) and AO-CAR-TPR cells (middle) at different ratios were shown while in the last row, values from the 1:1 effector:target ratios for all cell types are compared. Each symbol represents mean ± SEM of three independent experiments performed in triplicate. Statistical analysis is indicated for each condition vs. non-stimulated cells (* p < 0.05, ** p < 0.01, *** p < 0.001), ratio 2:1 vs. 1:1 for each condition (^# p < 0.05, ^## p < 0.01, ^### p < 0.001); ratio 2:1 vs. 10:1 for each condition (⁺ p < 0.05, ⁺⁺⁺ p < 0.001); WT-CAR vs. AO-CAR for each ratio (^x p < 0.05, ^xx p < 0.01, ^xxx p < 0.001).