Team:SMMU-China/Education

Introduction:

In this ‘proof of concept’, we aim to demonstrate that our project is conducted systematically and serves the designed purposes. The essence of our project lies in the application of synthetic cells——CAR-Macrophage in virus infection treatment. At the same time, the extremely low inflammatory cytokine release reduces the risk of cytokine storm, mediating the ‘silent’ virus clearance. These two features embodies the exclusive value of our CAR-Macrophage treatment.

Basic idea:

Macrophages are innate immune cells, and the innate immune system is the first line of defense against viral infection. Based on the former researches of using macrophages to fight against viruses, we assumed that CAR-Macrophages can be used in the fight against SARS-CoV-2. However, it is found that high inflammatory macrophage reaction has great damage on the host, especially in severe infections(including SARS-CoV-2) and cytokine release syndrome (CRS). Therefore we also focused on the safety problems of using of CAR-Macrophages to eliminate the virus. Considering that critical patients are often accompanied by a cytokine storm caused by the release of large amounts of proinflammatory cytokines, our project focused on targeted therapy in patients with severe COVID-19 and attempted to construct a "silent CAR" that can effectively remove virus particles without inflammatory response.

Construction of CAR-Macrophages:

The common CAR structure consists of 3 domains: the extracellular domain, the transmembrane domain and the intracellular domain. The extracellular domain targets at the distinctive S protein of SARS-CoV-2, the transmembrane domain permits the signal transduction and the intracellular domain activates the phagocytosis process. The extracellular domain is the single chain variable fragment of the viral structural spike (S) protein antibody.The sequence encoding the scFv generated from CR3022 was chemically synthesized. The human CD8α molecule constitutes the transmembrane region[1]. Four CAR tags constitute the intracellular domain: common gamma subunit of the Fc receptor (CARγ), MEGF10 (CARMEGF10), MERTK (CARMERTK) and CD3ζ(CARζ). Those fragments were subcloned into the pELNS vector. High-titer replication-defective lentiviruses were produced and concentrated. Lentiviral infection was used to stably express CAR constructs in THP-1 cells[2]. The cDNA sequences containing the various fusion constructs were cloned into a third-generation lentiviral vector in which the CMV promoter was replaced with the EF-1α promoter[3]. An extracellular MYC epitope was also cloned into the receptors to permit detection by flow cytometry.

Testing virus eliminating efficiency:

Firstly, we conducted phagocytosis assay to test the phagocytic capacity and conducted cell killing assay to examine the results. Then, we mimicked the SARS-CoV-2 internalization by utilizing SARS-CoV-2 S pseudotyped virions.

1.UTD or CAR-expressing THP-1 cells were cocultured with GFP+ 293T cells or GFP+ 293T-S (S+ 29 ) target cells for 4 h at 37 °C. The effector-to-target (E:T) ratio was 1:1, and 1 × 105 cells were used as both effector cells and target cells. After coculturing, the cells were harvested and stained with an anti-CD11b APC-Cy7-conjugated antibody (M1/70, BioLegend) and analyzed by FACS using a FACSCalibur flow cytometer (BD Biosciences). The percentage of phagocytosis was calculated based on the percent of GFP+ events within the CD11b+ population. Data are represented as the mean ± standard error of quadruplicate wells.The phagocytic potential of human macrophage THP-1 cell lines expressing different CAR receptors or a truncated CAR receptor (CARΔ) lacking the intracellular domain was measured with a cell-based assay. CAR-Macrophages, control untransduced (UTD) macrophages, CARMERTK and CARΔ did not show notable phagocytosis of 293T cells while CARMEGF10, CARγ and CARζ cells engulfed Spike-bearing 293T cells in an S-specific manner.

293T and 293T-S cells were used as targets in luciferase-based killing assays including control (UTD) or CAR macrophages. The effector-to-target (E:T) ratio was 10:1 for all the groups. Bioluminescence was measured using a Bio-Tek Synergy H1 microplate reader. The percentage of specific lysis was calculated on the basis of the experimental luciferase signal (total flux) relative to the signal of the target alone using the following formula: %Specific Lysis = [(Sample signal－Target alone signal)] / [Background signal－Target alone signal)] × 100. CAR-mediated macrophage phagocytosis was further confirmed by a luciferase-based killing assay, and our data showed that CARMEGF10, CARγ and CARζ cells eradicated S protein-expressing 293T cells in an antigen-specific manner. Interestingly, CARMERTK and UTD macrophages showed no difference in killing capacity. Our data further showed that all synthetic receptors had the ability to bind the S protein, therefore, the differences in phagocytosis and the lytic effect were not due to the affinity for the S protein.

SARS-CoV-2 S pseudotyped virions were pelleted (90 min at 14,000 rpm and 4 °C), and after removal of the supernatant, the pellets were resuspended in RPMI medium and incubated with phagocytes (THP-1 cells or CAR macrophages) at 37 °C for 1.5 h. After allowing time for phagocytosis, the cells were washed three times with PBS and incubated with Accutase (Innovative Cell Technologies) for 10 min at 37 °C, followed by a final wash in Accutase. Intracellular staining for the S protein was performed for 60 min on ice after using a fixation/permeabilization kit(eBioscience) and then analyzed using a FACSCalibur flow cytometer (BD Biosciences). The phagocytic score was determined by gating the samples on events representing cells and was calculated as follows: Percent S protein positive × median fluorescence intensity (MFI). CARMEGF10, CARγ, CARζ and CARMERTK macrophages showed similar virion internalization.

The virus eliminating efficiency of different types of CAR-Macrophages were ultimately confirmed by 3 phases experiments.

Antibody-mediated phagocytosis and internalization of virions are important mechanisms of antiviral activity performed by macrophages against pathogens, however, using the phagocytosis assay developed for SARS-CoV-2, we observed low levels of phagocytic activity when UTD cells directly contacted virions. Phagocytic activity was not significantly increased when CARΔ cells rather than UTD macrophages were the phagocytes in the assay, suggesting that the extracellular domain of the CAR alone is not sufficient to induce strong virion internalization. CARγ, CARMEGF10, and CARζ mediated similar significantly stronger levels of SARS-CoV-2 phagocytosis by THP-1 cells than CARΔ. Unexpectedly, we also observed relatively strong internalization of virions in CARMERTK cells, which did not show specific phagocytic or lytic effects on S protein-expressing 293T cells. Since all the CARs exhibited the ability to induce phagocytosis of SARS-CoV-2 virions while there was no evidence of infection, these experiments strongly suggest the clearance of SARS-CoV-2 virions by CAR macrophages.

Detecting inflammatory factors:

Cytokine analysis was performed on supernatants derived from cultures given the indicated treatments using a human cytokine 10-plex panel (Thermo Scientific) per the manufacturer’s instructions, with the panel results read on a Luminex Analyzer.Culture supernatants from THP-1 cells with different CARs treated with virions were further analyzed in a multiplex cytokine assay. Following SARS-CoV-2 treatment of THP-1 cells, we observed slightly increased secretion of the cytokines IL-6, IL-8 and TNF-α, but no discernable patterns could be confidently drawn for GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, and IFN-γ. CARΔ cells showed a cytokine profile similar to that of UTD macrophages. Notably, we observed not only strongly increased induction of IL-6, IL-8 and TNF-α but also induction of IFN-γ and IL-10 in SARS-CoV-2-treated CARγ and CARζ cells. However, for CARMERTK cells, we did not observe significant changes in cytokines.

Evaluating cell protective property:

We further used a transwell-based coculture model to evaluate the protective role of CAR macrophages in SARS-CoV-2 infection. All the CAR-expressing macrophages potently inhibited Vero E6 cell infection with the SARS-CoV-2-S pseudotyped virus. Interestingly, CARΔ cells showed no protective effect in the infection assay, although they had a similar capacity to bind to the S protein, suggesting that the intracellular signaling domain is necessary for virion clearance by CAR macrophages.

Avoiding potential risks:

Although there is currently no evidence that SARS-CoV-2 can infect THP-1 cells with or without IgG[4], THP-1 cells have been shown to support antibody-mediated enhancement of SARS-CoV infection in previous studies[5]. We therefore sought to determine whether synthetic receptors facilitate the entry of SARS-COV-2 into macrophages as host cells under the premise that the extracellular domain of the CAR constructs has the capacity to directly bind to the S protein. Replication-defective VSV particles bearing coronavirus S proteins faithfully reflect key features of host cell entry by coronaviruses, including SARS-CoV-2[6,7]. We therefore employed VSV pseudotypes bearing SARS-2-S to study the cell entry of SARS-CoV-2. Our data showed that Vero E6 cells were susceptible to entry driven by SARS-S. However, no evidence of infection was detected in THP-1 cells with or without synthetic receptors. The safety of our cell system withstands those strict tests and reviews.

Conclusion:

We demonstrate that our concept: a ‘silent CAR’ (CAR-Macrophage) can successfully eliminate SARS-CoV-2 while causing barely cytokine release. After constructing 4 types of , we conducted experiments concerning their phagocytic capacity, cell lytic effect and virion internalization property to demonstrate that all types of CAR-Macrophages show significant virus eliminating efficiency. Subsequently, we conducted cytokine analysis assay and observed strongly increased induction of cytokines in all CAR-Macrophages except for CARMERTK cells. Thus demonstrating that our concept to construct a "silent CAR" that can effectively remove virus particles without inflammatory response is realized. Furthermore, we demonstrated the protective role of CAR macrophages in SARS-CoV-2 infection by using coculture model, serving as an extended application on cell level. Finally, we ruled out the possibility that macrophages being infected by SARS-CoV-2, demonstrating the safety and reliability of our COVID-19 targeted therapy.

References:

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