Team:SMMU-China/Basic Parts

Background and structure of CAR-MEGF10:

The function of CAR-MEGF10 is the same as the CARγ, which increases the phagocytosis of the macrophages. And the structure is also highly similar to the CARγ, with the only difference lying in the intracellular domain of the CAR, in which the common γ subunit of Fc receptors is replaced by MEGF10.

The mechanism of CAR-MEGF10:

After the extracellular αS-scFv domain binds to the S protein of SARS-CoV-2, the intracellular cytosolic Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) are activated with the help of the Src family kinases. And the SH2 binding domains for the protein expressing SH2 matching domains will be further exposed due to the space conformation change. The protein expressing SH2 matching domains, protein Syk, a phagocytic signaling effector can then active the downstream signaling transduction cascades and initiate the phagocytosis in our CARγ-macrophages eventually.

Background and structure of the CAR-MERTK:

Previous studies have shown that the MER tyrosine kinase (MERTK) expressed on the surface of plasmid membrane of the macrophages can not only mediate the phagocytosis of the apoptotic cells and the virus particles but at the same time, reducing the secretion of the inflammatory factors. We consequently structured a CAR-MERTK containing the function domain of the MER tyrosine kinase (MERTK) to reprogram the phagocytosis of our macrophages.

Our CAR-MERTK is consisted of three domains, an αS-scFv extracellular domain targeting to the spike(S) protein of the SARS-CoV-2, a CD8 hinge and a CD8 transmembrane domain presented in the αCD19 CAR for T cells permitting the signal transduction, and an intracellular domain with the same structure of the intracellular part of MER tyrosine kinase (MERTK) acquiring the magical function of the MER tyrosine kinase (MERTK). Also, there is a Myc tag gene making the lentiviral transcription efficiency detectable.

The function of CAR-MERTK:

The function of the CAR-MERTK depends on the structure of CAR-MERTK. So, our CAR-MERTK can not only capture the SARS-CoV-2 in a targeted manner but it can also engulf the SARS-CoV-2 with low inflammatory factors secretion.

Characterization of the CAR-MERTK:

Validation of the CAR-MERTK expression on the macrophages:

We choose the lentiviral technology to transfect our CAR-MERTK to human macrophages THP-1 cells. The cDNA containing the CAR-MERTK gene was inserted to a third-generation lentiviral vector in which the CMV promotor was replaced by the EF-1α promoter to improve the transcription efficiency. We next confirmed the expression of the CAR-MERTK on the macrophage by detecting the fluorescence intensity of the co-transfected Myc tag through cytometry technology. The results showed that the lentiviral vector transfected THP-1 cells with high efficiency. (Fig 1b)

CAR-MERK macrophages can engulf SARS-CoV-2 S pseudotyped virions:

After confirming the expression of CAR-MERTK on the macrophages, we next tested whether the CAR-MERTK macrophages would perform the phagocytosis function as we expected. We first cocultured the CAR-MERTK macrophages with the SARS-CoV-2 S pseudotyped virions. It showed that the phagocytosis efficiency of the CAR-MERTK macrophages is relevant to the amount of SARS-CoV-2 S pseudotyped virions, or the quantity of the S protein inside the CAR-MERTK macrophages linearly. So, by detecting the fluorescence intensity through the cytometry technology after the intracellular staining for the S protein was performed, we finally acquired the phagocytosis efficiency of the CAR-MERTK macrophages. (Fig 2b)

The inflammatory reaction can be blocked when the CAR-MERTK macrophages engulf the SARS-CoV-2 S pseudotyped virions:

CAR-MERTK macrophages do not kill the S protein expressing autologous cells, alleviating the inflammatory damage to the body:

From the previous study we knew that the S protein would be expressed on the surface of the alveolar epithelial cell after infected by the SARS-CoV-2. There would be extra damage if our CAR-MERTK macrophages still attack the S protein expressing alveolar epithelial cells, aggravating the lung injury. But as mentioned before, our CAR-macrophages will engulf the SARS-CoV-2 in a silent and protective way. The demonstrating procedure is conducted as follows. We first constructed a targeted cell expressing S protein called Spike-bearing 293 cells through the lentiviral technology. Then, we cocultured the CAR-MERTK macrophages with the Spike-bearing 293 cells. The existence of intracellular SARS-CoV-2 S pseudotyped virions, or the presence of the S protein inside the CAR-MERTK macrophages was the key evidence to determine whether there was a phagocytosis of S protein expressing autologous cells. So, only by detecting the fluorescence intensity of the intracellular staining S protein could we acquire the expected results: the CAR-MERTK macrophages would not damage the Spike-bearing 293 cells and thus proving the potential safety of our CAR-MERTK macrophages. (Fig 1c,d)

CAR-MERTK macrophages engulfed the SARS-CoV-2 S pseudotyped virions without increasing inflammatory release:

Systematically speaking, an exemption of the S protein expressing alveolar epithelial cells is far from enough. The systemic damage of the SARS-CoV-2 is not only limited to the alveolar epithelial cells, but also some distant organs by inflammatory factors. So, our next goal was to confirm that our CAR-MERTK macrophages can engulf the SARS-CoV-2 in an inflammatory factor free manner. As the results showed, the inflammatory factors, including the GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, and IFN-γ, were relatively lower in the CAR-MERTK macrophages group. (Fig 2c)

Structure and function of the CARγ:

Researchers have identified that the common γ subunit of Fc receptors (FcRγ) have the potential ability to enhance the phagocytosis efficiency of the macrophages. We consequently constructed a chimeric antigen receptor containing the critical function domain of the FcRγ, expecting the permitting function of the FcRγ. There are three parts in our CAR, an αS-scFv extracellular domain targeting the spike(S) protein of the SARS-CoV-2, a CD8 hinge and a CD8 transmembrane domain presented in the αCD19 CAR for T cells permitting the signal transduction, a intracellular domain with the same structure of the FcRγ for the enhancement of the phagocytosis efficiency.

The mechanism of the CARγ:

The function domain of the CARγ mainly lies in the intracellular domain, namely the cytosolic Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). The cytosolic Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) in the intracellular domain of the CARγ will be phosphorylated by Src family kinases when the CARγ is stimulated by its ligands S protein. The expression of the S protein on the surface of the SARS-CoV-2, is actually a strong stimulus of the CARγ-macrophages. The activated Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) will expose the SH2 binding domains for the SH2 matching domain. And the SH2 matching domain contains the kinase ZAP70 and the Syk in the T cells and the macrophages respectively. Protein Syk, a phagocytic signaling effector can then active the downstream signaling transduction cascades and initiate the phagocytosis in our CARγ-macrophages eventually.

The structure and function of CAR ζ:

CAR ζ is another CAR aimed to increase the phagocytosis of the macrophages. And the structure is also similar to the other three CARs, with the difference in the intracellular domain which is replaced by CD ζ subunit of the T cell receptor.

The mechanism of CAR ζ:

The main function domain of the CAR ζ is the cytosolic Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) as mentioned before. When the ligands matches its receptor, the Src family kinases will consequently be phosphorylated and thus activating the cytosolic Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). The further signal pathway is the same as mentioned in CAR-MEGF10.

The luciferase transcribed from the luciferase expression cassettes can catalyze the oxidation of the luciferin. And the bioluminescence released from the redox reaction can be determined by the fluor tester quantitatively. The bioluminescence intensity is linearly dependent to the amount of the substrate. Therefore, we inserted the luciferase expression cassettes to the gene sequence of the vesicular stomatitis virus (VSV) pseudotyped virus to make the infection rate of SARS-CoV-2 pseudotyped virus be detected indirectly. When the gene of SARS-CoV-2 pseudotyped virus is released and then inserted to the host gene, the original edited luciferase expression cassettes can perform its function and illumine the infected cells.

Myc tag is a tag protein containing 11 amino acids, and it can formulate a stable antigen domain for specific detectable antibody when inserted into a complex gene sequence. We therefore equipped the Myc tag gene to our CARs to better measure the CARs expression quantity in our CAR-macrophage, testing the transcription efficiency of the lentiviral technology indirectly.

Structure of the spike(S) protein:

The spike(S) protein is a highly glycosylated protein embedded in the viral envelope of coronaviruses. There are three critical parts of the S protein: a γ carboxyglutamate-rich domain (Gla domain) in the amino terminus prepared for the binding with the phosphatidylserine located in the apoptotic cells or virus particles, four epidermal growth factor (EGF) like domains, and one sex hormone binding globulin (SHBG) domain which consists of two globular laminin G-like (LG) domains.

The function of the S protein in SARS-CoV-2 infection:

The spike protein of the SARS-CoV-2 plays a crucial role in penetrating host cells and initiating infection. There are two function domains of the S protein, the S1 and the S2. The S1 domain has the specialized ability to bind with the ACE2 of the alveolar epithelial cells. After the S protein on the surface of SARS-CoV-2 binds with the extracellular domain of the ACE2 on the alveolar epithelial cells, the extracellular domain of the ACE will be lysed and the remaining SARS-CoV-2-ACE complex will be internalized with the assist of the Clathrin. After the fusion between the lipid bilayers of the SARS-CoV-2 and the alveolar epithelial cell, the RNA of the SARS-CoV-2 can finally be released and inserted into the targeted gene sequence to conduct virus replication.

Considering the crucial role the S protein plays in the SARS-CoV-2 infection process, we are determined to construct a replication-defective pseudo- SARS-CoV-2 by expressing the S protein in the replication-defective vesicular stomatitis virus (Delta-G-VSV pseudovirus).

The sequence of the spike protein is from GenBank (YP_009724390), and we ordered the DNA from a synthesis company.

The transmembrane domain of the CAR performs a signal transduction role after the interaction between the S protein of pseudo- SARS-CoV-2 and the CARs. In this part, we used the CD8 hinge and the CD8 transmembrane domain presented in the αCD19 CAR for T cell to construct the transmembrane domain of our CAR-macrophage.

The αS-scFv, also named CR3022, is a scFv derived from an antibody recognizing the spike protein of the SARS-CoV-2. As the previous research reported, the αS-scFv has a striking affinity towards the S protein. We consequently adopted the αS-scFv to be the extracellular domain of our CARs to improve the phagocytosis efficiency of our CAR-macrophages.