Targeting SARS-CoV-2 with off-the-shelf CAR-NK cells

The current COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread and cause disease and death in many countries even today. At present, there is no effective treatment. A recent study published on the preprint server bioRxiv* in August 2020 reports the potential of a new class of drugs called CAR NK cells, which could be used off-the-shelf to treat this disease.

Current Therapies

Current medical interventions in COVID-19 comprise three types of therapy: antiviral treatments, immunomodulators, and supporting treatments. The first category includes remdesivir, lopinavir/ritonavir, chloroquine, hydroxychloroquine, and ribavirin, often used in combinations. Immune response reduction has been attempted using antibodies to the IL-6 receptors, corticosteroids, and non-steroidal anti-inflammatory drugs (NSAIDs). Supportive management includes mechanical ventilation and supplementary oxygen, as well as the use of convalescent plasma.

Immunotherapy Approaches

Multiple immunotherapeutic techniques are being tried out at present. A novel approach to modulating cell-based immunity involves the use of chimeric antigen receptor (CAR) technology with natural killer (NK) cells. These are isolated from peripheral blood and can be adapted to express CAR in order to treat multiple infectious and neoplastic conditions.

Previously, CAR T cells have been used in some tumor therapies. However, to treat COVID-19, the T cells have to be derived from affected individuals who have lymphopenia. On the other hand, the CAR NK cells can be sourced from a third party, allowing them to be used off-the-shelf, at reduced costs and with broader applicability.

Generation of CR3022-CAR-NK92MI cells. (a) Schematic design of CR3022-CAR in SFG retroviral vector. The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ. (b) Generation of CR3022-CAR-NK cells. 293T cells were transfected with SFG-CR3022- CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells. (c) Determination of CAR expression by flow cytometry. CR3022-CAR cells were harvested after 4-5 days then stained with anti-CD56 and CAR F(ab)2 domain [IgG (H+L)] for flow cytometry.
Generation of CR3022-CAR-NK92MI cells. (a) Schematic design of CR3022-CAR in SFG retroviral vector. The SFG retroviral vector contains the CR3022 single chain antibody fragment (clone 3), a human IgG1 CH2CH3 hinge region and CD28 transmembrane region, followed by the intracellular domains of co-stimulatory CD28, 4-1BB, and the intracellular domain of CD3ζ. (b) Generation of CR3022-CAR-NK cells. 293T cells were transfected with SFG-CR3022- CAR for 48-72 hours for CAR retrovirus packaging and transduced into NK92MI cells. (c) Determination of CAR expression by flow cytometry. CR3022-CAR cells were harvested after 4-5 days then stained with anti-CD56 and CAR F(ab)2 domain [IgG (H+L)] for flow cytometry.

Generating CAR-NK Cells

The current study used NK cells, which they then modified with a CAR molecule that binds specifically to the SARS-CoV-2 spike (S) protein. Prior research has shown the significant similarity between the genomes of this virus and the earlier SARS-CoV, as well as a cross-reactive neutralizing antibody CR3022. The researchers, therefore, transduced the scFv domain of the antibody into a viral vector containing IgG1 hinge and other parts of the immunoglobulin molecule. This antibody CR3022 has potent binding activity against the spike proteins of both viruses.

They first generated CR3022-CAR expression on the NK cells in a human NK-92 cell line using plasmids containing the chimeric antigen. After sorting the resulting cells using flow cytometry, the CR3022-expressing cells were maintained for 2 months to confirm that CAR was consistently expressed. Once the stable expression of CAR on the cell membrane was established, they confirmed that the pattern of receptor expression of the chimeric cells was similar to that of the parent NK cells.

Binding Activity to RBD and Pseudovirus

The researchers then evaluated how well these cells bound to the receptor-binding domain (RBD) of the virus. On co-incubation, they found that as expected, the CR3022-CAR-NK cells bound to the RBD protein.

They then looked at the binding activity of the CR3022-CAR-NK cells to pseudotyped SARS-CoV-2 viral particles. They found that just like the binding of the RBD to the natural receptor, ACE2, which promotes viral entry into the host cell, these chimeric NK cells also bound to the pseudoviruses. In fact, the pseudovirus binding to a cell culture line expressing human ACE2 was weaker than the binding to the CR3022-CAR-NK-92MI cells, which indicates a higher affinity of this cell line to the spike protein than its natural ACE2 receptor.

Activation of CR3022-CAR-NK cells by Spike RBD

The investigators then expressed the RBD on a cell line expressing the ACE2 receptor, which was activated after culturing with these cells. They then examined the efficiency of binding and activation of CR3022-CAR-NK cells by these target cells. They found that that the activation molecule CD107 on the surface of the CR3022-CAR-NK cells increased when cultured with the ACE2- and RBD-expressing cells.

The levels of the activation products TNF-alpha and perforin also shot up in these CR3022-CAR-NK-92MI cells after co-culturing with these 293T-hACE2 cells expressing the spike RBD target. They observed that CR3022-CAR-NK cells were able to kill cells infected with SARS-CoV-2, supporting this modality for clinical use.

Implications

The study proves, “CR3022-CAR-NK cells can be activated by SARS-CoV-2 spike protein receptor-binding domain expressing infected target cells and specifically kill their susceptible target cells.” The cells used in the study have malignant potential and must be irradiated before use in patients to prevent them from taking permanent root in the recipient. Secondly, natural SARS-CoV-2 particles were not tested, nor was an animal model used. Such studies will be required to demonstrate the relevance of these observations in the clinical setting, by showing the effectiveness and safety of the CR3022-CAR-NK cells.

This study could promote more advanced preclinical studies and perhaps the production of a line of universally applicable readymade CAR-NK-based immunotherapy for COVID-19.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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