In our preliminary study we established a manufacturing system to culture SARS-Coronavirus-2 (SARS-CoV-2) antigen specific T cells by collecting whole blood from patients who have been cured by COVID-19. In addition, we collected blood from healthy donors who have not been exposed to SARS-CoV-2 as a control and was able to generate SARS-CoV-2 antigen specific T cells.

Thus, in this study, we will recruit both COVID-19 recovered and unexposed individuals to generate SARS-CoV-2 antigen specific T cells in a GMP cell production facility to use the final product for IND approval and for future use in clinical trials.

COVID-19 is an acute viral respiratory disease caused by SARS-CoV-2 virus infection. Main symptoms include fever and respiratory symptoms. There are currently no vaccines or treatments for the infection and only treatments that ameliorate patient's symptoms are available. The definition of a COVID-19 patients is an individual who has been confirmed to be infected with an infectious disease according to examination diagnostic criteria regardless of clinical conditions. Diagnostic tests include PCR testing and virus testing. An individual can be released or considered treated when both the clinical and examination criteria are met. The clinical standard is to have no fever with all clinical symptoms improved. Also, PCR results must be negative twice ever 24 hours. Nonetheless, in the absence of specific antiviral or preventive vaccines for SARS-CoV2, the development of new therapeutic agents are urgent.
For viral diseases, it is essential to establish antiviral immunity to be cured and maintained long term. In fact, it has been confirmed by ELISPOT assays that immunity against SARS-CoV2 was established in a cured patient. Therefore, it provides evidence that virus-specific T cells can be amplified after SARS-CoV2 infection and could be used as a therapeutic agent if these immune cells can be amplified.
Among immune cells, cytotoxic T lymphocytes (CTLs) have the ability to recognize and destroy virus infected cells through MHC molecules. Because CTLs can be isolated and expanded from patients or donors, preclinical and clinical studies have ben extensively performed on the role of immune cells against antiviral protective effects in vivo. However, existing CTL production methods are complicated and expensive taking up to 8-12 weeks involving the production of antigen presenting cells and the use of genetic modification. Therefore, it cannot be used for patients who need it for immediate and urgent use.
In our study, we have developed a technology for producing dual targeting killer cells (DTK cells) by amplifying small amounts of antigen-specific T cells present in the peripheral blood. Our cell culture process involves strategic use of cytokines and peptide mixtures to amplify virus specific T cells. This method has many advantages over previous methods as it can be produced at a low cost without the requirement of antigen presenting cells and genetic modification and viral infection. In addition, it is expected to be target cells that have been modified with various mechanisms of action because it contains NKT cell subtypes that are MHC-independent. and antigen-specific CTLs that are MHC-dependent.

Others

(Human resource study- Cell therapy study (Blood collection))

Blood collection of whole blood 300cc or leukapheresis 250cc, once
Periphieral blood mononuclear cells are isolated and the cells are expanded in culture using cytokines for 21 days to generate antigen specific immune cells.

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Target Number of Participant

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Blood collection of whole blood 300cc or leukapheresis 250cc,once
Periphieral blood mononuclear cells are isolated from healthy individuals and the cells are expanded in culture using cytokines for 21 days to generate antigen specific immune cells.

Arm Label

Target Number of Participant

Arm Type

Arm Description

Blood collection of whole blood 300cc or leukapheresis 250cc, once
Peripheral blood mononuclear cells are isolated from patients treated with COVID-19 disease and the cells are expanded ub cyktyre using cytokines for 21 days to generate antigen specific immune cells.

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Description

For COVID-19 treated individuals:
1. Those who consent to the study
2. Aged 20 - to 65 years old
3. Individual who has been diagnosed with COVID-19 and released from quarantine, that is, the person who meets all the criteria for public release by the CDC:
A. (clinical standard): no fever and no clinical symptoms
B. (diagnostic criteria) PCR test results are negative twice every 24 hours

For health donors:
1. Those who consent
2. Aged 20 to 65 years old
3. Those who do not have any fever or respiratory symptoms
4. Negative for Anti-SARS-CoV-2 Ab IgG antibodies

1. Those who do not consent
2. Those under 20 or over 65 years of age
3. Positive for Anti-SARS-CoV-2 IgG antibodies
4. Positive for HIV, syphilis or hepatitis B and C carriers.
5. Those with any active infections requiring systemic medical treatment
6. Those who have been diagnosed with any other serious diseases

Expression of CD3 T cell marker in culture-expanded virus antigen specific T cells by flow cytometry

Day +21 of cell culture

Expression of CD14 monocyte and CD19 B cell marker in culture-expanded virus antigen specific T cells by flow cytometry

Day +21 of cell culture

Determining antigen specificity by detecting IFN-gamma secretion of culture expanded virus antigen-specific T cells against coronavirus-19 peptides through ELISPOT assay

Day +21 of cell culture

Fold expansion following culture of SARS-CoV-2 virus antigen peptide mixture treated peripheral nucleated cells

Day +21 of cell culture

Characterization of cultured cells (Phenotypical analysis of cultured cells through flow cytometry)

Day +21 of cell culture