CAR/TCR Copy Number Detection Kit, Accurately Monitoring The Vector Copy Number In Cell Therapy Products
Introduction to CAR-T & TCR-T Cell Therapy
Cell therapy refers to obtaining living cells with specific functions from a person's own or allogeneic sources, and then injecting them into the human body after in vitro manipulation to achieve the purpose of treating a certain disease. Depending on the type of cells, it can be divided into immune cell therapy, stem cell therapy and other cell therapies. Both CAR-T and TCR-T are relatively mainstream specific immune cell therapies, and their cells come from the same source, peripheral blood mononuclear cells (PBMC), autologous or allogeneic cells.
The mechanism of action of CAR-T cell therapy is to use lentivirus (LV) as a delivery vector to transfer the CAR gene sequence to the T cell genome, so that the T cells can specifically recognize and bind to tumor cells, and then kill tumor cells by releasing factors such as perforin, thereby achieving the purpose of treatment. It has the advantages of good tumor killing effect, long-lasting therapeutic effect, and is suitable for the elderly and combined with other drugs.
The mechanism of action of TCR-T cell therapy is to first isolate T cells from the patient's blood or tumor tissue, then isolate TCRα and β chains from a single T cell clone, insert them into a lentiviral or retroviral vector, and then perform viral transfection of T cells to introduce the target TCRαβ sequence into T cells to obtain TCR-T cells that can specifically recognize tumor antigens. These modified T cells are then expanded in vitro to obtain sufficient numbers for treatment and re-infusion into the patient. TCR-T therapy has the advantages of strong targeting, permeability and stability.
Both CAR-T and TCR-T have received extensive attention and research because they can express specific receptors and target specific cells such as tumor cells. However, while these two therapies fight tumors and alleviate patients' diseases, they can also cause side effects such as cytokine storms and neurotoxicity. Therefore, domestic and foreign regulatory agencies such as the National Medical Products Administration have formulated a series of guiding principles and guidelines for immune cell therapy products to regulate and guide the research and development, application and evaluation of immune cell therapy products. CAR/TCR copy number detection concept and regulatory requirements The copy number refers to the number of copies of a gene (which can be a plasmid) in the genome of a certain organism. Single copy means that there is only one copy of the gene in the genome of the organism, and multiple copies means that there are multiple copies.
It is known that CAR-T and TCR-T cell therapy are to construct the target gene containing CAR or TCRαβ sequence into lentiviral or retroviral vectors, and then infect T cells with the virus to obtain CAR-T or TCR-T cells. These vector genes are integrated into the T cell genome. On the one hand, it shows that the T cells have been modified with CAR or TCR genes. On the other hand, it is a safety indicator. The integration may bring about the risk of secondary tumors such as activation of oncogenes or inactivation of tumor suppressor genes. Although the current vector design has greatly reduced the risk of integration, this risk has not been completely eliminated, so it is necessary to detect the number of viral vector copies integrated into the cell genome.
In response to the potential risks brought by CAR and TCR gene integration, drug regulatory agencies have issued corresponding guidance documents. The US FDA released a draft guidance on "Research and Development Considerations for Chimeric Antigen Receptor (CAR) T Cell Therapy" on March 15, 2022, mentioning that vector copy number transgene integration may change the expression of cellular genes and lead to tumorigenicity. Therefore, transgene integration in the preparation is an important safety parameter for measuring the release of cells such as CAR-T and TCR-T. In June 2018, the “Key Points for Consideration in Quality Control Testing and Non-clinical Research of CAR-T Cell Therapy Products” issued by the China Food and Drug Inspection Institute clearly stipulates that the CAR gene copy number should be ≤ 5 copies/cell. CAR/TCR gene copy number detectionAt present, real-time fluorescence quantitative PCR (qPCR) is widely used in gene expression, copy number determination and pathogen detection research, etc. This method can accurately quantify the number of nucleic acid target sequences of DNA or RNA in the sample. Therefore, the qPCR method is also widely used in the detection industry of transgenic copy number such as CAR and TCR.
The detection of CAR/TCR gene copy number mainly includes the CAR-T or TCR-T cell suspension harvesting stage and the regular peripheral blood follow-up monitoring stage of patients receiving CAR-T or TCR-T cell therapy.
In response to the above situation, Yeasen Biotechnology independently developed a CAR/TCR gene copy number detection kit, which uses multiple fluorescent probe qPCR to detect the copy number of CAR or TCR genes in the genome of CAR-T or TCR-T cells and single copy genes (SCG) in human cells. It also developed a host cell residual DNA sample pretreatment kit and a matching automated nucleic acid extraction instrument. The following figure provides a detailed introduction to the CAR/TCR gene copy number detection workflow:
The mechanism of action of CAR-T cell therapy is to use lentivirus (LV) as a delivery vector to transfer the CAR gene sequence to the T cell genome, so that the T cells can specifically recognize and bind to tumor cells, and then kill tumor cells by releasing factors such as perforin, thereby achieving the purpose of treatment. It has the advantages of good tumor killing effect, long-lasting therapeutic effect, and is suitable for the elderly and combined with other drugs.
The mechanism of action of TCR-T cell therapy is to first isolate T cells from the patient's blood or tumor tissue, then isolate TCRα and β chains from a single T cell clone, insert them into a lentiviral or retroviral vector, and then perform viral transfection of T cells to introduce the target TCRαβ sequence into T cells to obtain TCR-T cells that can specifically recognize tumor antigens. These modified T cells are then expanded in vitro to obtain sufficient numbers for treatment and re-infusion into the patient. TCR-T therapy has the advantages of strong targeting, permeability and stability.
Both CAR-T and TCR-T have received extensive attention and research because they can express specific receptors and target specific cells such as tumor cells. However, while these two therapies fight tumors and alleviate patients' diseases, they can also cause side effects such as cytokine storms and neurotoxicity. Therefore, domestic and foreign regulatory agencies such as the National Medical Products Administration have formulated a series of guiding principles and guidelines for immune cell therapy products to regulate and guide the research and development, application and evaluation of immune cell therapy products. CAR/TCR copy number detection concept and regulatory requirements The copy number refers to the number of copies of a gene (which can be a plasmid) in the genome of a certain organism. Single copy means that there is only one copy of the gene in the genome of the organism, and multiple copies means that there are multiple copies.
It is known that CAR-T and TCR-T cell therapy are to construct the target gene containing CAR or TCRαβ sequence into lentiviral or retroviral vectors, and then infect T cells with the virus to obtain CAR-T or TCR-T cells. These vector genes are integrated into the T cell genome. On the one hand, it shows that the T cells have been modified with CAR or TCR genes. On the other hand, it is a safety indicator. The integration may bring about the risk of secondary tumors such as activation of oncogenes or inactivation of tumor suppressor genes. Although the current vector design has greatly reduced the risk of integration, this risk has not been completely eliminated, so it is necessary to detect the number of viral vector copies integrated into the cell genome.
In response to the potential risks brought by CAR and TCR gene integration, drug regulatory agencies have issued corresponding guidance documents. The US FDA released a draft guidance on "Research and Development Considerations for Chimeric Antigen Receptor (CAR) T Cell Therapy" on March 15, 2022, mentioning that vector copy number transgene integration may change the expression of cellular genes and lead to tumorigenicity. Therefore, transgene integration in the preparation is an important safety parameter for measuring the release of cells such as CAR-T and TCR-T. In June 2018, the “Key Points for Consideration in Quality Control Testing and Non-clinical Research of CAR-T Cell Therapy Products” issued by the China Food and Drug Inspection Institute clearly stipulates that the CAR gene copy number should be ≤ 5 copies/cell. CAR/TCR gene copy number detectionAt present, real-time fluorescence quantitative PCR (qPCR) is widely used in gene expression, copy number determination and pathogen detection research, etc. This method can accurately quantify the number of nucleic acid target sequences of DNA or RNA in the sample. Therefore, the qPCR method is also widely used in the detection industry of transgenic copy number such as CAR and TCR.
The detection of CAR/TCR gene copy number mainly includes the CAR-T or TCR-T cell suspension harvesting stage and the regular peripheral blood follow-up monitoring stage of patients receiving CAR-T or TCR-T cell therapy.
In response to the above situation, Yeasen Biotechnology independently developed a CAR/TCR gene copy number detection kit, which uses multiple fluorescent probe qPCR to detect the copy number of CAR or TCR genes in the genome of CAR-T or TCR-T cells and single copy genes (SCG) in human cells. It also developed a host cell residual DNA sample pretreatment kit and a matching automated nucleic acid extraction instrument. The following figure provides a detailed introduction to the CAR/TCR gene copy number detection workflow:
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