Advantages of retroviral vectors in CAR-NK cells
In recent years, the success of CAR-T therapy has made people see the great potential of immunotherapy, and has also become an important milestone in the history of human conquest of cancer. But at present, CAR-T therapy is mainly only for blood tumors and is easy to cause cytokine storms, which also encourages researchers to develop more effective cell therapies, and the advent of CAR-NK is regarded as a new generation of immunotherapy tools.
The so-called CAR-NK cell therapy is to express CAR on the surface of NK cells through genetic engineering technology to endow and enhance the targeted killing function of NK cells to tumors. NK cells belong to the intrinsic lymphocyte family, which is found in human peripheral blood, bone marrow, and tissues and organs, and can be identified by the absence of T cell receptor (TCR) and related CD3 molecules, as well as the expression of nerve cell adhesion molecules (also known as CD56). Fortunately, allogeneic NK cells do not express individual specific TCR, which is different from the way that T cells generate activation signals through TCR-specific recognition of MHC-antigen-peptide complexes and then activation. NK cells can kill target cells without antigen stimulation, antigen presentation and autologous MHC restriction. The risk of developing graft-versus-host disease in clinical therapy is much lower than that of allogeneic T cell therapy. In addition, based on the "self-loss" recognition mode of NK cells, whether it is transfused alone or combined with hematopoietic stem cell transplantation, donor NK cells that are incompatible with the recipient's KIR gene have stronger anti-tumor effects, and are more suitable to be developed as universal immune cell therapy products, which is conducive to large-scale industrial application.
With advances in gene modification technology, many methods have been used to produce CAR-NK. One of the main challenges of CAR-NK cell therapy is the inefficiency of gene transfer. Compared with other gene transfer methods, retrovirus transduction is widely used, efficient and has no obvious toxicity. Specific advantages are reflected in the following aspects:
一、Retroviruses have a relatively neutral integration spectrum and are less genotoxic
Retrovirus vectors most commonly used in clinical applications include vectors derived from human immunodeficiency virus-1 (lentiviral vector) and Moloni mouse leukemia virus (gamma retrovirus vector). Integration group analysis showed that γ-retrovirus vectors preferentially integrated near transcription initiation sites, CpG islands, and cancer-affecting genes, while lentiviral vectors tended to integrate in actively transcribed genes [1]. In contrast, alpha retrovirus vectors have a relatively neutral integrated spectrum, which is more conducive to clinical research [2]. However, previous studies have used alpha-retrovirus vectors carrying viral coding sequences and complete long terminal repeats (LTR). While viral coding sequences are potentially immunogenic in host cells and increase the risk of carrier mobilization, intact LTRS containing transcriptional elements are capable of activating cellular genes and contributing to insertion mutagenesis [1].
At present, gamma and alpha retroviral self-inactivation (SIN) vectors have been developed to remove enhancer and promoter elements from LTR, and an advanced split packaging system has been established, in addition to removing virus coding sequences and retroviral splicting sites from the vectors to solve the problem of insertional mutagenesis [6]. In addition, the likelihood of the formation of replication-capable retroviruses (RCRS) has been reduced through a variety of strategies, including deletion of genes required for self-replication and reduction of overlapping sequences of viral genomes and vector generating cells, which have been used in many clinical studies [3, 4].
二、Retroviruses have high efficiency in NK cell transduction
Retroviruses have been used as gene therapy vectors for decades. A possible explanation for NK cells' resistance to retrovirus transduction may stem from the cells' inherent defense mechanisms against viral infection. Strategies to improve the efficiency of retroviral transduction include preactivating NK cells with cytokines (IL-2) and K562 cells to express membrane-bound IL-21. The transfection efficiency of retrovirus vectors into NK cells is 27%-52%, and the transduction efficiency of retroviruses encoding secreted IL-15 or membrane bound IL-15 (mIL-15) genes can reach 70% [3].
Different types of retroviruses were used to produce CAR-NK cells. Compared with gamma retroviruses and lentiviruses, RD114α retroviruses showed higher transduction efficiency in primary NK cells. Among different retroviruses, α-retrovirus vectors showed superior performance than γ-retrovirus vectors, with a transduction efficiency of 90% in NK cell lines and greater than 60% in stimulated primary NK cells.
By the end of 2021, 20 studies using CAR-NK cell lines and 15 studies using primary NK cells have applied retroviruses. In addition, in a recent Phase I clinical trial, CD19 CAR-NK cells transduced by retroviruses were used to treat CD19+ non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this study, 73 percent of patients responded, and seven out of eight patients achieved a complete response. In addition, at all dose levels, CAR-NK responded rapidly within 30 days after infusion. After one year of follow-up, amplified CAR-NK cells could still be detected. The copy number of CAR-NK DNA in peripheral blood remained stable for up to one year after infusion, indicating for the first time that retroviral transduced CAR-NK cells can survive in vivo for a long time [5].
三、Retroviruses can achieve consistent, economical, large-scale vector production through stable packaging cell lines
With an increasing number of clinical trials using gamma reverse transcription and lentiviral vectors for stable genome-modified gene therapy, the need for clinically applicable retroviral vector batches is increasing, indicating the importance of economical vector production for future gene therapy trials. The use of stable packaged cell lines holds the promise of consistent, economical, large-scale vector production compared to instantaneous transfection, with the potential to treat more patients.
The unique replication ability of retroviruses allows the transgene to stably integrate into the target genome and express CAR for a long time, but only in dividing NK cells. In general, retrovirus vectors can be generated by transient transfection or stable packaging cell lines. These two methods have obvious advantages and disadvantages in terms of establishment, flexibility, repeatability, scaling possibilities and quality assurance. For instantaneous production, all components of the packaging system are delivered instantaneously into the packaging cell in the form of plasmids. This allows the flexibility to change the carrier composition. In contrast, the establishment of a stable packaged cell line requires the stable integration of all components into the genome of the virus-producing packaged cell line, although the initial work is time-consuming and requires technical platform building and activation. However, the generation of viral vectors from stable packaged cell lines can guarantee a high level of product consistency from batch to batch, while instantaneously generated viral particles are subject to significant fluctuations. Due to the need to achieve manufacturing specification (GMP) grade plasmids, upgrading the instantaneous transfection production process is costly and technically demanding. Therefore, from a regulatory, production-related, and economic perspective, the generation of viral vectors from stable packaged cell lines is preferred [4].
四、Retroviruses can enhance the efficiency of gene transfer to NK cells through false typing
Retrovirus pseudotyping refers to the incorporation of glycoproteins from other enveloped viruses onto the viral vector, allowing the retrovirus vector to transport a wider range of cells and tissues. One way to enhance NK cell transduction is to select the best pseudotype envelope protein. For the time being. Previous studies have proposed the successful strategy of using RD114-TR pseudoretrovirus particles in combination with Vectofusin-1 to genetically modify PBMC derived NK cells to obtain high-cytotoxic CD19-CAR-NK cells with high yield. Moreover, α-retrovirus-transduced CAR-NK cells have higher cytotoxicity than lentivirus-transduced CAR-NK cells [6]. It has been suggested that the level of gene transfer of RD114 / TR pseudovirus particles is higher than that of VSVg pseudovirus particles in α and γ retrovirus environment. This transduction effect may be due in part to the expression of neutral amino acid transporter 2 (ASCT-2, SLC1A5), which RD114/TR uses to enter cells. The use of RD114 / TR pseudotype alpha retrovirus particles in combination with Retronectin mediated transduction can stabilize transduction levels above 90% in NKL cell lines and up to 60% in primary NK cells [2].
Reference：
[1] Suerth, Julia D et al. “Alpharetroviral vectors: from a cancer-causing agent to a useful tool for human gene therapy. "Viruses vol. 6,12 4811-38. 5 Dec. 2014, Doi: 10.3390 / v6124811
[2]Suerth, J.D., Morgan, M.A., Kloess, S. et al. Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors. J Mol Med 94, 83 -- 93 (2016).
[3]Matosevic, Sandro. “Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies.” Journal of immunology research vol. 2018 4054815.17 Sep. 2018, doi:10.1155/2018/4054815
[4]Matosevic, Sandro. “Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies.” Journal of immunology research vol. 2018 4054815.17 Sep. 2018, doi:10.1155/2018/4054815
[5]Franks SE, Wolfson B, Hodge JW. Natural Born Killers: NK Cells in Cancer Therapy. Cancers (Basel). 2020 Jul 31; 12(8):2131. doi: 10.3390/cancers12082131.
[6] Muller, Stephan et al. “High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.” Frontiers in immunology vol. 10 3123. 24 Jan. 2020, doi: 10.3389 / fimmu. 2019.03123
Disclaimer: Shenzhen Cell Valley is committed to the research of cell and gene therapy, in order to promote emerging technologies, so that more people understand the new development of biomedicine. The content of this article is only used for information exchange, and the platform remains neutral on the content, statements and opinions of the article, and does not represent the position and views of Shenzhen Cell Valley. The relevant information in this article should not be used as a diagnosis or treatment, is not a substitute for professional medical advice, and the company's website will not assume any responsibility. The final interpretation of the content of the above statement belongs to the company's website, this statement will apply to the company's website all the time to share the article, thank you for your cooperation! Copyright description: The copyright of the article belongs to Shenzhen Cell Valley, individuals are welcome to forward to the circle of friends, media or institutions without authorization, reproduced in any form to other platforms, will be regarded as infringement. For reprinting, please contact email: contact@sz-cell.com
The so-called CAR-NK cell therapy is to express CAR on the surface of NK cells through genetic engineering technology to endow and enhance the targeted killing function of NK cells to tumors. NK cells belong to the intrinsic lymphocyte family, which is found in human peripheral blood, bone marrow, and tissues and organs, and can be identified by the absence of T cell receptor (TCR) and related CD3 molecules, as well as the expression of nerve cell adhesion molecules (also known as CD56). Fortunately, allogeneic NK cells do not express individual specific TCR, which is different from the way that T cells generate activation signals through TCR-specific recognition of MHC-antigen-peptide complexes and then activation. NK cells can kill target cells without antigen stimulation, antigen presentation and autologous MHC restriction. The risk of developing graft-versus-host disease in clinical therapy is much lower than that of allogeneic T cell therapy. In addition, based on the "self-loss" recognition mode of NK cells, whether it is transfused alone or combined with hematopoietic stem cell transplantation, donor NK cells that are incompatible with the recipient's KIR gene have stronger anti-tumor effects, and are more suitable to be developed as universal immune cell therapy products, which is conducive to large-scale industrial application.
With advances in gene modification technology, many methods have been used to produce CAR-NK. One of the main challenges of CAR-NK cell therapy is the inefficiency of gene transfer. Compared with other gene transfer methods, retrovirus transduction is widely used, efficient and has no obvious toxicity. Specific advantages are reflected in the following aspects:
一、Retroviruses have a relatively neutral integration spectrum and are less genotoxic
Retrovirus vectors most commonly used in clinical applications include vectors derived from human immunodeficiency virus-1 (lentiviral vector) and Moloni mouse leukemia virus (gamma retrovirus vector). Integration group analysis showed that γ-retrovirus vectors preferentially integrated near transcription initiation sites, CpG islands, and cancer-affecting genes, while lentiviral vectors tended to integrate in actively transcribed genes [1]. In contrast, alpha retrovirus vectors have a relatively neutral integrated spectrum, which is more conducive to clinical research [2]. However, previous studies have used alpha-retrovirus vectors carrying viral coding sequences and complete long terminal repeats (LTR). While viral coding sequences are potentially immunogenic in host cells and increase the risk of carrier mobilization, intact LTRS containing transcriptional elements are capable of activating cellular genes and contributing to insertion mutagenesis [1].
At present, gamma and alpha retroviral self-inactivation (SIN) vectors have been developed to remove enhancer and promoter elements from LTR, and an advanced split packaging system has been established, in addition to removing virus coding sequences and retroviral splicting sites from the vectors to solve the problem of insertional mutagenesis [6]. In addition, the likelihood of the formation of replication-capable retroviruses (RCRS) has been reduced through a variety of strategies, including deletion of genes required for self-replication and reduction of overlapping sequences of viral genomes and vector generating cells, which have been used in many clinical studies [3, 4].
二、Retroviruses have high efficiency in NK cell transduction
Retroviruses have been used as gene therapy vectors for decades. A possible explanation for NK cells' resistance to retrovirus transduction may stem from the cells' inherent defense mechanisms against viral infection. Strategies to improve the efficiency of retroviral transduction include preactivating NK cells with cytokines (IL-2) and K562 cells to express membrane-bound IL-21. The transfection efficiency of retrovirus vectors into NK cells is 27%-52%, and the transduction efficiency of retroviruses encoding secreted IL-15 or membrane bound IL-15 (mIL-15) genes can reach 70% [3].
Different types of retroviruses were used to produce CAR-NK cells. Compared with gamma retroviruses and lentiviruses, RD114α retroviruses showed higher transduction efficiency in primary NK cells. Among different retroviruses, α-retrovirus vectors showed superior performance than γ-retrovirus vectors, with a transduction efficiency of 90% in NK cell lines and greater than 60% in stimulated primary NK cells.
By the end of 2021, 20 studies using CAR-NK cell lines and 15 studies using primary NK cells have applied retroviruses. In addition, in a recent Phase I clinical trial, CD19 CAR-NK cells transduced by retroviruses were used to treat CD19+ non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this study, 73 percent of patients responded, and seven out of eight patients achieved a complete response. In addition, at all dose levels, CAR-NK responded rapidly within 30 days after infusion. After one year of follow-up, amplified CAR-NK cells could still be detected. The copy number of CAR-NK DNA in peripheral blood remained stable for up to one year after infusion, indicating for the first time that retroviral transduced CAR-NK cells can survive in vivo for a long time [5].
三、Retroviruses can achieve consistent, economical, large-scale vector production through stable packaging cell lines
With an increasing number of clinical trials using gamma reverse transcription and lentiviral vectors for stable genome-modified gene therapy, the need for clinically applicable retroviral vector batches is increasing, indicating the importance of economical vector production for future gene therapy trials. The use of stable packaged cell lines holds the promise of consistent, economical, large-scale vector production compared to instantaneous transfection, with the potential to treat more patients.
The unique replication ability of retroviruses allows the transgene to stably integrate into the target genome and express CAR for a long time, but only in dividing NK cells. In general, retrovirus vectors can be generated by transient transfection or stable packaging cell lines. These two methods have obvious advantages and disadvantages in terms of establishment, flexibility, repeatability, scaling possibilities and quality assurance. For instantaneous production, all components of the packaging system are delivered instantaneously into the packaging cell in the form of plasmids. This allows the flexibility to change the carrier composition. In contrast, the establishment of a stable packaged cell line requires the stable integration of all components into the genome of the virus-producing packaged cell line, although the initial work is time-consuming and requires technical platform building and activation. However, the generation of viral vectors from stable packaged cell lines can guarantee a high level of product consistency from batch to batch, while instantaneously generated viral particles are subject to significant fluctuations. Due to the need to achieve manufacturing specification (GMP) grade plasmids, upgrading the instantaneous transfection production process is costly and technically demanding. Therefore, from a regulatory, production-related, and economic perspective, the generation of viral vectors from stable packaged cell lines is preferred [4].
四、Retroviruses can enhance the efficiency of gene transfer to NK cells through false typing
Retrovirus pseudotyping refers to the incorporation of glycoproteins from other enveloped viruses onto the viral vector, allowing the retrovirus vector to transport a wider range of cells and tissues. One way to enhance NK cell transduction is to select the best pseudotype envelope protein. For the time being. Previous studies have proposed the successful strategy of using RD114-TR pseudoretrovirus particles in combination with Vectofusin-1 to genetically modify PBMC derived NK cells to obtain high-cytotoxic CD19-CAR-NK cells with high yield. Moreover, α-retrovirus-transduced CAR-NK cells have higher cytotoxicity than lentivirus-transduced CAR-NK cells [6]. It has been suggested that the level of gene transfer of RD114 / TR pseudovirus particles is higher than that of VSVg pseudovirus particles in α and γ retrovirus environment. This transduction effect may be due in part to the expression of neutral amino acid transporter 2 (ASCT-2, SLC1A5), which RD114/TR uses to enter cells. The use of RD114 / TR pseudotype alpha retrovirus particles in combination with Retronectin mediated transduction can stabilize transduction levels above 90% in NKL cell lines and up to 60% in primary NK cells [2].
Reference：
[1] Suerth, Julia D et al. “Alpharetroviral vectors: from a cancer-causing agent to a useful tool for human gene therapy. "Viruses vol. 6,12 4811-38. 5 Dec. 2014, Doi: 10.3390 / v6124811
[2]Suerth, J.D., Morgan, M.A., Kloess, S. et al. Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors. J Mol Med 94, 83 -- 93 (2016).
[3]Matosevic, Sandro. “Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies.” Journal of immunology research vol. 2018 4054815.17 Sep. 2018, doi:10.1155/2018/4054815
[4]Matosevic, Sandro. “Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies.” Journal of immunology research vol. 2018 4054815.17 Sep. 2018, doi:10.1155/2018/4054815
[5]Franks SE, Wolfson B, Hodge JW. Natural Born Killers: NK Cells in Cancer Therapy. Cancers (Basel). 2020 Jul 31; 12(8):2131. doi: 10.3390/cancers12082131.
[6] Muller, Stephan et al. “High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.” Frontiers in immunology vol. 10 3123. 24 Jan. 2020, doi: 10.3389 / fimmu. 2019.03123
Disclaimer: Shenzhen Cell Valley is committed to the research of cell and gene therapy, in order to promote emerging technologies, so that more people understand the new development of biomedicine. The content of this article is only used for information exchange, and the platform remains neutral on the content, statements and opinions of the article, and does not represent the position and views of Shenzhen Cell Valley. The relevant information in this article should not be used as a diagnosis or treatment, is not a substitute for professional medical advice, and the company's website will not assume any responsibility. The final interpretation of the content of the above statement belongs to the company's website, this statement will apply to the company's website all the time to share the article, thank you for your cooperation! Copyright description: The copyright of the article belongs to Shenzhen Cell Valley, individuals are welcome to forward to the circle of friends, media or institutions without authorization, reproduced in any form to other platforms, will be regarded as infringement. For reprinting, please contact email: contact@sz-cell.com
