Application of safety switch in cell therapy
一、Safety switch of cell therapy
After autologous or allogeneic cell infusion, a large number of immune cells are activated and prolifed rapidly, resulting in excessive cascade release of cytokines such as IL-6, IL-1, IL-12, TNF-α, IFN-α, and GM-CSF, leading to symptoms such as fever, hypotension, capillary leakage, hypoxia, and end-organ dysfunction. Also known as cytokine release syndrome (CRS). In order to cope with the emergence of CRS in patients after receiving cell therapy, there are two approaches: on the one hand, predictive biomarkers of CRS and neurotoxicity can be detected, and timely response can be made through intensive monitoring; The other is to design safer cell therapies, and the consensus strategy is to put a "safety switch" on cells, treat them under human control, and step on the brake before adverse effects occur, which will help reduce the toxic side effects.
As technology has developed, it has become feasible to reversibly control cellular products by providing or removing small molecules, providing protein-based regulators, or by physical stimuli such as light, ultrasound, or heat [1].
二、The type of safety switch
(一) Use of CID technology platform
CID is Chemical Induced Dimerization. The so-called CID technology platform (Chemical Induced Dimerization technology) is to introduce specific DNA molecules into target cells, so that target cells can express CID proteins (such as FKBP12) and target proteins. Then the dimerization is induced by adding small molecule compound CID, so that the corresponding CID protein can play its function. The CID protein consists of the signal region and the binding region, and the addition of a dimeric inducer (such as rimiducid) can bind it to the CID binding region into a dimer, and the dimer will start the cascade of downstream signals.
Caspase-9 is the activation enzyme of apoptosis signaling pathway, which will produce signaling cascade reaction and eventually lead to apoptosis. In adoptive cell therapy, iCasp9 can be transduced to control cell life activity. iCasp9 consists of the CID protein sequence via the linker and caspase9 sequences (FIG. 1B), and exposure to dimerization induced drugs causes rapid death of cells expressing this structure, alleviating severe side effects of treatment.
Figure 2. Lenalidomide regulates the degradation of CAR structure [3]
Figure 3. Tetracycline regulatory system of E. coli [4]
After autologous or allogeneic cell infusion, a large number of immune cells are activated and prolifed rapidly, resulting in excessive cascade release of cytokines such as IL-6, IL-1, IL-12, TNF-α, IFN-α, and GM-CSF, leading to symptoms such as fever, hypotension, capillary leakage, hypoxia, and end-organ dysfunction. Also known as cytokine release syndrome (CRS). In order to cope with the emergence of CRS in patients after receiving cell therapy, there are two approaches: on the one hand, predictive biomarkers of CRS and neurotoxicity can be detected, and timely response can be made through intensive monitoring; The other is to design safer cell therapies, and the consensus strategy is to put a "safety switch" on cells, treat them under human control, and step on the brake before adverse effects occur, which will help reduce the toxic side effects.
As technology has developed, it has become feasible to reversibly control cellular products by providing or removing small molecules, providing protein-based regulators, or by physical stimuli such as light, ultrasound, or heat [1].
二、The type of safety switch
(一) Use of CID technology platform
CID is Chemical Induced Dimerization. The so-called CID technology platform (Chemical Induced Dimerization technology) is to introduce specific DNA molecules into target cells, so that target cells can express CID proteins (such as FKBP12) and target proteins. Then the dimerization is induced by adding small molecule compound CID, so that the corresponding CID protein can play its function. The CID protein consists of the signal region and the binding region, and the addition of a dimeric inducer (such as rimiducid) can bind it to the CID binding region into a dimer, and the dimer will start the cascade of downstream signals.
Caspase-9 is the activation enzyme of apoptosis signaling pathway, which will produce signaling cascade reaction and eventually lead to apoptosis. In adoptive cell therapy, iCasp9 can be transduced to control cell life activity. iCasp9 consists of the CID protein sequence via the linker and caspase9 sequences (FIG. 1B), and exposure to dimerization induced drugs causes rapid death of cells expressing this structure, alleviating severe side effects of treatment.
Figure 1. iCasp9 system regulates apoptosis [2]
(二) The use of targeted protein degradation technology
One team created a molecular switch to regulate the activity of CAR-T cells by administering lenalidomide, a commonly used anticancer drug [3]. They took advantage of a novel technique known as targeted protein degradation, a pathway through which a handful of drugs, including lenalidomide, work by targeting the degradation of specific proteins. This mechanism was used to modify the small protein tag. When the degradation tag was attached to the CAR, during the administration of lenalidomide, lenalidomide would bind to the protein tag of the CAR structure, inducing CRL4CrBN-mediated ubiquitination and proteasomal degradation, and the labeled CAR would be degraded, thereby preventing T cells from recognizing cancer cells. Since T cells continue to manufacture CAR proteins, after the withdrawal of lenalidomide, the newly generated CAR proteins gradually accumulate, and CAR T cells recover anti-tumor function.
One team created a molecular switch to regulate the activity of CAR-T cells by administering lenalidomide, a commonly used anticancer drug [3]. They took advantage of a novel technique known as targeted protein degradation, a pathway through which a handful of drugs, including lenalidomide, work by targeting the degradation of specific proteins. This mechanism was used to modify the small protein tag. When the degradation tag was attached to the CAR, during the administration of lenalidomide, lenalidomide would bind to the protein tag of the CAR structure, inducing CRL4CrBN-mediated ubiquitination and proteasomal degradation, and the labeled CAR would be degraded, thereby preventing T cells from recognizing cancer cells. Since T cells continue to manufacture CAR proteins, after the withdrawal of lenalidomide, the newly generated CAR proteins gradually accumulate, and CAR T cells recover anti-tumor function.
Figure 2. Lenalidomide regulates the degradation of CAR structure [3]
(三) Use tetracycline (Tet) to regulate the system
Simply put, the tetracycline (Tet) regulatory expression system is to control the expression of the target protein by inducing drugs such as Tet to change the conformation of the regulatory protein. In the bacterial system, the tetracycline repressor protein (tetR) will normally bind to the TEtracycline resistance operon (tetO), inhibiting the transcription of the downstream resistance gene tetA. When tetracycline or tetracycline analogues such as doxycycline (Dox) are present, tetR will bind to TEtracycline instead of tetO, resulting in the expression of downstream resistance gene tetA, and TEtracycline will be transported out of the cell through tetA protein, thus enabling bacteria to acquire drug resistance [4].
Simply put, the tetracycline (Tet) regulatory expression system is to control the expression of the target protein by inducing drugs such as Tet to change the conformation of the regulatory protein. In the bacterial system, the tetracycline repressor protein (tetR) will normally bind to the TEtracycline resistance operon (tetO), inhibiting the transcription of the downstream resistance gene tetA. When tetracycline or tetracycline analogues such as doxycycline (Dox) are present, tetR will bind to TEtracycline instead of tetO, resulting in the expression of downstream resistance gene tetA, and TEtracycline will be transported out of the cell through tetA protein, thus enabling bacteria to acquire drug resistance [4].
Figure 3. Tetracycline regulatory system of E. coli [4]
According to the characteristics, it can be divided into activation system Tet-on and inhibitory system Tet-off.
Tet-off principle: tTA is a protein derived from the fusion of TetR and the viral transcriptional activation domain VP16. In the absence of Dox, tTA and TRE (7 duplicated TetO sequences) bind to initiate downstream gene expression. In the presence of Dox, the conformation of tTA is changed, and tTA will shed on the TRE, resulting in downstream gene expression shutdown.
Tet-on principle: rtTA is a fusion protein of rTetR and VP16, and its phenotype is the opposite of tTA. rtTA is an anti-TET repressor (rTetR) and relies on the presence of tetracycline for induction, rather than inhibition. When Dox is absent, rtTA cannot bind TRE and downstream gene expression is turned off. When Dox is present, the conformation of rtTA changes, rtTA aggregates TRE, and downstream gene expression is turned on.
The tetracycline regulatory system can be used to precisely regulate the expression of target genes, so as to control whether cells express CAR structure or regulate cell death.
Reference
[1]Sahillioglu, A.C. and T.N. Schumacher, Safety switches for adoptive cell therapy. Curr Opin Immunol, 2022. 74: p. 190-198.
[2]Di Stasi, A., et al., Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med, 2011. 365(18): p. 1673-83.
[3]Jan, M., et al., Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide. Sci Transl Med, 2021. 13(575).
[4]https://2013.igem.org/Team:Bielefeld-Germany/Biosesafety/Biosesafety System M.
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@duanglink.com
Tet-off principle: tTA is a protein derived from the fusion of TetR and the viral transcriptional activation domain VP16. In the absence of Dox, tTA and TRE (7 duplicated TetO sequences) bind to initiate downstream gene expression. In the presence of Dox, the conformation of tTA is changed, and tTA will shed on the TRE, resulting in downstream gene expression shutdown.
Tet-on principle: rtTA is a fusion protein of rTetR and VP16, and its phenotype is the opposite of tTA. rtTA is an anti-TET repressor (rTetR) and relies on the presence of tetracycline for induction, rather than inhibition. When Dox is absent, rtTA cannot bind TRE and downstream gene expression is turned off. When Dox is present, the conformation of rtTA changes, rtTA aggregates TRE, and downstream gene expression is turned on.
The tetracycline regulatory system can be used to precisely regulate the expression of target genes, so as to control whether cells express CAR structure or regulate cell death.
Reference
[1]Sahillioglu, A.C. and T.N. Schumacher, Safety switches for adoptive cell therapy. Curr Opin Immunol, 2022. 74: p. 190-198.
[2]Di Stasi, A., et al., Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med, 2011. 365(18): p. 1673-83.
[3]Jan, M., et al., Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide. Sci Transl Med, 2021. 13(575).
[4]https://2013.igem.org/Team:Bielefeld-Germany/Biosesafety/Biosesafety System M.
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@duanglink.com
