Tumor cell immunotherapy -CAR-NK and CAR-M cell therapy
一、What is CAR cell therapy
T cells in the immune system can be targeted to kill tumor cells, and T cell killing tumor cells depends on specific recognition of tumor antigens. However, the recognition pattern of tumor antigens by T cell receptor (TCR) is limited by human Leukocyte antigen (HLA) molecules, and in the face of down-regulated expression of HLA molecules in tumor cells and immunosuppressed tumor microenvironment, It is difficult for TCR to activate T cells to play an antitumor role. In order to overcome the limitations of HLA molecules, an engineered molecule called chimeric antigen receptor (CAR) was created. The structure of CAR is similar to that of TCR, which consists of five parts: extracellular specific tumor antigen recognition domain, hinge domain, transmembrane domain, co-stimulatory domain and intracellular signal transduction domain. Extracellular specific tumor antigen recognition region can be composed of antibody single chain variable region (heavy and light chains of tumor antigen-specific antibodies) or nano antibodies, which can directly recognize tumor-associated antigens or specific antigens in a non-HLA molecular-restricted mode, and achieve specific tumor killing effect by transmitting signals through intracellular signal domain and activating T cells [1].
This kind of CAR targeting tumor-specific antigen or tumor-associated antigen is expressed on the surface of T cells through genetic engineering, and then expanded in vitro and then adoptive transfused into corresponding tumor patients is called CAR-T cell immunotherapy, and T cells expressing CAR molecules are called CAR-T cells (FIG. 1) [2]. Besides T cells, CAR can also be expressed on the surface of other immune cells, such as Natural Killer cells (NK), Macrophage, γδT cells, DC cells and other immune cells. This corresponds to CAR-NK cells, CAR-M cells, CAR-γδT cells, and CAR-DC cells.
T cells in the immune system can be targeted to kill tumor cells, and T cell killing tumor cells depends on specific recognition of tumor antigens. However, the recognition pattern of tumor antigens by T cell receptor (TCR) is limited by human Leukocyte antigen (HLA) molecules, and in the face of down-regulated expression of HLA molecules in tumor cells and immunosuppressed tumor microenvironment, It is difficult for TCR to activate T cells to play an antitumor role. In order to overcome the limitations of HLA molecules, an engineered molecule called chimeric antigen receptor (CAR) was created. The structure of CAR is similar to that of TCR, which consists of five parts: extracellular specific tumor antigen recognition domain, hinge domain, transmembrane domain, co-stimulatory domain and intracellular signal transduction domain. Extracellular specific tumor antigen recognition region can be composed of antibody single chain variable region (heavy and light chains of tumor antigen-specific antibodies) or nano antibodies, which can directly recognize tumor-associated antigens or specific antigens in a non-HLA molecular-restricted mode, and achieve specific tumor killing effect by transmitting signals through intracellular signal domain and activating T cells [1].
This kind of CAR targeting tumor-specific antigen or tumor-associated antigen is expressed on the surface of T cells through genetic engineering, and then expanded in vitro and then adoptive transfused into corresponding tumor patients is called CAR-T cell immunotherapy, and T cells expressing CAR molecules are called CAR-T cells (FIG. 1) [2]. Besides T cells, CAR can also be expressed on the surface of other immune cells, such as Natural Killer cells (NK), Macrophage, γδT cells, DC cells and other immune cells. This corresponds to CAR-NK cells, CAR-M cells, CAR-γδT cells, and CAR-DC cells.
Figure 1 Schematic diagram of CAR T cells
二、CAR-T therapy
In recent years, CAR-T therapy has made remarkable achievements in the treatment of malignant tumors, especially in the treatment of hematoma has shown excellent therapeutic effect, and is considered to be one of the most promising therapeutic methods to cure tumors. At present, the blood system tumors treated by CAR-T technology include acute myeloid leukemia, acute lymphoblastic leukemia, lymphoma, multiple myeloma, etc., and it also has certain effects in the treatment of solid tumors such as liver cancer and prostate cancer. As of September 2023, a total of 9 CAR T therapeutic drugs have been marketed worldwide, targeting CD19 and BCMA, mainly for the treatment of blood tumors. Among them, according to Gilead's financial report statistics in the first three quarters of 2023, Yescarta (Anti-CD19 CAR-T) and Tecartus (Anti-CD19 CAR-T) two cell products sales reached 1.402 billion US dollars (a total of about 10.2 billion yuan), its market potential can be seen.
Figure 2. How CAR T cells kill tumors
But there are also some challenges and deficiencies. One is the safety of clinical treatment. CAR-T therapy is prone to clinical cytotoxic side effects, and the most common CAR-T cell toxic side effects are cytokine release syndrome (CRS) and immune effector cell-related neurotoxic syndrome (ICANS) [3]. Severe CRS and ICANS have fatal risks, so how to improve the safety of CAR-T therapy and reduce the cytotoxic side effects has become an urgent problem in cell drug development. Second, due to various reasons [4] (see previous tweets for details), CAR-T therapy is not very effective in the treatment of solid tumors. The third is that because the current CAR-T therapy is personalized customized treatment, the production capacity is limited, and the cost is high, and most domestic patients can not pay.
Based on the various problems faced by autologous CAR-T therapy in solid tumors, people are urgently looking forward to the emergence of a universal, off-the-shelf cell therapy product that can be prepared on a large scale, and CAR-NK therapy may bring hope to achieve this goal.
三、CAR-NK therapy
Natural killer cells (NK cells) are derived from bone marrow lymphoid stem cells, accounting for 5% to 15% of adult peripheral blood lymphocytes, and are important innate immune cells, named for their non-specific cytotoxic effects. NK cells play a key role in tumor immune surveillance, killing tumor cells through direct lysis and secretion of cytokines, so the development of its anticancer function has become a hot spot in cancer research in recent years.
Unlike T cells that generate activation signals through TCR-specific recognition of MHC-antigen-peptide complexes, NK cells can kill target cells without antigen stimulation or antigen presentation, and are not restricted by autologous MHC. The surface of NK cells expresses a complex spectrum of receptors, mainly including killer immunoglobulin-like receptors (KIR), C-type lectin receptor family, and active natural cytotoxic receptors (NCRS) such as NKp30, NKp44, and NKp46. NK cells can be divided into inhibitory and activated receptors according to their functions, and interact with corresponding ligands to transmit inhibitory and activated signals respectively. The activation of NK cells is determined by the balance between the two signals [5]. Activated NK cells "destroy" target cells through a variety of mechanisms (Figure 3), such as: ① release perforin and granase, causing target cell lysis or apoptosis; ② Promote FasL expression and induce apoptosis of tumor cells; ③ Release TNF-α, IFN-γ, GM-CSF and chemokines (such as CCL1, CCL2, CCL3, etc.), recruit and activate other effector immune cells, etc.
Based on the various problems faced by autologous CAR-T therapy in solid tumors, people are urgently looking forward to the emergence of a universal, off-the-shelf cell therapy product that can be prepared on a large scale, and CAR-NK therapy may bring hope to achieve this goal.
三、CAR-NK therapy
Natural killer cells (NK cells) are derived from bone marrow lymphoid stem cells, accounting for 5% to 15% of adult peripheral blood lymphocytes, and are important innate immune cells, named for their non-specific cytotoxic effects. NK cells play a key role in tumor immune surveillance, killing tumor cells through direct lysis and secretion of cytokines, so the development of its anticancer function has become a hot spot in cancer research in recent years.
Unlike T cells that generate activation signals through TCR-specific recognition of MHC-antigen-peptide complexes, NK cells can kill target cells without antigen stimulation or antigen presentation, and are not restricted by autologous MHC. The surface of NK cells expresses a complex spectrum of receptors, mainly including killer immunoglobulin-like receptors (KIR), C-type lectin receptor family, and active natural cytotoxic receptors (NCRS) such as NKp30, NKp44, and NKp46. NK cells can be divided into inhibitory and activated receptors according to their functions, and interact with corresponding ligands to transmit inhibitory and activated signals respectively. The activation of NK cells is determined by the balance between the two signals [5]. Activated NK cells "destroy" target cells through a variety of mechanisms (Figure 3), such as: ① release perforin and granase, causing target cell lysis or apoptosis; ② Promote FasL expression and induce apoptosis of tumor cells; ③ Release TNF-α, IFN-γ, GM-CSF and chemokines (such as CCL1, CCL2, CCL3, etc.), recruit and activate other effector immune cells, etc.
Figure 3. How NK cells kill tumor cells
Compared with CAR-T therapy, CAR-NK therapy has the following advantages:
1)CAR-NK cells are widely sourced (Figure 4), and can be obtained from a variety of clinical samples or commercial cell lines, including NK92 cell lines, PBMC and iPSC, and CAR-NK cells can be used as "off-the-shelf" cell therapy products [6].
1)CAR-NK cells are widely sourced (Figure 4), and can be obtained from a variety of clinical samples or commercial cell lines, including NK92 cell lines, PBMC and iPSC, and CAR-NK cells can be used as "off-the-shelf" cell therapy products [6].
Figure 4 Comparison of the origin of NK cells and T cells
2) CAR-NK cells have a limited lifetime in circulation, so the risk of targeting/detumorization toxicity to normal tissues is relatively low;
3) Low risk of CRS after treatment with CAR-NK cells: Unlike CAR T cells, which continue to expand in vivo and produce pro-inflammatory factors such as IL6 and IL1β, NK cells mainly secrete IFN-γ and GM-CSF, and do not expand in vivo for several weeks, thus reducing the risk of CRS.
4) CAR-NK cells do not require rigorous HLA gene matching: Allogeneic NK cells do not express individual specific TCR, and the potential risk of causing graft-versus-host disease (GVHD) is much lower than that of CAR-T cell therapy;
5)NK cells kill tumor cells in a variety of ways. Car-modified NK cells can not only eliminate antigen-specific tumors in a CAR-dependent manner, but also effectively eradicate some heterogeneous tumors that do not express CAR-targeted antigens through a receptor-dependent mechanism of NK cells.
Table 1 Comparison of CAR-T and CAR-NK (Table source: Southwest Securities)
四、CAR-M therapy
Macrophages (M) have high heterogeneity, high plasticity, phagocytosis, antigen presentation and infiltration. Attempts by macrophages to fight cancer began decades ago, but efficacy uations of early clinical trials have shown that macrophage transplantation therapy has little effect, and macrophages need additional signals to guide their fight against tumors. Therefore, with the development potential and preparation process of CAR-T/NK as a reference template, researchers began to study the use of monocytes/macrophages as carrier cells for cellular immunotherapy, combined with the current booming chimeric antigen receptor technology and the increasingly mature gene engineering optimization technology, to transform monocytes/macrophages at the molecular level. Chimeric antigen receptor-macrophage (CAR-M) was prepared, and its phagocytosis and antigen presenting ability were enhanced by its high infiltrative ability, so as to be applied in the field of tumor. CAR-M is expected to become a new generation of protagonists in cellular immunotherapy.
Figure 5. Schematic diagram of macrophage killing tumor
Unlike CAR-T, CAR-M has three main advantages:
1) Firstly, macrophages can easily invade tumor microenvironment (TME) and phagocytic tumor cells, affect the proportion of tument-associated macrophages (TAM) in TME, and change the phenotype of TAM (from M2 to M1);
2) Secondly, macrophages can also up-regulate the expression of antigen-presenting mechanism related molecules, such as histocompatibility complex Class II (MHC-II), CD80 and CD86, which can act as antigen-presenting cells and activate adaptive immune response by cross-presenting phagocytosis of antigens;
3) Finally, the cycle of CAR-M has a time limit, and the non-tumor targeting toxicity is small.
CAR-M was first reported in 2020, and the vast majority of research is still in the scientific stage, with industrialization in its early stages. Although theoretically, CAR-M therapy has obvious advantages for solid tumors, it is still in its "infancy", and many difficulties still need to be overcome in order to truly join the clinical application, such as:
1) Source and amplification. Unlike CAR-T/NK, T cells and NK cells can be extracted from patients and expanded in large numbers in the laboratory, but macrophages are hardly expanded in vitro, and obtaining large numbers is a major challenge. However, immortalized or tumor cell lines do not represent the properties of primary cells well, and the study of CAR-M provides limited benefits.
2) Macrophages are naturally resistant to commonly used viral vectors. Macrophages are the body's first line of defense against cancer cells and viruses, and thus may develop resistance to commonly used standard viral vectors, resulting in the inability of viral vectors to efficiently infect macrophages for CAR-M preparation, which is one of the major challenges limiting the development of CAR-M therapies. Moreover, the transduced CAR-M needs positive sorting in order to better validate the downstream anti-tumor function.
At present, Shenzhen Cell Valley has made major breakthroughs in the preparation of CAR-NK and CAR-M, and we welcome predecessors in the field of CGT to exchange and study together and come to guide our work.
References:
[1] Zhao Yuan, Hu Wanli, Zhang Liansheng. Research progress and application prospect of chimeric antigen receptor therapy in hematologic tumor immunotherapy [J/CD]. Chinese Journal of Clinicians: Electronic Edition, 2014,8 (6) : 1158-1161.
[2]Chen YJ, Abila B, Mostafa Kamel Y. CAR-T: What Is Next? Cancers (Basel). 2023 Jan 21; 15 (3) : 663.
[3] Li He, Ren Jiahui, Shi Yinghui et al. Advances in the traditional field of CAR-T cell therapy [J]. China Medical Guide,2022,24(10):966-973. (in Chinese)
[4] Dang Kechun, Wang Jinwei. Challenges and strategies of CAR-T cells in the treatment of solid tumors [J]. Pharmaceutical and Clinical Research, 2019,30(06):529-535. (in Chinese)
[5] Liu Hongxiu, Han Yanqiu. Research progress of CAR-NK cells in the treatment of hematologic tumors [J]. Journal of Clinical Hematology, 2019, 36(1) : 76-80.
[6]Lu H, Zhao X, Li Z, Hu Y, Wang H. From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies. Front Oncol. 2021 Aug 6; 7 20501.
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
3) Low risk of CRS after treatment with CAR-NK cells: Unlike CAR T cells, which continue to expand in vivo and produce pro-inflammatory factors such as IL6 and IL1β, NK cells mainly secrete IFN-γ and GM-CSF, and do not expand in vivo for several weeks, thus reducing the risk of CRS.
4) CAR-NK cells do not require rigorous HLA gene matching: Allogeneic NK cells do not express individual specific TCR, and the potential risk of causing graft-versus-host disease (GVHD) is much lower than that of CAR-T cell therapy;
5)NK cells kill tumor cells in a variety of ways. Car-modified NK cells can not only eliminate antigen-specific tumors in a CAR-dependent manner, but also effectively eradicate some heterogeneous tumors that do not express CAR-targeted antigens through a receptor-dependent mechanism of NK cells.
| CAR-T | CAR-NK | |
| Main cell source | PBMC；Autologous or allogeneic cells | Peripheral blood NK cells；Cord blood-derived NK cells；NK-92 cell line；Pluripotent stem cell-derived NK cells |
| Transduction efficiency | High | Low |
| In vivo persistence | Long-term presence in the body | Disappears rapidly after mediating antitumor effects |
| Introducing a suicide gene | Need | Not needed in most cases |
| Identify tumor patterns | CAR dependence | CAR dependence and non-CAR dependence |
| Side effect | Allogeneic T cells may cause GVHD；GRS； | Usually manifests as controllable immune side effects, such as fever |
| Convenience | Poor：HLA matching is necessary | Better: No HLA matching required, cells come from a wider range of sources |
| Can it be mass produced? | NO | YES |
Table 1 Comparison of CAR-T and CAR-NK (Table source: Southwest Securities)
四、CAR-M therapy
Macrophages (M) have high heterogeneity, high plasticity, phagocytosis, antigen presentation and infiltration. Attempts by macrophages to fight cancer began decades ago, but efficacy uations of early clinical trials have shown that macrophage transplantation therapy has little effect, and macrophages need additional signals to guide their fight against tumors. Therefore, with the development potential and preparation process of CAR-T/NK as a reference template, researchers began to study the use of monocytes/macrophages as carrier cells for cellular immunotherapy, combined with the current booming chimeric antigen receptor technology and the increasingly mature gene engineering optimization technology, to transform monocytes/macrophages at the molecular level. Chimeric antigen receptor-macrophage (CAR-M) was prepared, and its phagocytosis and antigen presenting ability were enhanced by its high infiltrative ability, so as to be applied in the field of tumor. CAR-M is expected to become a new generation of protagonists in cellular immunotherapy.
Figure 5. Schematic diagram of macrophage killing tumor
Unlike CAR-T, CAR-M has three main advantages:
1) Firstly, macrophages can easily invade tumor microenvironment (TME) and phagocytic tumor cells, affect the proportion of tument-associated macrophages (TAM) in TME, and change the phenotype of TAM (from M2 to M1);
2) Secondly, macrophages can also up-regulate the expression of antigen-presenting mechanism related molecules, such as histocompatibility complex Class II (MHC-II), CD80 and CD86, which can act as antigen-presenting cells and activate adaptive immune response by cross-presenting phagocytosis of antigens;
3) Finally, the cycle of CAR-M has a time limit, and the non-tumor targeting toxicity is small.
CAR-M was first reported in 2020, and the vast majority of research is still in the scientific stage, with industrialization in its early stages. Although theoretically, CAR-M therapy has obvious advantages for solid tumors, it is still in its "infancy", and many difficulties still need to be overcome in order to truly join the clinical application, such as:
1) Source and amplification. Unlike CAR-T/NK, T cells and NK cells can be extracted from patients and expanded in large numbers in the laboratory, but macrophages are hardly expanded in vitro, and obtaining large numbers is a major challenge. However, immortalized or tumor cell lines do not represent the properties of primary cells well, and the study of CAR-M provides limited benefits.
2) Macrophages are naturally resistant to commonly used viral vectors. Macrophages are the body's first line of defense against cancer cells and viruses, and thus may develop resistance to commonly used standard viral vectors, resulting in the inability of viral vectors to efficiently infect macrophages for CAR-M preparation, which is one of the major challenges limiting the development of CAR-M therapies. Moreover, the transduced CAR-M needs positive sorting in order to better validate the downstream anti-tumor function.
At present, Shenzhen Cell Valley has made major breakthroughs in the preparation of CAR-NK and CAR-M, and we welcome predecessors in the field of CGT to exchange and study together and come to guide our work.
References:
[1] Zhao Yuan, Hu Wanli, Zhang Liansheng. Research progress and application prospect of chimeric antigen receptor therapy in hematologic tumor immunotherapy [J/CD]. Chinese Journal of Clinicians: Electronic Edition, 2014,8 (6) : 1158-1161.
[2]Chen YJ, Abila B, Mostafa Kamel Y. CAR-T: What Is Next? Cancers (Basel). 2023 Jan 21; 15 (3) : 663.
[3] Li He, Ren Jiahui, Shi Yinghui et al. Advances in the traditional field of CAR-T cell therapy [J]. China Medical Guide,2022,24(10):966-973. (in Chinese)
[4] Dang Kechun, Wang Jinwei. Challenges and strategies of CAR-T cells in the treatment of solid tumors [J]. Pharmaceutical and Clinical Research, 2019,30(06):529-535. (in Chinese)
[5] Liu Hongxiu, Han Yanqiu. Research progress of CAR-NK cells in the treatment of hematologic tumors [J]. Journal of Clinical Hematology, 2019, 36(1) : 76-80.
[6]Lu H, Zhao X, Li Z, Hu Y, Wang H. From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies. Front Oncol. 2021 Aug 6; 7 20501.
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
