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■ Author|Gu Yu■ On-duty editor|Xingyuanzhixi is different from CAR-T cells.
NK cells have a short life cycle and limited expansion in the body, which improves their safety.
Even the adoptive treatment of allogeneic cells is relatively safe.
There is almost no CRS and neurotoxicity, so that NK cells can be made into off-the-shelf products, increasing the accessibility of cell therapy
.
CAR-T currently uses autologous cell transplantation.
In addition to autologous NK cells, CAR-NK can also use NK cell lines, stem cells to differentiate NK, and cord blood NK cells
.
Peripheral blood NK cells (PB-NK) NK cells can be separated relatively easily from the patient itself (autologous PB-NK) or healthy donors (allogeneic PB-NK), so most (31/33) preclinical CAR- NK research uses PB-NK
.
For cancer immunotherapy, autologous NK cells are usually not very effective because they are functionally silenced when they encounter their own MHC antigens
.
In addition, the function of patient-derived NK cells is often impaired by underlying diseases or preventive treatments
.
Therefore, allogeneic PB-NK is usually the first choice for immunotherapy, but T lymphocytes in the product need to be carefully removed because these cells can induce GVHD
.
Due to the low number of NK cells in peripheral blood, NK cells are routinely expanded after isolation
.
The treatment of NK cells is usually infused at 105-108 cells/kg, so up to 1010 NK cells are needed for a patient weighing 100kg
.
The main advantage of PB-NK cells for cancer immunotherapy is that the cells are mature and do not need to undergo a long period of differentiation
.
On the other hand, the transduction efficiency of PB-NK is relatively low, and prolonging the culture time often leads to shortening and depletion of telomerase, resulting in reduced cytotoxicity
.
One possible limitation of cord blood-derived NK cells (UCB-NK) PB-NK cells is the dependence on the availability of healthy donors
.
Newly diagnosed patients often have their own NK cells that do not meet the needs of clinical treatment.
The cell viability and cytotoxicity of mature PB-NK cells cryopreserved are significantly reduced, which limits the clinical use of CAR-NK
.
Umbilical cord blood is a relatively stable source of NK cells
.
Generally speaking, due to the high proliferation ability of these cells, only 10% of a cord blood unit can produce a pure cell pool of almost more than 109 NK cells in two weeks, which is usually suitable for one treatment cycle
.
Another advantage of UCB-NK cells is that the haplotype of the sample can be determined at the time of collection, allowing the generation of a cell bank, and NK cells that do not match HLA can be selected as needed
.
In a clinical study conducted by MD Anderson Cancer Center (NCT03056339), umbilical cord blood-derived CD19 CAR-NK cells treated 11 cases of relapsed or refractory hematological tumors, 8 cases (73%) responded, and 7 cases reached CR (Reference 2)
.
The shortcomings of stem cell differentiation NK cells PB-NK and UCB-NK as the cell source for CAR-NK development include the natural heterogeneity between donors, resulting in differences in the performance of NK cell products
.
Therefore, the expansion of stem cell-derived NK cells is an attractive alternative to the development of standardized therapeutic products
.
It has proved feasible to produce clinical therapeutic scale NK cells from human embryonic stem cells (hESC) or induced pluripotent stem cells (induced PSC)
.
The commonly used hESC/iPSC cells are usually preferable to using stem cells from bone marrow biopsy, G-CSF mobilization, or human embryos
.
In addition to the ethical issues associated with the use of these source cells, the results of NK cell induction starting from primary stem cells are difficult to predict and are often not as effective
.
It takes 3-5 weeks to generate NK cells from hESC or induced pluripotent stem cells.
NK cells derived from H9 hESC cells show a limited allogeneic immune response, while compared with UCB-NK, they show a more mature cytotoxic phenotype
.
On the other hand, iPSC-derived NK cells have been shown to have anti-tumor functions, and have recently been used to generate CAR-NK cells
.
After optimizing the activation domain for iPSC-derived NK cells, mesothelin targeting iPSC CAR-NK cells proved to be as effective as CAR-T cells
.
In the mouse xenograft model, compared with CAR-NK treatment, CAR-iPSC-NK treated tumor-bearing mice showed less pathogenic organ damage and lower IFN-γ and IL-6 levels
.
CAR-iPSC-NK (Cell Stem Cell.
2018;23(2):181–92.
) However, when iPSC CAR-NK is applied to the clinic, there are still some challenges: First, in the mouse model, it is necessary to stop exogenous administration.
After cytokines, CAR-iPSC-NK cells stop proliferating in the body
.
Systemic administration of cytokines in a clinical setting is highly undesirable because it is not only expensive, but also potentially dangerous
.
Secondly, iPSC-derived cells always have the potential for malignant transformation
.
Finally, iPSC-derived cells are potentially immunogenic, which may lead to the destruction of embryonic stem cells and even lead to adverse immune responses such as cytokine release storms
.
NK cell lines The above three sources of NK cells have a major common disadvantage: obtaining a large number of NK cells is relatively cumbersome and time-consuming
.
In general, cell lines can bypass most of these problems because they are easy to maintain and expand
.
Therefore, it is not surprising that 72 experimental studies have reported the use of NK cell lines to develop CAR-NK cells
.
More than 80% of the studies used the NK-92 cell line derived from lymphoma
.
Among the six existing NK cell lines, NK-92 has high anti-tumor activity and direct cytotoxicity
.
When NK-92 cells are transfected with CD16, the cell line can also trigger antibody-dependent cell-mediated cytotoxicity (ADCC)
.
For CAR-NK cell development, NK-92 cells have another major advantage: the cell line can also be easily genetically modified using non-viral methods, including electroporation
.
So far, NK-92 pre-clinical research has involved AML, lymphoma (CD19), myeloma (CS1), prostate cancer (EpCAM), prostate cancer (CAR, breast cancer (Her2), GD2), neuroblasts Tumor (GD2), Glioblastoma (EGFR) and Ovarian Cancer (Mesothelin)
.
One of the main disadvantages of the NK-92 cell line is that these cells are of tumor origin and are aneuploid
.
Therefore, they need to be irradiated before infusion to prevent proliferation in the body and limit their lifespan
.
In the phase I trial, the administration of NK-92 proved to be safe, with only mild reactions
.
The head-to-head comparison of primary CAR-NK cells and NK-92 cells is limited
.
In a study using CdD23 to target CAR, it was shown that primary NK cells are not as efficient as NK-92 cells in eradicating AML cells in vitro (Reference 5)
.
This may be due to the low surface expression level of CAR in primary NK cells
.
However, due to the ability of NK-92 cells to replicate, whether the irradiated CAR-NK-92 cells are better than the primary CAR-NK cells in terms of overall patient survival rate still needs to wait for future clinical results
.
The current clinical trials of NK-92 cell line are: NCT03383978, NCT02839954, NCT02742727, NCT02892695, NCT03940833, NCT03941457, NCT02944162, NCT04052061, NCT04050709
.
The editor concludes that CAR-NK has the potential to become an off-the-shelf cell therapy product and has received more and more attention.
Currently, more than 20 clinical trials have begun
.
Understanding the four sources of CAR-NK cells, as well as the advantages and disadvantages of each source, will help you choose the right NK cell raw materials and design and produce products that meet the purpose of treatment
.
Four sources of CAR-NK and their advantages and disadvantages (Clin Transl Immunology.
2021 Apr 28;10(4):e1274.
) Main references Daher M, Melo Garcia L, Li Y, Rezvani K.
CAR-NK cells: the next wave of cellular therapy for cancer.
Clin Transl Immunology.
2021 Apr 28;10(4):e1274.
Liu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, Nassif Kerbauy L, Overman B, Thall P, Kaplan M, et al.
Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors.
N Engl J Med.
2020;382(6):545–53Li Y, Hermanson DL, Moriarity BS, Kaufman DS.
Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity.
Cell Stem Cell.
2018;23(2):181–92.
Lu H, Zhao X, Li Z, Hu Y and Wang H (2021) From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies.
Front.
Oncol.
11:720501.
doi: 10.
3389/fonc.
2021.
720501Kloess S, Oberschmidt O,Dahlke J, Vu XK, Neudoerf C, Kloos A, Gardlowski T, Matthies N, Heuser M, Meyer J, et al.
Preclinical assessment of suitable natural killer cell sources for chimeric antigen receptor natural killer-based “Of-the-Shelf” acute myeloid leukemia immunotherapies.
Hum Gene Ther.
2019;30(4):381–401
NK cells have a short life cycle and limited expansion in the body, which improves their safety.
Even the adoptive treatment of allogeneic cells is relatively safe.
There is almost no CRS and neurotoxicity, so that NK cells can be made into off-the-shelf products, increasing the accessibility of cell therapy
.
CAR-T currently uses autologous cell transplantation.
In addition to autologous NK cells, CAR-NK can also use NK cell lines, stem cells to differentiate NK, and cord blood NK cells
.
Peripheral blood NK cells (PB-NK) NK cells can be separated relatively easily from the patient itself (autologous PB-NK) or healthy donors (allogeneic PB-NK), so most (31/33) preclinical CAR- NK research uses PB-NK
.
For cancer immunotherapy, autologous NK cells are usually not very effective because they are functionally silenced when they encounter their own MHC antigens
.
In addition, the function of patient-derived NK cells is often impaired by underlying diseases or preventive treatments
.
Therefore, allogeneic PB-NK is usually the first choice for immunotherapy, but T lymphocytes in the product need to be carefully removed because these cells can induce GVHD
.
Due to the low number of NK cells in peripheral blood, NK cells are routinely expanded after isolation
.
The treatment of NK cells is usually infused at 105-108 cells/kg, so up to 1010 NK cells are needed for a patient weighing 100kg
.
The main advantage of PB-NK cells for cancer immunotherapy is that the cells are mature and do not need to undergo a long period of differentiation
.
On the other hand, the transduction efficiency of PB-NK is relatively low, and prolonging the culture time often leads to shortening and depletion of telomerase, resulting in reduced cytotoxicity
.
One possible limitation of cord blood-derived NK cells (UCB-NK) PB-NK cells is the dependence on the availability of healthy donors
.
Newly diagnosed patients often have their own NK cells that do not meet the needs of clinical treatment.
The cell viability and cytotoxicity of mature PB-NK cells cryopreserved are significantly reduced, which limits the clinical use of CAR-NK
.
Umbilical cord blood is a relatively stable source of NK cells
.
Generally speaking, due to the high proliferation ability of these cells, only 10% of a cord blood unit can produce a pure cell pool of almost more than 109 NK cells in two weeks, which is usually suitable for one treatment cycle
.
Another advantage of UCB-NK cells is that the haplotype of the sample can be determined at the time of collection, allowing the generation of a cell bank, and NK cells that do not match HLA can be selected as needed
.
In a clinical study conducted by MD Anderson Cancer Center (NCT03056339), umbilical cord blood-derived CD19 CAR-NK cells treated 11 cases of relapsed or refractory hematological tumors, 8 cases (73%) responded, and 7 cases reached CR (Reference 2)
.
The shortcomings of stem cell differentiation NK cells PB-NK and UCB-NK as the cell source for CAR-NK development include the natural heterogeneity between donors, resulting in differences in the performance of NK cell products
.
Therefore, the expansion of stem cell-derived NK cells is an attractive alternative to the development of standardized therapeutic products
.
It has proved feasible to produce clinical therapeutic scale NK cells from human embryonic stem cells (hESC) or induced pluripotent stem cells (induced PSC)
.
The commonly used hESC/iPSC cells are usually preferable to using stem cells from bone marrow biopsy, G-CSF mobilization, or human embryos
.
In addition to the ethical issues associated with the use of these source cells, the results of NK cell induction starting from primary stem cells are difficult to predict and are often not as effective
.
It takes 3-5 weeks to generate NK cells from hESC or induced pluripotent stem cells.
NK cells derived from H9 hESC cells show a limited allogeneic immune response, while compared with UCB-NK, they show a more mature cytotoxic phenotype
.
On the other hand, iPSC-derived NK cells have been shown to have anti-tumor functions, and have recently been used to generate CAR-NK cells
.
After optimizing the activation domain for iPSC-derived NK cells, mesothelin targeting iPSC CAR-NK cells proved to be as effective as CAR-T cells
.
In the mouse xenograft model, compared with CAR-NK treatment, CAR-iPSC-NK treated tumor-bearing mice showed less pathogenic organ damage and lower IFN-γ and IL-6 levels
.
CAR-iPSC-NK (Cell Stem Cell.
2018;23(2):181–92.
) However, when iPSC CAR-NK is applied to the clinic, there are still some challenges: First, in the mouse model, it is necessary to stop exogenous administration.
After cytokines, CAR-iPSC-NK cells stop proliferating in the body
.
Systemic administration of cytokines in a clinical setting is highly undesirable because it is not only expensive, but also potentially dangerous
.
Secondly, iPSC-derived cells always have the potential for malignant transformation
.
Finally, iPSC-derived cells are potentially immunogenic, which may lead to the destruction of embryonic stem cells and even lead to adverse immune responses such as cytokine release storms
.
NK cell lines The above three sources of NK cells have a major common disadvantage: obtaining a large number of NK cells is relatively cumbersome and time-consuming
.
In general, cell lines can bypass most of these problems because they are easy to maintain and expand
.
Therefore, it is not surprising that 72 experimental studies have reported the use of NK cell lines to develop CAR-NK cells
.
More than 80% of the studies used the NK-92 cell line derived from lymphoma
.
Among the six existing NK cell lines, NK-92 has high anti-tumor activity and direct cytotoxicity
.
When NK-92 cells are transfected with CD16, the cell line can also trigger antibody-dependent cell-mediated cytotoxicity (ADCC)
.
For CAR-NK cell development, NK-92 cells have another major advantage: the cell line can also be easily genetically modified using non-viral methods, including electroporation
.
So far, NK-92 pre-clinical research has involved AML, lymphoma (CD19), myeloma (CS1), prostate cancer (EpCAM), prostate cancer (CAR, breast cancer (Her2), GD2), neuroblasts Tumor (GD2), Glioblastoma (EGFR) and Ovarian Cancer (Mesothelin)
.
One of the main disadvantages of the NK-92 cell line is that these cells are of tumor origin and are aneuploid
.
Therefore, they need to be irradiated before infusion to prevent proliferation in the body and limit their lifespan
.
In the phase I trial, the administration of NK-92 proved to be safe, with only mild reactions
.
The head-to-head comparison of primary CAR-NK cells and NK-92 cells is limited
.
In a study using CdD23 to target CAR, it was shown that primary NK cells are not as efficient as NK-92 cells in eradicating AML cells in vitro (Reference 5)
.
This may be due to the low surface expression level of CAR in primary NK cells
.
However, due to the ability of NK-92 cells to replicate, whether the irradiated CAR-NK-92 cells are better than the primary CAR-NK cells in terms of overall patient survival rate still needs to wait for future clinical results
.
The current clinical trials of NK-92 cell line are: NCT03383978, NCT02839954, NCT02742727, NCT02892695, NCT03940833, NCT03941457, NCT02944162, NCT04052061, NCT04050709
.
The editor concludes that CAR-NK has the potential to become an off-the-shelf cell therapy product and has received more and more attention.
Currently, more than 20 clinical trials have begun
.
Understanding the four sources of CAR-NK cells, as well as the advantages and disadvantages of each source, will help you choose the right NK cell raw materials and design and produce products that meet the purpose of treatment
.
Four sources of CAR-NK and their advantages and disadvantages (Clin Transl Immunology.
2021 Apr 28;10(4):e1274.
) Main references Daher M, Melo Garcia L, Li Y, Rezvani K.
CAR-NK cells: the next wave of cellular therapy for cancer.
Clin Transl Immunology.
2021 Apr 28;10(4):e1274.
Liu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, Nassif Kerbauy L, Overman B, Thall P, Kaplan M, et al.
Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors.
N Engl J Med.
2020;382(6):545–53Li Y, Hermanson DL, Moriarity BS, Kaufman DS.
Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity.
Cell Stem Cell.
2018;23(2):181–92.
Lu H, Zhao X, Li Z, Hu Y and Wang H (2021) From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies.
Front.
Oncol.
11:720501.
doi: 10.
3389/fonc.
2021.
720501Kloess S, Oberschmidt O,Dahlke J, Vu XK, Neudoerf C, Kloos A, Gardlowski T, Matthies N, Heuser M, Meyer J, et al.
Preclinical assessment of suitable natural killer cell sources for chimeric antigen receptor natural killer-based “Of-the-Shelf” acute myeloid leukemia immunotherapies.
Hum Gene Ther.
2019;30(4):381–401
