Abstract
Chimeric antigen receptors (CARs) offer a promising new approach for targeting B cell malignancies through the immune system. Despite the proven effectiveness of CAR T cells targeting CD19 and CD22 in hematological malignancies, it is imperative to note that their production remains a highly complex process. Unlike T cells, NK cells eliminate targets in a non-antigen-specific manner while avoiding graft vs. host disease (GvHD). CAR-NK cells are considered safer than CAR-T cells because they have a shorter lifespan and produce less toxic cytokines. Due to their unlimited ability to proliferate in vitro, NK-92 cells can be used as a source for CAR-engineered NK cells. We found that CARs created from the m971 anti-CD22 mAb, which specifically targets a proximal CD22 epitope, were more effective at anti-leukemic activity compared to those made with other binding domains. To further enhance the anti-leukemic capacity of NK cells, we used lentiviral transduction to generate the m971-CD28-CD3ζ NK-92. CD22 is highly expressed in B cell lymphoma. To evaluate the potential of targeting CD22, Raji cells were selected as CD22-positive cells. Our study aimed to investigate CD22 as a potential target for CAR-NK-92 therapy in the treatment of B cell lymphoma. We first generated m971-CD28-CD3ζ NK-92 that expressed a CAR for binding CD22 in vitro. Flow cytometric analysis was used to evaluate the expression of CAR. The 7AAD determined the cytotoxicity of the m971-CD28-CD3ζ NK-92 towards target lymphoma cell lines by flow cytometry assay. The ELISA assay evaluated cytokine production in CAR NK-92 cells in response to target cells. The m971-CD28-CD3ζ NK-92 cells have successfully expressed the CD22-specific CAR. m971-CD28-CD3ζ NK-92 cells efficiently lysed CD22-expressing lymphoma cell lines and produced large amounts of cytokines such as IFN-γ and GM-CSF but a lower level of IL-6 after coculturing with target cells. Based on our results, it is evident that transferring m971-CD28-CD3ζ NK-92 cells could be a promising immunotherapy for B cell lymphoma.
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Abbreviations
- CARs :
-
Chimeric antigen receptors
- GvHD :
-
Graft vs. host disease
- scFv :
-
Single-chain variable fragment
- NK :
-
Natural killer
- CRS :
-
Cytokine release syndrome
- GM-CSF :
-
Granulocyte-macrophage colony-stimulating factor
- NCBI :
-
National Cell Bank of Iran
- HEK293T :
-
Human embryonic kidney cells
- IBRC :
-
Iranian Biological Resource Center
- DMEM :
-
Dulbeccoʼs modified Eagleʼs medium
- FBS :
-
Fetal bovine serum
- PCR :
-
Polymerase chain reaction
- MOI :
-
Multiplicity of infection
- ELISA :
-
Enzyme-linked immunosorbent assay
- TH-1 :
-
T helper-1
- TNFα :
-
Tumor necrosis factor α
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We would like to acknowledge our colleagues for their helpful assistance in this study.
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MS and MHM designed and contributed to the concept of the experiments; MAD performed experiments and collected data; MSZ discussed the results and strategy and interpretation of data; MS and MHM supervised, directed, and managed the study; All authors read and approved the final manuscript.
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This project was approved by Ethical Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.RETECH.REC.1402.158) (Approval date: 11/06/2023).
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11626_2024_895_MOESM2_ESM.tif
Supplementary file2 Fig. S2: the effect of CD22-CAR NK-92 cells on the CFSE-labeled target cells by a fluorescence microscope. A: the left one showed CFSE-labeled Raji cells alone. The right one showed CFSE-labeled Raji cells were co-incubated with CD22-CAR NK-92 cells for 24 h at E: T ratios of 10:1. B: the left one showed CFSE-labeled K562 cells alone. The right one showed CFSE-labeled K562 cells were co-incubated with CD22-CAR NK-92 cells for 24 h at E: T ratios of 10:1. (TIF 8566 KB)
11626_2024_895_MOESM3_ESM.tif
Supplementary file3 Fig. S3: Flow cytometry plots showing the gating strategy used in cytotoxicity experiments. A: the cytotoxic activity of untransduced NK-92 cells against Raji. Each row showed different E: T ratios of 1:2, 1:1, 2:1, 5:1, and 10:1 respectively. B: CD22-CAR NK-92 -transduced cells against Raji. Each row showed different E: T ratios of 1:2, 1:1, 2:1, 5:1, and 10:1 respectively. (TIF 4010 KB)
11626_2024_895_MOESM4_ESM.tif
Supplementary file4 Fig. S4: Flow cytometry plot showing the gating strategy used in cytotoxicity experiments. A: the cytotoxic activity of untransduced NK-92 cells against K562. Each row showed different E: T ratios of 1:2, 1:1, 2:1, 5:1, and 10:1 respectively. B: CD22-CAR NK-92 -transduced cells against K562. Each row showed different E: T ratios of 1:2, 1:1, 2:1, 5:1, and 10:1 respectively. (TIF 3859 KB)
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Abbaszade Dibavar, M., Soleimani, M., Mohammadi, M.H. et al. High yield killing of lymphoma cells by anti-CD22 CAR-NK cell therapy. In Vitro Cell.Dev.Biol.-Animal 60, 321–332 (2024). https://doi.org/10.1007/s11626-024-00895-2
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DOI: https://doi.org/10.1007/s11626-024-00895-2