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Cited by 35CAR-T cell therapy researchImmune Cell Function and InteractionMonoclonal and Polyclonal Antibodies Research

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Single VHH-directed BCMA CAR-NK cells for multiple myeloma

Open access
Ren, Quan, Zu, Yingling, Su, Hongchang, Lu, Qiumei, Xiang, Bin, Luo, Yanping, Zhang, Jishuai, Song, Yongping·2023·Experimental Hematology & Oncology
DOI 10.1186/s40164-023-00461-8PMC PMC10680242
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Abstract

Natural killer (NK) cells are promising alternatives for the production of “off-the-shelf” CAR products, posing a lower risk of cytokine release syndrome (CRS) than CAR-T cells. We synthesized four single VHH-directed anti-BCMA CARs, incorporating various intracellular regions (2B4 versus CD28) and hinge domains (CD28 versus IgG1) and ectopically producing IL-15. NK cells derived from peripheral blood (PB) were expanded ex vivo by K562-mbIL21 feeder cells. Stable CAR transduction was obtained through lentiviral transduction with the BaEV-Rless pseudotyped lentiviral vector. BCMA-CD28-IL15 CAR-NK cells with ectopic expression of IL-15 exhibited superior cytotoxicity were compared to BCMA-CD28 CAR-NK cells lacking IL-15 and BCMA-hIgG1-IL15 CAR-NK cells with an IgG1 hinge domain. We further assessed the cytotoxic capabilities of BCMA-2B4-IL15 CAR-NK cells with 2B4 intracellular domain. The BCMA-CD28-IL15 CAR-NK cells revealed stronger cytotoxicity and higher cytokine secretion against BCMA+ tumor cells than BCMA-2B4-IL15 CAR-NK cells in vitro. In the MM.1S-Luc mouse model, BCMA-CD28-IL15 CAR-NK inhibited the growth of tumor cells and prolonged mouse survival. These results show that the single VHH-directed BCMA CAR-NK cells exhibited remarkable specific killing ability, making them a potential candidate for immunotherapy in multiple myeloma treatment.

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Fig. 1 BCMA-CD28-IL15-engineered NK cells showed enhanced antitumor activity compared to BCMA-CD28 CAR-NK cells. (A) Schematic diagrams of BCMA CAR-NK constructs. (B) The fold expansion curve of NK cells, BCMA-CD28-IL15 CAR-NK cells, and BCMA-CD28 CAR-NK cells. (C) BCMA CAR expression of NK cells on day 14. (D) The cytotoxic activity of BCMA-CD28-IL15 CAR-NK cells vs. BCMA-CD28 CAR-NK cells and ex vivo-expanded NK cells against MM.1S and Daudi cells using a lactate dehydrogenase release assay (n = 3; ****, P < 0.0001). The numbers of effector cells were calculated as CAR-positive cells. NK cells were used to adjust the different CAR-positive cells. (E) MM.1S and Daudi cells were cocultured with BCMA-CD28-IL15 CAR-NK cells, BCMA-CD28 CAR-NK cells, or NK cells at an E:T ratio of 1:2 for 24 h. IFN-γ, granzyme B, and IL-15 secretion in supernatants was measured by ELISA (n = 3; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001)
Fig. 1 BCMA-CD28-IL15-engineered NK cells showed enhanced antitumor activity compared to BCMA-CD28 CAR-NK cells. (A) Schematic diagrams of BCMA CAR-NK constructs. (B) The fold expansion curve of NK cells, BCMA-CD28-IL15 CAR-NK cells, and BCMA-CD28 CAR-NK cells. (C) BCMA CAR expression of NK cells on day 14. (D) The cytotoxic activity of BCMA-CD28-IL15 CAR-NK cells vs. BCMA-CD28 CAR-NK cells and ex vivo-expanded NK cells against MM.1S and Daudi cells using a lactate dehydrogenase release assay (n = 3; ****, P < 0.0001). The numbers of effector cells were calculated as CAR-positive cells. NK cells were used to adjust the different CAR-positive cells. (E) MM.1S and Daudi cells were cocultured with BCMA-CD28-IL15 CAR-NK cells, BCMA-CD28 CAR-NK cells, or NK cells at an E:T ratio of 1:2 for 24 h. IFN-γ, granzyme B, and IL-15 secretion in supernatants was measured by ELISA (n = 3; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001)CC BY · PubMed Central
Fig. 2 In vivo antitumor activity of BCMA-CD28-IL15 CAR-NK cells in the MM.1S-Luc transplanted NCG mouse model. (A) Schematic diagram of the mouse in vivo study. The NCG mice received an IV injection of 2.0 × 106 MM.1S-Luc cells on day 0. Nine days after tumor inoculation, the mice were randomly divided into three groups (n = 3 for the control and mock-NK groups; n = 4 for the BCMA-CD28-IL15 CAR-NK group) according to the average radiance of the bioluminescence imaging. The mice were intravenously administered cryoprotectant (a solvent control), mock-NK cells, and BCMA-CD28-IL15 CAR-NK cells on day 9. (B) Bioluminescence images on days 9, 13, 20, 27, and 34. (C) Statistical analysis of the bioluminescence intensity on different days (****, P < 0.001; ns, no significance). (D) Body weight of each group measured on different days (****, P < 0.0001; ns, no significance). (E) Kaplan–Meier survival curves of mice in vivo. A statistical analysis of survival between groups was performed using the log-rank test. Statistical significance in survival rates was obtained after Bonferroni correction for multiple comparisons (*, P < 0.05)
Fig. 2 In vivo antitumor activity of BCMA-CD28-IL15 CAR-NK cells in the MM.1S-Luc transplanted NCG mouse model. (A) Schematic diagram of the mouse in vivo study. The NCG mice received an IV injection of 2.0 × 106 MM.1S-Luc cells on day 0. Nine days after tumor inoculation, the mice were randomly divided into three groups (n = 3 for the control and mock-NK groups; n = 4 for the BCMA-CD28-IL15 CAR-NK group) according to the average radiance of the bioluminescence imaging. The mice were intravenously administered cryoprotectant (a solvent control), mock-NK cells, and BCMA-CD28-IL15 CAR-NK cells on day 9. (B) Bioluminescence images on days 9, 13, 20, 27, and 34. (C) Statistical analysis of the bioluminescence intensity on different days (****, P < 0.001; ns, no significance). (D) Body weight of each group measured on different days (****, P < 0.0001; ns, no significance). (E) Kaplan–Meier survival curves of mice in vivo. A statistical analysis of survival between groups was performed using the log-rank test. Statistical significance in survival rates was obtained after Bonferroni correction for multiple comparisons (*, P < 0.05)CC BY · PubMed Central

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Research works printed but not linked: 10

  1. Zhang Y, Zhou W, Yang J, Yang J, Wang W. Chimeric antigen receptor engineered natural killer cells for cancer therapy. Exp Hematol Oncol. 2023;12:70. · DOI
  2. Mazinani M, Rahbarizadeh F. New cell sources for CAR-based immunotherapy. Biomarker Res. 2023;11:49. · DOI
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  4. Han L, Zhang J-S, Zhou J, Zhou K-S, Xu B-L, Li L-L, et al. Single VHH-directed BCMA CAR-T cells cause remission of relapsed/refractory Multiple Myeloma. Leukemia. 2021;35(10):3002–6. · DOI
  5. Colamartino ABL, Lemieux W, Bifsha P, Nicoletti S, Chakravarti N, Sanz J, et al. Efficient and robust NK-Cell Transduction with Baboon Envelope Pseudotyped Lentivector. Front Immunol. 2019;10:2873. · DOI
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  9. Xu Y, Liu Q, Zhong M, Wang Z, Chen Z, Zhang Y, et al. 2B4 costimulatory domain enhancing cytotoxic ability of anti-CD5 chimeric antigen receptor engineered natural killer cells against T cell malignancies. J Hematol Oncol. 2019;12(1):49. · DOI
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Metadata

Authors
Ren, Q., Zu, Y., Su, H., Lu, Q., Xiang, B., Luo, Y., Zhang, J., & Song, Y.
Affiliations
  • Zhengzhou University (CN)
  • Shenzhen Pregene Biopharma Co., Ltd. (China) (CN)
Corresponding author
Song, Yongping
ORCID
Venue
Experimental Hematology & Oncology
Publisher
Springer Science and Business Media LLC
Published
2023-11-27
Volume
12
Issue
1
Pages
98-98
ISSN
2162-3619 (ISSN-L)
DOI
10.1186/s40164-023-00461-8
PMC
PMC10680242
OpenAlex
W4389049606
Cited by
35
References
2
Citations by year
2026: 8 · 2025: 21 · 2024: 6
FWCI
4.25
Type
journal article
Language
en
Open access
Licence
Locations
  • Experimental Hematology and Oncology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Zhengzhou University
Topics
  • Health Sciences › Medicine › Oncology › CAR-T cell therapy research
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Cell Function and Interaction
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
Keywords
BCMA, multiple myeloma, CAR-NK cells, natural killer cells, CD28, 2B4, IL-15, hinge domain, lentiviral transduction, cytokine release syndrome

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