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Arjomandnejad M, Rahbarizadeh F. Impact of CAR Structure in Nanobody-Based Constructs on the Cytotoxic Function of CAR-T Cells Targeting CD19⁺ Cells. bloodj 2025; 22 (3) :227-237
URL: http://bloodjournal.ir/article-1-1595-en.html
Impact of CAR Structure in Nanobody-Based Constructs on the Cytotoxic Function of CAR-T Cells Targeting CD19⁺ Cells
Motahareh Arjomandnejad , Fatemeh Rahbarizadeh *
Keywords: CAR T-Cell Therapy, Single-Chain Antibodies, CD19 Antigen
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      References:
  1. Rheingold SR, Bhojwani D, Ji L, Xu X, Devidas M, Kairalla JA, et al. Determinants of survival after first relapse of acute lymphoblastic leukemia: a Children's Oncology Group study. Leukemia 2024; 38(11): 2382-94. [DOI:10.1038/s41375-024-02395-4] [PMID] []
  2. Iqbal M, Kharfan-Dabaja MA.  Relapse  of   Hodgkin lymphoma after autologous hematopoietic cell transplantation: A current management perspective. Hematol Oncol Stem Cell Ther 2021; 14(2): 95-103. [DOI:10.1016/j.hemonc.2020.05.011] [PMID]
  3. Zhou D, Zhu X, Xiao Y. CAR-T cell combination therapies in hematologic malignancies. Exp Hematology Oncol 2024; 13(1): 69. [DOI:10.1186/s40164-024-00536-0] [PMID] []
  4. Cuenca M, Peperzak V. Advances and Perspectives in the Treatment of B-Cell Malignancies. Cancers (Basel) 2021; 13(9): 2266. [DOI:10.3390/cancers13092266] [PMID] []
  5. Maleki EH, Bahrami AR, Matin MM. Cancer cell cycle heterogeneity as a critical determinant of therapeutic resistance. Genes Dis 2024; 11(1): 189-204. [DOI:10.1016/j.gendis.2022.11.025] [PMID] []
  6. Mazinani M, Rahbarizadeh F. New cell sources for CAR-based immunotherapy. Biomark Res 2023; 11(1): 49. [DOI:10.1186/s40364-023-00482-9] [PMID] []
  7. Ramírez-Chacón A, Betriu-Méndez S, Bartoló-Ibars A, González A, Martí M, Juan M. Ligand-based CAR-T cell: Different strategies to drive T cells in future new treatments. Front Immunol 2022; 13: 932559. [DOI:10.3389/fimmu.2022.932559] [PMID] []
  8. Tang L, Huang Z, Mei H, Hu Y. Immunotherapy in hematologic malignancies: achievements, challenges and future prospects. Signal Transduct Target Ther 2023; 8(1): 306. [DOI:10.1038/s41392-023-01521-5] [PMID] []
  9. Rajabzadeh A, Rahbarizadeh F, Ahmadvand D, Kabir Salmani M, Hamidieh AA. A VHH-Based Anti-MUC1 Chimeric Antigen Receptor for Specific Retargeting of Human Primary T Cells to MUC1-Positive Cancer Cells. Cell J 2021; 22(4): 502-13. [DOI:10.1186/s12860-021-00397-z] [PMID] []
  10. Nasiri F, Safarzadeh Kozani P, Rahbarizadeh F. T-cells engineered with a novel VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal efficacy to their FMC63-based counterparts. Front Immunol 2023; 14: 1063838. [DOI:10.3389/fimmu.2023.1063838] [PMID] []
  11. Jamnani FR, Rahbarizadeh F, Shokrgozar MA, Mahboudi F, Ahmadvand D, Sharifzadeh Z, et al. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: Towards tumor-directed   oligoclonal     T     cell    therapy. Biochim Biophys Acta 2014; 1840(1): 378-86. [DOI:10.1016/j.bbagen.2013.09.029] [PMID]
  12. Banihashemi SR, Hosseini AZ, Rahbarizadeh F, Ahmadvand D. Development of specific nanobodies (VHH) for CD19 immuno-targeting of human B-lymphocytes. Iran J Basic Med Sci 2018; 21(5): 455-64.
  13. Sena-Esteves M, Gao G. Production of High-Titer Retrovirus and Lentivirus Vectors. Cold Spring Harb Protoc 2018; 2018(4). [DOI:10.1101/pdb.prot095687] [PMID]
  14. Arjomandnejad M, Sylvia K, Blackwood M, Nixon T, Tang Q, Muhuri M, et al. Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells. Mol Ther Methods Clin Dev 2021; 23: 490-506. [DOI:10.1016/j.omtm.2021.10.010] [PMID] []
  15. Brown CE, Wright CL, Naranjo A, Vishwanath RP, Chang WC, Olivares S, et al. Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing. J Immunol Methods 2005; 297(1-2): 39-52. [DOI:10.1016/j.jim.2004.11.021] [PMID]
  16. Benmebarek MR, Karches CH, Cadilha BL, Lesch S, Endres S, Kobold S. Killing Mechanisms of Chimeric Antigen Receptor (CAR) T Cells. Int J Mol Sci 2019;
  17. 20(6): 1283. [DOI:10.3390/ijms20061283] [PMID] []
  18. Safarzadeh Kozani P, Naseri A, Mirarefin SMJ, Salem F, Nikbakht M, Evazi Bakhshi S, et al. CAR-T cells for cancer immunotherapy. Biomark Res 2022; 10(1): 24. [DOI:10.1186/s40364-022-00371-7] [PMID] []
  19. Bannas P, Hambach J, Koch-Nolte F. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol 2017; 8: 1603. DOI:10.3389/fimmu.2017.01603] [PMID] []
  20. Mazinani M, Rahbarizadeh F. CAR-T cell potency: from structural elements to vector backbone components. Biomark Res 2022; 10(1): 70. [DOI:10.1186/s40364-022-00417-w] [PMID] []
  21. Bao C, Gao Q, Li LL, Han L, Zhang B, Ding Y, et al. The Application of Nanobody in CAR-T Therapy. Biomolecules 2021; 11(2): 238. [DOI:10.3390/biom11020238] [PMID] []
  22. Mao R, Kong W, He Y. The affinity of antigen-binding domain on the antitumor efficacy of CAR T cells: Moderate is better. Front Immunol 2022; 13: 1032403. [DOI:10.3389/fimmu.2022.1032403] [PMID] []
  23. Hirobe S, Imaeda K, Tachibana M, Okada N. The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity. Int J Mol Sci 2022; 23(7): 4056. [DOI:10.3390/ijms23074056] [PMID] []
  24. Qin L, Lai Y, Zhao R, Wei X, Weng J, Lai P, et al. Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells. J Hematol Oncol 2017; 10(1): 68. [DOI:10.1186/s13045-017-0437-8] [PMID] []
  25. Fujiwara K, Tsunei A, Kusabuka H, Ogaki E, Tachibana M, Okada N. Hinge and Transmembrane Domains of Chimeric Antigen Receptor Regulate Receptor Expression and Signaling Threshold. Cells 2020; 9(5): 1182. [DOI:10.3390/cells9051182] [PMID] []
  26. Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 2015; 3(2): 125-35. [DOI:10.1158/2326-6066.CIR-14-0127] [PMID] []
  27. Li N, Quan A, Li D, Pan J, Ren H, Hoeltzel G, et al. The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer. Nat Commun 2023; 14(1): 1986. [DOI:10.1038/s41467-023-37616-4] [PMID] []
  28. Zhao  Z,  Condomines  M, van der  Stegen   SJC,  Perna F, Kloss CC, Gunset G, et al. Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T Cells. Cancer Cell 2015; 28(4): 415-28. [DOI:10.1016/j.ccell.2015.09.004] [PMID] []
  29. Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D, et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 2009; 17(8): 1453-64. [DOI:10.1038/mt.2009.83] [PMID] []
  30. Gomes-Silva D, Mukherjee M, Srinivasan M, Krenciute G, Dakhova O, Zheng Y, et al. Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent. Cell Rep 2017; 21(1): 17-26. [DOI:10.1016/j.celrep.2017.09.015] [PMID] []
  31. Long AH, Haso WM, Shern JF, Wanhainen KM, Murgai M, Ingaramo M, et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 2015; 21(6): 581-90. [DOI:10.1038/nm.3838] [PMID] []


Impact of CAR Structure in Nanobody-Based Constructs on the Cytotoxic Function of CAR-T Cells Targeting CD19⁺ Cells
‎
Motahareh Arjomandnejad1         , Fatemeh Rahbarizadeh1


1Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Received: 2025/08/10
Accepted: 2025/09/07
 





http://dx.doi.org/10.61186/bloodj.22.1.54
    



Citation:
Arjomandnejad M, Rahbarizadeh F. Impact of CAR Structure in Nanobody-Based Constructs on the Cytotoxic Function of CAR-T Cells Targeting CD19⁺ Cells. J Iran Blood Transfus. 2025: 22 (3): 227-237
    



Correspondence: Rahbarizadeh F., Proffessor of Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University.
P.O.Box: 14115-111, Tehran, Iran.
Tel: (+9821) 82883884
E-mail:
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1- Acridine Orange
1- Biological safety cabinet
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Rahbarif@modares.ac.ir  		 A B S T R A C T
Background and Objectives
In recent years, chimeric antigen receptors (CAR-T) therapy has revolutionized the treatment of hematologic malignancies, particularly B cell-derived cancers such as ALL and non-Hodgkin lymphomas. Most CARs employ single-chain variable fragments (scFvs) as the antigen-recognition domain. However, limitations including low stability, tendency to aggregate, and high immunogenicity have directed attention toward alternatives such as nanobodies (VHHs). In this study, CAR-T cells equipped with an anti-CD19 nanobody receptor were designed and constructed, and their specific ability to recognize and eliminate CD19⁺ cells was evaluated in vitro.
Materials and Methods
In this experimental study, the structure of a CAR containing an anti-CD19 nanobody was designed in both second- and third-generation formats with CD28 and 4-1BB domains. The constructs were cloned into a lentiviral vector and transduced into human T cells. Transduction efficiency was assessed via flow cytometry. Functional evalualation of the engineered cells following exposure to CD19⁺ target cells was evaluated in terms of activation marker expression, cytokine secretion (IFN-γ and interleukin-2), and cytotoxic activity. Experimental data were analyzed using GraphPad Prism software, and group comparisons were performed with one-way and two-way ANOVA tests as well as the t-test.
Results
The generated CAR-T cells showed robust receptor expression and, upon encountering CD19⁺ cells, exhibited specific cytokine responses and cytotoxic activity. In contrast, no responses were observed against CD19⁻ cells. Functional differences observed  differences between constructs with short and long hinge regions, as well as between second- and third-generation CARs, highlighted the importance of structural design in optimizing CAR-T cell efficacy.
Conclusions 
This study, conducted by the first research team in Iran to develop nanobody-based anti-CD19 CAR constructs, highlights the potential of nanobody integration as a safe and effective alternative to scFv-CARs. The findings offer a practical frame work for improving CAR-T cell therapies.
Key words: CAR T-Cell Therapy, Single-Chain Antibodies, CD19 Antigen
 
Copyright © 2025 Journal of Iranian Blood Transfusion, Published by Blood Transfusion Research Center.
This work is licensed under a Creative Common Attribution-Non Commercial 4.0 International license.






 
Type of Study: Research | Subject: Hematology and Oncology
References
1. Rheingold SR, Bhojwani D, Ji L, Xu X, Devidas M, Kairalla JA, et al. Determinants of survival after first relapse of acute lymphoblastic leukemia: a Children's Oncology Group study. Leukemia 2024; 38(11): 2382-94. [DOI:10.1038/s41375-024-02395-4] [PMID] []
2. Iqbal M, Kharfan-Dabaja MA. Relapse of Hodgkin lymphoma after autologous hematopoietic cell transplantation: A current management perspective. Hematol Oncol Stem Cell Ther 2021; 14(2): 95-103. [DOI:10.1016/j.hemonc.2020.05.011] [PMID]
3. Zhou D, Zhu X, Xiao Y. CAR-T cell combination therapies in hematologic malignancies. Exp Hematology Oncol 2024; 13(1): 69. [DOI:10.1186/s40164-024-00536-0] [PMID] []
4. Cuenca M, Peperzak V. Advances and Perspectives in the Treatment of B-Cell Malignancies. Cancers (Basel) 2021; 13(9): 2266. [DOI:10.3390/cancers13092266] [PMID] []
5. Maleki EH, Bahrami AR, Matin MM. Cancer cell cycle heterogeneity as a critical determinant of therapeutic resistance. Genes Dis 2024; 11(1): 189-204. [DOI:10.1016/j.gendis.2022.11.025] [PMID] []
6. Mazinani M, Rahbarizadeh F. New cell sources for CAR-based immunotherapy. Biomark Res 2023; 11(1): 49. [DOI:10.1186/s40364-023-00482-9] [PMID] []
7. Ramírez-Chacón A, Betriu-Méndez S, Bartoló-Ibars A, González A, Martí M, Juan M. Ligand-based CAR-T cell: Different strategies to drive T cells in future new treatments. Front Immunol 2022; 13: 932559. [DOI:10.3389/fimmu.2022.932559] [PMID] []
8. Tang L, Huang Z, Mei H, Hu Y. Immunotherapy in hematologic malignancies: achievements, challenges and future prospects. Signal Transduct Target Ther 2023; 8(1): 306. [DOI:10.1038/s41392-023-01521-5] [PMID] []
9. Rajabzadeh A, Rahbarizadeh F, Ahmadvand D, Kabir Salmani M, Hamidieh AA. A VHH-Based Anti-MUC1 Chimeric Antigen Receptor for Specific Retargeting of Human Primary T Cells to MUC1-Positive Cancer Cells. Cell J 2021; 22(4): 502-13. [DOI:10.1186/s12860-021-00397-z] [PMID] []
10. Nasiri F, Safarzadeh Kozani P, Rahbarizadeh F. T-cells engineered with a novel VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal efficacy to their FMC63-based counterparts. Front Immunol 2023; 14: 1063838. [DOI:10.3389/fimmu.2023.1063838] [PMID] []
11. Jamnani FR, Rahbarizadeh F, Shokrgozar MA, Mahboudi F, Ahmadvand D, Sharifzadeh Z, et al. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: Towards tumor-directed oligoclonal T cell therapy. Biochim Biophys Acta 2014; 1840(1): 378-86. [DOI:10.1016/j.bbagen.2013.09.029] [PMID]
12. Banihashemi SR, Hosseini AZ, Rahbarizadeh F, Ahmadvand D. Development of specific nanobodies (VHH) for CD19 immuno-targeting of human B-lymphocytes. Iran J Basic Med Sci 2018; 21(5): 455-64.
13. Sena-Esteves M, Gao G. Production of High-Titer Retrovirus and Lentivirus Vectors. Cold Spring Harb Protoc 2018; 2018(4). [DOI:10.1101/pdb.prot095687] [PMID]
14. Arjomandnejad M, Sylvia K, Blackwood M, Nixon T, Tang Q, Muhuri M, et al. Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells. Mol Ther Methods Clin Dev 2021; 23: 490-506. [DOI:10.1016/j.omtm.2021.10.010] [PMID] []
15. Brown CE, Wright CL, Naranjo A, Vishwanath RP, Chang WC, Olivares S, et al. Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing. J Immunol Methods 2005; 297(1-2): 39-52. [DOI:10.1016/j.jim.2004.11.021] [PMID]
16. Benmebarek MR, Karches CH, Cadilha BL, Lesch S, Endres S, Kobold S. Killing Mechanisms of Chimeric Antigen Receptor (CAR) T Cells. Int J Mol Sci 2019; 20(6): 1283. [DOI:10.3390/ijms20061283] [PMID] []
17. Safarzadeh Kozani P, Naseri A, Mirarefin SMJ, Salem F, Nikbakht M, Evazi Bakhshi S, et al. CAR-T cells for cancer immunotherapy. Biomark Res 2022; 10(1): 24. [DOI:10.1186/s40364-022-00371-7] [PMID] []
18. Bannas P, Hambach J, Koch-Nolte F. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol 2017; 8: 1603. [DOI:10.3389/fimmu.2017.01603] [PMID] []
19. Mazinani M, Rahbarizadeh F. CAR-T cell potency: from structural elements to vector backbone components. Biomark Res 2022; 10(1): 70. [DOI:10.1186/s40364-022-00417-w] [PMID] []
20. Bao C, Gao Q, Li LL, Han L, Zhang B, Ding Y, et al. The Application of Nanobody in CAR-T Therapy. Biomolecules 2021; 11(2): 238. [DOI:10.3390/biom11020238] [PMID] []
21. Mao R, Kong W, He Y. The affinity of antigen-binding domain on the antitumor efficacy of CAR T cells: Moderate is better. Front Immunol 2022; 13: 1032403. [DOI:10.3389/fimmu.2022.1032403] [PMID] []
22. Hirobe S, Imaeda K, Tachibana M, Okada N. The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity. Int J Mol Sci 2022; 23(7): 4056. [DOI:10.3390/ijms23074056] [PMID] []
23. Qin L, Lai Y, Zhao R, Wei X, Weng J, Lai P, et al. Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells. J Hematol Oncol 2017; 10(1): 68. [DOI:10.1186/s13045-017-0437-8] [PMID] []
24. Fujiwara K, Tsunei A, Kusabuka H, Ogaki E, Tachibana M, Okada N. Hinge and Transmembrane Domains of Chimeric Antigen Receptor Regulate Receptor Expression and Signaling Threshold. Cells 2020; 9(5): 1182. [DOI:10.3390/cells9051182] [PMID] []
25. Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 2015; 3(2): 125-35. [DOI:10.1158/2326-6066.CIR-14-0127] [PMID] []
26. Li N, Quan A, Li D, Pan J, Ren H, Hoeltzel G, et al. The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer. Nat Commun 2023; 14(1): 1986. [DOI:10.1038/s41467-023-37616-4] [PMID] []
27. Zhao Z, Condomines M, van der Stegen SJC, Perna F, Kloss CC, Gunset G, et al. Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T Cells. Cancer Cell 2015; 28(4): 415-28. [DOI:10.1016/j.ccell.2015.09.004] [PMID] []
28. Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D, et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 2009; 17(8): 1453-64. [DOI:10.1038/mt.2009.83] [PMID] []
29. Gomes-Silva D, Mukherjee M, Srinivasan M, Krenciute G, Dakhova O, Zheng Y, et al. Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent. Cell Rep 2017; 21(1): 17-26. [DOI:10.1016/j.celrep.2017.09.015] [PMID] []
30. Long AH, Haso WM, Shern JF, Wanhainen KM, Murgai M, Ingaramo M, et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 2015; 21(6): 581-90. [DOI:10.1038/nm.3838] [PMID] []
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