Volume 22, Issue 3 (Autumn 2025)
bloodj 2025, 22(3): 227-237 |
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Ethics code: IR.TMU.REC.1394.300
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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
URL: http://bloodjournal.ir/article-1-1595-en.html
Abstract: (247 Views)
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.
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.
Type of Study: Research |
Subject:
Hematology and Oncology
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