CD19-CAR ENGINEERING OF MONOCYTES WITH MYELOID-DERIVED SIGNALING DOMAINS ENHANCES ANTITUMOR ACTIVITY AGAINST NEOPLASTIC B-CELL LINES
- 1Universidade Estadual Paulista (UNESP), Araraquara, SP, Brazil
Abstract
CD19-targeted chimeric antigen receptor (CAR) therapy has significantly improved the treatment of relapsed or refractory B-cell malignancies. Nevertheless, its clinical application is associated with side effects such as cytokine release syndrome and neurotoxicity, while conventional CAR-T therapy requires individualized autologous cell manufacturing. These limitations have stimulated the investigation of alternative immune-cell platforms, including genetically engineered monocytes and macrophages, which may provide distinct effector functions and complementary mechanisms of antitumor activity. In this context, we sought to establish a monocytic CD19-CAR platform based on THP-1 cells and investigate whether costimulatory domains derived from myeloid activation receptors, Dectin-1 and FcγRI, could promote antitumor activity against B-cell malignancies. To this end, THP-1 cells were genetically modified with lentiviral vectors encoding CD19-CAR constructs containing Dectin-1, FcγRI, or CD137/CD3ζ signaling domains. CD19-positive Nalm-6 and Daudi cells were engineered to express luciferase to enable the assessment of tumor-cell viability by bioluminescence. THP-1 cells were transduced at an MOI of 5, and CAR-expressing cells were enriched by EGFP-based cell sorting. CAR surface expression was assessed by flow cytometry using an anti-F(ab')₂ antibody or Protein L-PE. Antitumor activity was subsequently evaluated by co-culturing untransduced or CAR-engineered THP-1 cells with Nalm-6-Luc or Daudi-Luc target cells at effector-to-target ratios of 1:1, 3:1, and 5:1 for 6 and 24 h. CD19-8H-Dectin-1-CAR, CD19-8H/TM-FcγRI-CAR, and CD19-8H/TM-137ζ-CAR THP-1 cells achieved transduction efficiencies of 75.6%, 87.6%, and 64%, respectively. Following EGFP-based sorting, approximately 99% of cells were EGFP-positive, and all CAR constructs showed robust surface expression. In functional assays, CD19-8H/TM-137ζ-CAR THP-1 cells significantly decreased the bioluminescence of both Nalm-6-Luc and Daudi-Luc cells after 6 h. Notably, THP-1 cells expressing Dectin-1- or FcγRI-based CARs produced a sustained reduction in Nalm-6-Luc bioluminescence at both 6 and 24 h. Additionally, their antitumor effect against Daudi-Luc cells was detected only at 6 h. Collectively, these findings demonstrate that engineering THP-1 monocytic cells with CD19-CARs incorporating Dectin-1 or FcγRI signaling domains confers effective antitumor activity against in vitro models of B-cell acute lymphoblastic leukemia and Burkitt lymphoma. The enhanced and sustained response observed against Nalm-6 cells supports the potential of myeloid-derived signaling domains as an alternative strategy for the development of CAR-engineered myeloid cells targeting hematological malignancies. Importantly, these results also establish THP-1 cells as a practical and reproducible screening platform for the comparative evaluation and optimization of myeloid CAR designs before their investigation in primary human monocytes and macrophages differentiated, facilitating the identification of promising constructs for subsequent translational validation.
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BibTeX
@article{silva2026cd19car,
title = {{CD19-CAR ENGINEERING OF MONOCYTES WITH MYELOID-DERIVED SIGNALING DOMAINS ENHANCES ANTITUMOR ACTIVITY AGAINST NEOPLASTIC B-CELL LINES}},
author = {Victor Gabriel Santos SILVA and Isabele Held LEMOS and Pedro Augusto Macedo TEIXEIRA and Nathália Ferreira FREGONEZI and Gustavo Matheus AMARO and Milena Barrocali Araujo MELO and Júlia Garcia GUIMARãES and Thiago Aparecido SILVA},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.106835},
url = {https://doi.org/10.1016/j.htct.2026.106835},
note = {CC BY},
}
RIS
TY - JOUR
TI - CD19-CAR ENGINEERING OF MONOCYTES WITH MYELOID-DERIVED SIGNALING DOMAINS ENHANCES ANTITUMOR ACTIVITY AGAINST NEOPLASTIC B-CELL LINES
AU - Victor Gabriel Santos SILVA
AU - Isabele Held LEMOS
AU - Pedro Augusto Macedo TEIXEIRA
AU - Nathália Ferreira FREGONEZI
AU - Gustavo Matheus AMARO
AU - Milena Barrocali Araujo MELO
AU - Júlia Garcia GUIMARãES
AU - Thiago Aparecido SILVA
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.106835
UR - https://doi.org/10.1016/j.htct.2026.106835
AB - CD19-targeted chimeric antigen receptor (CAR) therapy has significantly improved the treatment of relapsed or refractory B-cell malignancies. Nevertheless, its clinical application is associated with side effects such as cytokine release syndrome and neurotoxicity, while conventional CAR-T therapy requires individualized autologous cell manufacturing. These limitations have stimulated the investigation of alternative immune-cell platforms, including genetically engineered monocytes and macrophages, which may provide distinct effector functions and complementary mechanisms of antitumor activity. In this context, we sought to establish a monocytic CD19-CAR platform based on THP-1 cells and investigate whether costimulatory domains derived from myeloid activation receptors, Dectin-1 and FcγRI, could promote antitumor activity against B-cell malignancies. To this end, THP-1 cells were genetically modified with lentiviral vectors encoding CD19-CAR constructs containing Dectin-1, FcγRI, or CD137/CD3ζ signaling domains. CD19-positive Nalm-6 and Daudi cells were engineered to express luciferase to enable the assessment of tumor-cell viability by bioluminescence. THP-1 cells were transduced at an MOI of 5, and CAR-expressing cells were enriched by EGFP-based cell sorting. CAR surface expression was assessed by flow cytometry using an anti-F(ab')₂ antibody or Protein L-PE. Antitumor activity was subsequently evaluated by co-culturing untransduced or CAR-engineered THP-1 cells with Nalm-6-Luc or Daudi-Luc target cells at effector-to-target ratios of 1:1, 3:1, and 5:1 for 6 and 24 h. CD19-8H-Dectin-1-CAR, CD19-8H/TM-FcγRI-CAR, and CD19-8H/TM-137ζ-CAR THP-1 cells achieved transduction efficiencies of 75.6%, 87.6%, and 64%, respectively. Following EGFP-based sorting, approximately 99% of cells were EGFP-positive, and all CAR constructs showed robust surface expression. In functional assays, CD19-8H/TM-137ζ-CAR THP-1 cells significantly decreased the bioluminescence of both Nalm-6-Luc and Daudi-Luc cells after 6 h. Notably, THP-1 cells expressing Dectin-1- or FcγRI-based CARs produced a sustained reduction in Nalm-6-Luc bioluminescence at both 6 and 24 h. Additionally, their antitumor effect against Daudi-Luc cells was detected only at 6 h. Collectively, these findings demonstrate that engineering THP-1 monocytic cells with CD19-CARs incorporating Dectin-1 or FcγRI signaling domains confers effective antitumor activity against in vitro models of B-cell acute lymphoblastic leukemia and Burkitt lymphoma. The enhanced and sustained response observed against Nalm-6 cells supports the potential of myeloid-derived signaling domains as an alternative strategy for the development of CAR-engineered myeloid cells targeting hematological malignancies. Importantly, these results also establish THP-1 cells as a practical and reproducible screening platform for the comparative evaluation and optimization of myeloid CAR designs before their investigation in primary human monocytes and macrophages differentiated, facilitating the identification of promising constructs for subsequent translational validation.
SN - 2531-1379
SN - 2531-1387
ER -