Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy
BackgroundDespite the curative potential, high cost of manufacturing and the toxicities limits the wider access of Chimeric Antigen Receptor (CAR) T cell therapy in global medicine. CARs are modular synthetic antigen receptors integrating the single-chain variable fragment (scFv) of an immunoglobuli...
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Frontiers Media S.A.
2025-05-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1545549/full |
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| author | Muthuganesh Muthuvel Muthuganesh Muthuvel Thamizhselvi Ganapathy Trent Spencer Sunil S. Raikar Saravanabhavan Thangavel Alok Srivastava Alok Srivastava Alok Srivastava Sunil Martin |
| author_facet | Muthuganesh Muthuvel Muthuganesh Muthuvel Thamizhselvi Ganapathy Trent Spencer Sunil S. Raikar Saravanabhavan Thangavel Alok Srivastava Alok Srivastava Alok Srivastava Sunil Martin |
| author_sort | Muthuganesh Muthuvel |
| collection | DOAJ |
| description | BackgroundDespite the curative potential, high cost of manufacturing and the toxicities limits the wider access of Chimeric Antigen Receptor (CAR) T cell therapy in global medicine. CARs are modular synthetic antigen receptors integrating the single-chain variable fragment (scFv) of an immunoglobulin molecule to the TCR signaling. CARs allow HLA independent, T cell mediated destruction of tumor cells independent of tumor associated-HLA downregulation and survive within the patient as ‘living drug.’ Here we report a safer approach for engineering alpha beta T cells with anti- CD19-CD28ζ CAR using self-inactivating (SIN) lentiviral vectors for adoptive immunotherapy.Methodαβ T cells from the peripheral blood (PB) were lentivirally transduced with CAR construct containing hinge domain from CD8α, transmembrane and co-stimulatory domain from CD28 along with signaling domain from CD3ζ and driven by human UBC promoter. The cells were pre-stimulated through CD3/CD28 beads before lentiviral transduction. Transduction efficiency, fold expansion and phenotype were monitored for the CAR T cells expanded for 10–12 days. The antigen-specific tumor-killing capacity of CD19 CAR T cells was assessed against a standard CD19 expressing NALM6 cell lines with a flow cytometry-based assay optimized in the lab.Results and conclusionWe have generated high titer lentiviral vectors of CAR with a titer of 9.85 ± 2.2×107 TU/ml (mean ± SEM; n=9) generating a transduction efficiency of 27.57 ± 2.4%. (n=7) at an MOI of 10 in total T cells. The product got higher CD8+ to CD4+ CAR T cell ratio with preponderance of an effector memory phenotype on day 07 and day 12. The CAR-T cells expanded (148.4 ± 29 fold; n=7) in serum free media with very high viability (87.8 ± 2.2%; n=7) on day 12. The antitumor functions of CD19 CAR T cells as gauged against percentage lysis of NALM6 cells at a 1:1 ratio is 27.68 ± 6.87% drawing up to the release criteria. CAR T cells produced IFNγ (11.23 ± 1.5%; n=6) and degranulation marker CD107α (34.82 ± 2.08%; n=5) in an antigen-specific manner. Furthermore, the sequences of WPRE, GFP, and P2A were removed from the CAR construct to enhance safety. These CAR T cells expanded up to 21.7 ± 5.53 fold with 82.7±5.43% viability (n=4).ConclusionWe have generated, validated, and characterized a reproducible indigenous workflow for generating anti-CD19 CAR T cells in vitro. This approach can be used for targeting cancer and autoimmune diseases in which CD19+ B lineage cells cause host damage. |
| format | Article |
| id | doaj-art-ed3ab307f84f44cf97cdc0e05b4c7210 |
| institution | Kabale University |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Immunology |
| spelling | doaj-art-ed3ab307f84f44cf97cdc0e05b4c72102025-08-20T03:48:50ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-05-011610.3389/fimmu.2025.15455491545549Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapyMuthuganesh Muthuvel0Muthuganesh Muthuvel1Thamizhselvi Ganapathy2Trent Spencer3Sunil S. Raikar4Saravanabhavan Thangavel5Alok Srivastava6Alok Srivastava7Alok Srivastava8Sunil Martin9Laboratory of Synthetic Immunology, Cancer Research Division, Biotechnology Research Innovation Council- Rajiv Gandhi Centre for Biotechnology (BRIC - RGCB), Department of Biotechnology, Thiruvananthapuram, IndiaManipal Academy of Higher Education (MAHE), Manipal, Karnataka, IndiaCenter for Stem Cell Research (CSCR), Christian Medical College (CMC) Vellore, Velllore, IndiaCell and Gene Therapy Program, Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University School of Medicine, Atlanta, GA, United StatesCell and Gene Therapy Program, Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University School of Medicine, Atlanta, GA, United StatesCenter for Stem Cell Research (CSCR), Christian Medical College (CMC) Vellore, Velllore, IndiaCenter for Stem Cell Research (CSCR), Christian Medical College (CMC) Vellore, Velllore, IndiaDepartment of Hematology, Christian Medical College (CMC) Vellore, Velllore, IndiaHaematology Research Unit, St. John’s Research Institute, St. John’s National Academy of Health Sciences, Bengaluru, Karnataka, IndiaLaboratory of Synthetic Immunology, Cancer Research Division, Biotechnology Research Innovation Council- Rajiv Gandhi Centre for Biotechnology (BRIC - RGCB), Department of Biotechnology, Thiruvananthapuram, IndiaBackgroundDespite the curative potential, high cost of manufacturing and the toxicities limits the wider access of Chimeric Antigen Receptor (CAR) T cell therapy in global medicine. CARs are modular synthetic antigen receptors integrating the single-chain variable fragment (scFv) of an immunoglobulin molecule to the TCR signaling. CARs allow HLA independent, T cell mediated destruction of tumor cells independent of tumor associated-HLA downregulation and survive within the patient as ‘living drug.’ Here we report a safer approach for engineering alpha beta T cells with anti- CD19-CD28ζ CAR using self-inactivating (SIN) lentiviral vectors for adoptive immunotherapy.Methodαβ T cells from the peripheral blood (PB) were lentivirally transduced with CAR construct containing hinge domain from CD8α, transmembrane and co-stimulatory domain from CD28 along with signaling domain from CD3ζ and driven by human UBC promoter. The cells were pre-stimulated through CD3/CD28 beads before lentiviral transduction. Transduction efficiency, fold expansion and phenotype were monitored for the CAR T cells expanded for 10–12 days. The antigen-specific tumor-killing capacity of CD19 CAR T cells was assessed against a standard CD19 expressing NALM6 cell lines with a flow cytometry-based assay optimized in the lab.Results and conclusionWe have generated high titer lentiviral vectors of CAR with a titer of 9.85 ± 2.2×107 TU/ml (mean ± SEM; n=9) generating a transduction efficiency of 27.57 ± 2.4%. (n=7) at an MOI of 10 in total T cells. The product got higher CD8+ to CD4+ CAR T cell ratio with preponderance of an effector memory phenotype on day 07 and day 12. The CAR-T cells expanded (148.4 ± 29 fold; n=7) in serum free media with very high viability (87.8 ± 2.2%; n=7) on day 12. The antitumor functions of CD19 CAR T cells as gauged against percentage lysis of NALM6 cells at a 1:1 ratio is 27.68 ± 6.87% drawing up to the release criteria. CAR T cells produced IFNγ (11.23 ± 1.5%; n=6) and degranulation marker CD107α (34.82 ± 2.08%; n=5) in an antigen-specific manner. Furthermore, the sequences of WPRE, GFP, and P2A were removed from the CAR construct to enhance safety. These CAR T cells expanded up to 21.7 ± 5.53 fold with 82.7±5.43% viability (n=4).ConclusionWe have generated, validated, and characterized a reproducible indigenous workflow for generating anti-CD19 CAR T cells in vitro. This approach can be used for targeting cancer and autoimmune diseases in which CD19+ B lineage cells cause host damage.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1545549/fullCD19 CAR T cellsB lineage malignanciesserum free mediaSIN vectorNALM-6 |
| spellingShingle | Muthuganesh Muthuvel Muthuganesh Muthuvel Thamizhselvi Ganapathy Trent Spencer Sunil S. Raikar Saravanabhavan Thangavel Alok Srivastava Alok Srivastava Alok Srivastava Sunil Martin Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy Frontiers in Immunology CD19 CAR T cells B lineage malignancies serum free media SIN vector NALM-6 |
| title | Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy |
| title_full | Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy |
| title_fullStr | Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy |
| title_full_unstemmed | Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy |
| title_short | Engineering safe anti-CD19-CD28ζ CAR T cells with CD8a hinge domain in serum-free media for adoptive immunotherapy |
| title_sort | engineering safe anti cd19 cd28ζ car t cells with cd8a hinge domain in serum free media for adoptive immunotherapy |
| topic | CD19 CAR T cells B lineage malignancies serum free media SIN vector NALM-6 |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1545549/full |
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