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Enhancing CAR-T cells: unleashing lasting impact potential with phytohemagglutinin activation in in vivo leukemia model

Cancer Gene Therapy volume 31, pages 387–396 (2024)Cite this article

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Abstract

Chimeric antigen receptor T (CAR-T) cell therapy holds great promise as an innovative immunotherapeutic approach for cancer treatment. To optimize the production and application of CAR-T cells, we evaluated the in vivo stability and efficacy capacities of CAR-T cells developed under different conditions. In this study, CAR-T cells were activated using Phytohemagglutinin (PHA) or anti-CD3&anti-CD28 and were compared in an in vivo CD19+B-cell cancer model in mouse groups. Our results demonstrated that CAR-T cells activated with PHA exhibited higher stability and anti-cancer efficacy compared to those activated with anti-CD3&anti-CD28. Specifically, CAR19BB-T cells activated with PHA exhibited continuous proliferation and long-term persistence without compromising their anti-cancer efficacy. Kaplan–Meier survival analysis revealed prolonged overall survival in the CAR-T cell-treated groups compared to the only tumor group. Furthermore, specific LTR-targeted RT-PCR analysis confirmed the presence of CAR-T cells in the treated groups, with significantly higher levels observed in the CAR19BB-T (PHA) group compared to other groups. Histopathological analysis of spleen, kidney, and liver tissue sections indicated reduced inflammation and improved tissue integrity in the CAR-T cell-treated groups. Our findings highlight the potential benefits of using PHA as a co-stimulatory method for CAR-T cell production, offering a promising strategy to enhance their stability and persistence. These results provide valuable insights for the development of more effective and enduring immunotherapeutic approaches for cancer treatment. CAR-T cells activated with PHA may offer a compelling therapeutic option for advancing cancer immunotherapy in clinical applications.

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Fig. 1: Injection administration schedule.
Fig. 2: In vivo CAR-T cell efficacy studies in ALL nude/NSG mouse models.
Fig. 3: Time-dependent visualization of CAR19BB-T and CAR1928-T-cell efficacy in an in vivo ALL cancer model in mice (7–63 days).
Fig. 4: In vivo quantification of fLuciferase bioluminescence, mCherry, and GFP fluorescence.
Fig. 5: Kaplan–Meier survival analysis and In vivo PCR detection of CAR-T cells.
Fig. 6: Hemogram and biochemical analysis in ALL cancer model mice after CAR-T cell therapy.
Fig. 7: Histopathological analysis of the Spleen, Kidney, and Liver in the Only tumor and CAR-T cell groups.

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Additional data beyond the data found in the article are available from the corresponding author on reasonable request.

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Funding

All funding for the project was supported by the Presidency of Turkish Health Institutes (TUSEB) with grant number 4293.

Author information

Author notes

  1. These authors contributed equally: Berranur Sert, Gamze Gulden.

Authors and Affiliations

  1. Molecular Biology, Institute of Science and Technology, Üsküdar University, Istanbul, Turkey

    Berranur Sert, Gamze Gulden, Tarik Teymur, Yasin Ay & Tahire Yurdakul

  2. Transgenic Cell Technologies and Epigenetic Application and Research Center (TRGENMER), Üsküdar University, Istanbul, Turkey

    Berranur Sert, Gamze Gulden, Tarik Teymur, Yasin Ay, Onur Mert Unaldi, Elanur Guzenge, Hamza Emir Erdil & Cihan Tastan

  3. Department of Genetics and Bioengineering, Faculty of Engineering, Cell and Gene Therapy Excellence Center, Yeditepe University, Istanbul, Turkey

    Raife Dilek Turan

  4. Molecular Biology and Genetics Department, Faculty of Engineering and Natural Science, Üsküdar University, Istanbul, Turkey

    Onur Mert Unaldi, Elanur Guzenge, Hamza Emir Erdil, Sevim Isik, Pinar Oz & Cihan Tastan

  5. Stem Cell Studies Application and Research Center (USKOKMER), Üsküdar University, Istanbul, Turkey

    Sevim Isik & Tahire Yurdakul

  6. Neuropsychopharmacology Application and Research Center (NPFUAM) Neurochemıstry Laboratory Üsküdar University, Istanbul, Turkey

    Pinar Oz & Osman Kamali

  7. NP Brain Hospital, Istanbul, Turkey

    Ilknur Bozkurt & Nevzat Tarhan

  8. Acıbadem Mehmet Ali Aydınlar University, Animal Application and Research Center, İstanbul, Turkey

    Samed Ozer

  9. Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Turkey

    Ercument Ovali

  10. Faculty of Humanities and Social Sciences, Üsküdar University, Istanbul, Turkey

    Nevzat Tarhan

Authors
  1. Berranur Sert
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Contributions

BS, GG, TT, YA, CT designed experiments. BS, GG, TT, YA, RDT, OMU, EG, HEE, CT performed experiments and acquired data. BS, GG, TT, YA, RDT, CT analyzed data and interpreted results. BS, GG, TT, YA, RDT, SO, CT performed animal treatments. BS, GG, TT, YA, RDT, OMU, EG, HEE, SI, PO, TA, OK, CT performed the histopathological analysis of tissues. IB, OMU performed clinical specimen collection and data following. CT supervised the work, acquired funds and performed the administration of the project. CT supervised the statistical analysis of the data. All authors wrote, reviewed and edited the manuscript.

Corresponding author

Correspondence to Cihan Tastan.

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Competing interests

CT, BS, GG, TT, and YA are inventors of patent applications (pending) including “Allogeneic CAR-T cells expressing bispecific chimeric antigen receptors (CARs) targeting CD19 along with CD20 or CD22” (PCT/TR2023/050541 and 2022/009703) at Turkish Patent and Trademark Office. No other author has a competing interest except for these authors.

Ethics approval and consent to participate

In vitro and in vivo studies were approved by the Acibadem University and Acibadem Healthcare Institutions Medical Research Ethics Committee (ATADEK-2019-17/31). All animal studies have been approved by Acıbadem University Animal Experiments Local Ethics Committee (ACU-HADYEK; 11.13.2019).

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Sert, B., Gulden, G., Teymur, T. et al. Enhancing CAR-T cells: unleashing lasting impact potential with phytohemagglutinin activation in in vivo leukemia model. Cancer Gene Ther 31, 387–396 (2024). https://doi.org/10.1038/s41417-023-00709-9

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