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322 Preclinical assessment of allogeneic Anti-CD5 γδ CAR-T therapy for T-Cell malignancies
  1. Seung Rok Yu,
  2. Hyeong Ji Lee,
  3. Jeong Hoon Jeong,
  4. Hyeon Jeong Cho,
  5. Hyo Bhin Lee,
  6. Hyung Cheol Kim and
  7. Young Ho Lee
  1. Curocell Inc., Daejeon, Daejeon, Republic of Korea
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background CAR-T cell therapy has revolutionized the treatment of B-cell malignancies. However, the development of CAR-T therapies for T-cell malignancies has been slower, partially due to technical difficulties in T-cell antigen targeting CAR-T production such as fratricide and contamination of malignant T cells.1 Although allogeneic αβ CAR-T cells from healthy donors provide blast-free treatment options, complications like GvHD and host rejection can arise. In this context, utilizing healthy donor-derived γδ T cells, free of the risk of GVHD and tumor contamination, presents a promising option for allogeneic CAR-T cell therapy.2 In this study, we introduce allogeneic anti-CD5 γδ CAR T cells for treating T-cell malignancies.

Methods We employed bio-panning techniques to screen novel fully human single-chain variable fragments (scFvs) capable of recognizing the CD5 antigen. Next, we genetically modified γδ T cells obtained from a healthy donor by introducing functional CAR constructs, which incorporated CD5-specific scFv-fused 4–1BB/CD3z sequences and assessed thefunctionality and phenotypic features of anti-CD5 γδ CAR T cells through in vitro functional assays. We also evaluated the preclinical potency of these CAR T cells using immunodeficient mice models engrafted with CD5+ T cell lymphoma cell lines.

Results Several fully human scFvs that target CD5 were successfully identified and two scFv clones (A2 and C7) were utilized in generating anti-CD5 γδ CAR-T cells (γδCAR5) and tested. A2 γδCAR5 exhibited higher cell expansion and CAR-positive portion compared to C7 γδCAR5, although A2 γδCAR5 displayed higher exhaustion phenotypes. A2 γδCAR5 demonstrated robust cytokine production and cytotoxic activity in vitro. Furthermore, A2 γδCAR5 exhibited superior anti-tumor activity in a CD5+ T cell lymphoma xenograft model (HUT-78) compared to C7 γδCAR5. Interestingly, in the context of αβ T cells, C7 αβCAR5 showed higher cell expansion and greater anti-tumor effect than A7 αβCAR5, implying that the exhaustion of A2 CAR5 is more severe in the context of αβ T cells, compared to γδ T cells.

Conclusions Our successful generation and evaluation of anti-CD5 γδ CAR T cells provide the foundation for developing allogeneic CAR T cell therapy as a promising treatment of T cell malignancies.


  1. Safarzadeh Kozani, Pouya, Pooria Safarzadeh Kozani, and Fatemeh Rahbarizadeh. ‘CAR-T cell therapy in T-cell malignancies: Is success a low-hanging fruit?.’ Stem cell research & therapy 2021;12(1):1–17.

  2. Saura-Esteller, José, et al. ‘Gamma delta T-cell based cancer immunotherapy: past-present-future.’ Frontiers in Immunology 2022:2649.

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