AbstractChimeric antigen receptor (CAR) T-cell therapy has been used with unprecedented clinical responses in relapsed/refractory B-ALL or B-NHL, MM patients. However, CAR-T therapy has not yet been proven effective in other hematological malignancies, such as T cell lymphoma and leukemia (T-NHL/T-ALL) and acute myeloid leukemia (AML). Developing CAR-T cells for patients with T-cell malignancies remains challenging as most CARs targeting T-lineage antigens, such as CD5, CD7, and CD3, target themselves, potentially resulting in fratricide of CAR-T cells. Thus, there is a need for technical approaches to alleviate the fratricide of CAR-T to treat T-cell malignancies successfully. As part of the approach, we studied whether the affinity of CAR and cognate antigen expression of CAR-T cells influence the intensity of CAR-T`s fratricide and hypothesized that if a single chain Fragment variable (scFv) with a low intensity of fratricide is applied to T-lineage antigen-downregulated CAR-T cells, the effector function of CAR-T cell targeting T cell antigen can be maximized. For this, we screened full human scFvs targeting CD5, a pan T-cell marker, tested the affinity of scFvs for CD5 antigen, and generated anti-CD5 CAR-T cells (CAR5). We evaluated the intensity of in vitro fratricide in CAR5 based on cell expansion and cytokine secretion in vitro and tested their anti-tumor activity in a CD5+ T-ALL xenograft model (Jurkat). In addition, to minimize CAR recognition to a cognate antigen expressed in CAR-T cells, we also generated CD5-downregulated CAR5s (CD5-KD CAR5) using a short hairpin RNA cassette integrated into an anti-CD5 CAR vector to test if the downregulation of CD5 in CAR5s can reduce the fratricide. We found that a CAR5 clone (C7CAR5) with the lowest level of fratricide in vitro, showed superior anti-tumor activity compared to other CAR5s in vivo and that CD5-downregulated C7 CAR5 (CD5-KD C7CAR5) further reduced the fratricide, maximizing the anti-tumor activity. In conclusion, we demonstrate that high-affinity CAR may not always show superior functionality in T cell malignancy CAR-T therapy, explaining one or more strategies to alleviate the fratricide are required. These studies provide the foundation for developing CD5 CART products to treat relapsed T-cell malignancies.Citation Format: Lee Young Ho, Seung Rok Yu, Jeong Hoon Jeong, Hyeong Ji Lee, Hyeon Jeong Cho, Hyung Cheol Kim. Mitigating the CD5 CAR-CD5 interaction enhances the functionality of CD5 CAR-T cells by alleviating the T-cell fratricide. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4086.