Abstract CD5-positive T-cell malignancies, including T-cell acute lymphoblastic leukemia and peripheral T-cell lymphoma, remain challenging to treat due to severe fratricide during CAR-T manufacturing and a lack of effective therapeutic options. Previous work generated two anti-CD5 CAR-T constructs (CAR5) containing unique CD5 binders with distinct affinities, designated A2CAR5 (high affinity) and C7CAR5 (low affinity), to evaluate how binding strength influences CAR-T cell functionality. A2CAR5 exhibited robust antigen engagement but suffered from severe fratricide accompanied by excessive cytokine release, whereas C7CAR5, with lower affinity, largely avoided fratricide while retaining potent cytotoxicity both in vitro and in vivo. However, attempts to improve the functionality of either construct through CD5 knockdown resulted in only limited enhancement. The present study builds upon these findings by applying CD5 and TRAC gene editing to the high-affinity CAR5 to further enhance its efficacy and safety. A comparative evaluation of A2CAR5 and C7CAR5 with CD5 knockout (KO) was conducted to assess affinity-dependent function, while a CD5/TRAC double knockout (dKO) was designed to develop an optimized allogeneic CAR5 platform. Since allogeneic CAR-T cells may exhibit limited persistence following infusion, achieving potent initial activity is critical for therapeutic success. CD5 KO effectively abolished fratricide in A2CAR5 cells, normalizing IFN-γ and TNF-α secretion to levels comparable to those of non-transduced T cells. Surface marker analysis revealed that fratricide led to a progressive increase in the proportion of CAR+ cells over time, whereas CD5 KO eliminated this abnormal enrichment, reduced exhaustion marker expression, and preserved a favorable memory phenotype. Following CD5 KO, A2CAR5 consistently demonstrated superior functional performance compared to C7CAR5, exhibiting stronger cytotoxic activity specifically against CD5+ target cells and greater functional persistence under repetitive antigen exposure. To achieve efficient disruption of both CD5 and TRAC, a sequential targeting approach was adopted, which proved more effective and consistent than conventional double knockout methods. In conclusion, these findings indicate that CD5 knockout reduces fratricide and limits T-cell exhaustion, supporting the development of optimized allogeneic CAR5 cells through the integration of binder design and gene editing. Future work will incorporate AAV6-mediated CAR knock-in at the TRAC locus to further enhance genomic precision and product consistency. Citation Format: Seojeong Kim, Jeong Hoon Jeong, Jihyun Park, Youngho Lee, Hyung Cheol Kim, Hyoung Jin Kang, Matthew H. Porteus, Steven Feldman, Youngil Koh. Integration of affinity-dependent modulation and CD5/TRAC knockout generates fratricide-resistant and highly cytotoxic anti-CD5 CAR-T cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3705.
Kim et al. (Fri,) studied this question.