T cell malignancies are a group of hematologic cancers with high recurrence and mortality rates. CD5 is highly expressed in ∼85% of T cell malignancies, although normal expression of CD5 is restricted to thymocytes, T cells, and B1 cells. However, CD5 expression on chimeric antigen receptor (CAR)-T cells leads to CAR-T cell fratricide. Once this limitation is overcome, CD5-targeting CAR-T therapy could be an attractive strategy to treat T cell malignancies. Here, we report the selection of novel CD5-targeting fully human heavy-chain variable (FHV_H) domains for the development of a biepitopic CAR, termed FHV_H3/V_H1, containing FHV_H1 and FHV_H3, which were validated to bind different epitopes of the CD5 antigen. To prevent fratricide in CD5 CAR-T cells, we optimized the manufacturing procedures of a CRISPR-Cas9-based CD5 knockout (CD5KO) and lentiviral transduction of anti-CD5 CAR. In vitro and in vivo functional comparisons demonstrated that biepitopic CD5KO FHV_H3/V_H1 CAR-T cells exhibited enhanced and longer lasting efficacy; produced moderate levels of cytokine secretion; showed similar specificity profiles as either FHV_H1, FHV_H3, or the clinically tested H65; and is therefore suitable for further development.

T细胞恶性肿瘤是一组具有高复发率和死亡率的血液系统癌症。CD5 在约 85% 的 T 细胞恶性肿瘤中高度表达，尽管 CD5 的正常表达仅限于胸腺细胞、T 细胞和 B1 细胞。然而，嵌合抗原受体 (CAR)-T 细胞上的 CD5 表达导致 CAR-T 细胞自相残杀。一旦克服了这一限制，靶向 CD5 的 CAR-T 疗法可能是治疗 T 细胞恶性肿瘤的一种有吸引力的策略。在这里，我们报告了新的 CD5 靶向全人类重链可变 (FHV _H ) 结构域的选择，用于开发双表位 CAR，称为 FHV _H 3/V _H 1，包含 FHV _H 1 和 FHV _H3，经验证可结合 CD5 抗原的不同表位。为了防止 CD5 CAR-T 细胞自相残杀，我们优化了基于 CRISPR-Cas9 的 CD5 敲除 (CD5KO) 和抗 CD5 CAR 慢病毒转导的制造程序。体外和体内功能比较表明，双表位 CD5KO FHV _H 3/V _H 1 CAR-T 细胞表现出增强且更持久的功效；产生中等水平的细胞因子分泌；_{显示出与 FHV H} 1、FHV _H 3 或临床测试的 H65相似的特异性特征；因此适合进一步发展。