Department/Center of Hematology-oncology, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China.
Pediatric Leukemia Diagnostic and Therapeutic Technology Research Center of Zhejiang Province, National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China.
J Transl Med. 2024 May 21;22(1):482. doi: 10.1186/s12967-024-05254-z.
Cellular immunotherapy, represented by the chimeric antigen receptor T cell (CAR-T), has exhibited high response rates, durable remission, and safety in vitro and in clinical trials. Unfortunately, anti-CD19 CAR-T (CART-19) treatment alone is prone to relapse and has a particularly poor prognosis in relapsed/refractory (r/r) B-ALL patients. To date, addressing or reducing relapse remains one of the research priorities to achieve broad clinical application.
We manufactured second generation CART-19 cells and validated their efficacy and safety in vitro and in vivo. Through co-culture of Nalm-6 cells with short-term cultured CART-19 cells, CD19-negative Nalm-6 cells were detected by flow cytometry, and further investigation of the relapsed cells and their resistance mechanisms was evaluated in vitro.
In this study, we demonstrated that CART-19 cells had enhanced and specific antileukemic activities, and the survival of B-ALL mouse models after CART-19 treatment was significantly prolonged. We then shortened the culture time and applied the serum-free culture to expand CAR-T cells, followed by co-culturing CART-19 cells with Nalm-6 cells. Surprisingly, we observed the proliferation of CD19-negative Nalm-6 cells around 28 days. Identification of potential resistance mechanisms showed that the relapsed cells express truncated CD19 proteins with decreased levels and, more importantly, CAR expression was detected on the relapsed cell surface, which may ultimately keep them antigen-negative. Furthermore, it was validated that CART-22 and tandem CART-22/19 cells could effectively kill the relapsed cells, but neither could completely eradicate them.
We successfully generated CART-19 cells and obtained a CD19-negative refractory relapsed B-ALL cell line, providing new insights into the underlying mechanisms of resistance and a new in vitro model for the treatment of r/r B-ALL patients with low antigen density.
嵌合抗原受体 T 细胞（CAR-T）为代表的细胞免疫疗法，在体外和临床试验中均表现出高反应率、持久缓解和安全性。不幸的是，抗 CD19 CAR-T（CART-19）单独治疗易复发，且在复发/难治性（r/r）B-ALL 患者中的预后尤其差。迄今为止，解决或降低复发率仍然是实现广泛临床应用的研究重点之一。
我们制造了第二代 CART-19 细胞，并在体外和体内验证了它们的功效和安全性。通过将 Nalm-6 细胞与短期培养的 CART-19 细胞共培养，通过流式细胞术检测到 CD19 阴性的 Nalm-6 细胞，并进一步评估了体外复发细胞及其耐药机制。
在这项研究中，我们证明了 CART-19 细胞具有增强和特异性的抗白血病活性，并且 CART-19 处理后 B-ALL 小鼠模型的存活时间明显延长。然后，我们缩短了培养时间并应用无血清培养来扩增 CAR-T 细胞，然后将 CART-19 细胞与 Nalm-6 细胞共培养。令人惊讶的是，我们在大约 28 天观察到 CD19 阴性的 Nalm-6 细胞的增殖。潜在耐药机制的鉴定表明，复发细胞表达截短的 CD19 蛋白，水平降低，更重要的是，CAR 表达在复发细胞表面检测到，这可能最终使它们抗原阴性。此外，验证了 CART-22 和串联 CART-22/19 细胞可以有效杀死复发细胞，但都不能完全清除它们。
我们成功地生成了 CART-19 细胞，并获得了 CD19 阴性难治性复发 B-ALL 细胞系，为耐药的潜在机制提供了新的见解，并为治疗抗原密度低的 r/r B-ALL 患者提供了新的体外模型。
