Fan Dongmei, Li Zhenzhen, Zhang Xiaolong, Yang Yuqi, Yuan Xiangfei, Zhang Xiuli, Yang Ming, Zhang Yizhi, Xiong Dongsheng
State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, People's Republic of China.
School of Pharmacy, Tianjin Medical University, Tianjin, 300070, People's Republic of China.
J Hematol Oncol. 2015 Feb 28;8:18. doi: 10.1186/s13045-015-0109-5.
Leukemic stem cells (LSCs) are frequently seen as a cause of treatment failure and relapse in patients with acute myeloid leukemia (AML). Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs. The identification of targets on the cell surface of LSCs is getting more and more attention. Among these, CD123, also known as the interleukin-3 (IL3)-receptor α chain, has been identified as a potential immunotherapeutic target due to its overexpression on LSCs in AML as well as on AML blasts, rather than normal hematopoietic stem cells.
We constructed a CD123-targeted fusion protein antiCD3Fv-⊿IL3, with one binding site for T cell antigen receptor (TCRCD3) and the other for CD123, by recombinant gene-engineering technology. Cysteine residues were introduced into the V domains of the antiCD3Fv segment to enhance its stability by locking the two chains of Fv together with disulfide covalent bonds. The stability and cytotoxicity of the two fusion proteins were detected in vitro and in vivo.
Both fusion proteins were produced and purified from Escherichia coli 16C9 cells with excellent yields in fully active forms. High-binding capability was observed between these two fusion proteins and human IL3R, leading to the specific lysis of CD123-expressing cell lines KG1a; also, mononuclear cells from primary AML patients were inhibited in a colony forming assay in vitro, presumably by redirecting T lymphocytes in vitro. In addition, they displayed an antileukemic activity against KG1a xenografts in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, especially disulfide-stabilized (ds)-antiCD3Fv-⊿IL3 for its improved stability.
These results suggest that both fusion proteins display the antileukemic activity against CD123-expressing cell lines as well as leukemic progenitors in vitro and in vivo, especially ds-antiCD3Fv-⊿IL3. They could be the promising candidates for future immunotherapy of AML.
白血病干细胞（LSCs）常被视为急性髓系白血病（AML）患者治疗失败和复发的原因。因此，成功治疗AML的新策略应旨在根除LSCs。LSCs细胞表面靶点的识别越来越受到关注。其中，CD123，也称为白细胞介素-3（IL3）受体α链，因其在AML的LSCs以及AML原始细胞上过度表达，而在正常造血干细胞上不表达，已被确定为一个潜在的免疫治疗靶点。
我们通过重组基因工程技术构建了一种靶向CD123的融合蛋白抗CD3Fv-ΔIL3，其一个结合位点针对T细胞抗原受体（TCRCD3），另一个针对CD123。将半胱氨酸残基引入抗CD3Fv片段的V结构域，通过二硫键共价键将Fv的两条链锁定在一起，以增强其稳定性。在体外和体内检测了这两种融合蛋白的稳定性和细胞毒性。
两种融合蛋白均从大肠杆菌16C9细胞中产生并纯化，产量优异，呈完全活性形式。观察到这两种融合蛋白与人IL3R之间具有高结合能力，导致表达CD123的细胞系KG1a发生特异性裂解；此外，原发性AML患者的单核细胞在体外集落形成试验中受到抑制，推测是通过在体外重定向T淋巴细胞实现的。此外，它们对非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠的KG1a异种移植瘤显示出抗白血病活性，特别是二硫键稳定的（ds）-抗CD3Fv-ΔIL3，因其稳定性提高。
这些结果表明，两种融合蛋白在体外和体内均对表达CD123的细胞系以及白血病祖细胞显示出抗白血病活性，尤其是ds-抗CD3Fv-ΔIL3。它们可能是未来AML免疫治疗的有希望的候选药物。
