急性髓细胞白血病（AML）是一种难治性，复发率和死亡率高的血液系统恶性肿瘤。白血病干细胞（LSC）的残留被认为是AML耐药和复发的根源。前期研究显示CD45RA在AML-LSC上高表达，可作为AML治疗的一个良好靶点。申请者前期成功构建靶向CD45RA的单抗，并获得其人源化抗体，但该抗体存在靶向杀伤作用不强的局限性。而双特异性抗体分子能克服上述局限，故本次研究首先构建人源化双特异性抗体分子CD3xCD45RA和CD16xCD45RA，并初步研究上述抗体功能与作用机制；由于单一靶标在治疗难治/复发性AML时可能不足，而CD33分子在多数AML患者肿瘤细胞及AML-LSC上高表达，故本研究同时探索构建人源化三特异性抗体CD33xCD3xCD45RA和CD33xCD16xCD45RA，通过体内外实验研究其靶向杀伤特性、安全性与有效性及相应的分子机制，为AML治疗性新药的开发打下理论与实验基础。

Acute myeloid leukemia (AML) is a common type of acute hematologic malignancy, with a high refractory or relapsed rate, maybe due to incomplete eradication of the leukemia stem cells (LSC). The CD45RA antigen is a specific marker for AML-LSC. Previously, we have generated a new anti-CD45RA humanized antibody hscFv3A4-Fc, with a high affinity and a targeting killing activity on tumor cells in our laboratory. To enhance the clinical efficacy of hscFv3A4-Fc antibody, we will humanize the anti-CD3 and anti-CD16 antibodies, then construct humanized bispecific antibodies CD3xCD45RA and CD16xCD45RA targeting both CD45RA and CD3 or CD16 antigens, since the bispecific antibody could have superior properties to its parental mAb alone or even a combination of both. As CD33 is expressed on the majority of myeloid blasts and LSC, we will construct humanized trispecific antibodies CD33xCD3xCD45RA and CD33xCD16xCD45RA targeting both CD45RA and CD33 with effector of T or NK cells. The effect and molecular mechnism will be explored both in bispecific and trispecific antibodies targeting for AML cells in vitro. Furthermore, we will develop a AML mouse model to estimate the safety and efficiency of targeting AML with the bispecific antibodies or trispecific antibodies.