新颖靶向AML-LSC的人源化3A4-CD3双特性抗体基因的优化表达及靶向杀伤机制研究

Relapsed/refractory acute leukemia is a serious clinical problem and leukemia stem cell (LSC) is believed to be the cause of relapse. CD3-CD19 bispecific T cell engager (BiTE) has being demonstrated very promising treatment outcomes for patients with r/r B lineage malignancies, however, early relapses occurs frequently after BiTE treatment, which may be caused by the induction of the human anti-mouse immunoglobulin antibody (HAMA). Our previous studies have showed that 3A4, a novel murine IgG1k antibody (designated as CD45RA by HLDA-7) was generated in our laboratory. This antibody can recognize almost all relapsed AML cells and about 90% of AML-LSC but not react with clinically important normal cell components which may offer a potential therapeutic target for those patients. However, no data regarding CD45RA or CD45RA-CD3 BiTE as targeting activity have yet been reported in the world. This project will focus on the genetic design, optimization and expression of mouse and humanized 3A4-CD3 BiTE antibody in Chinese hamster ovary (CHO) cells and the targeting activity and its mechanism of this BiTE antibody will be studied systemically using a series of cellular and moleculobiological approaches and mouse model, which is of great scientific significance and clinical application potential in the near future.

复发难治性白血病是临床难题，白血病干细胞(LSC)是其复发的根源。CD3-CD19双特异性抗体（CD3-CD19 BiTE）治疗B系肿瘤的临床试验结果良好，但重复应用易耐药，可能与鼠源性BiTE易诱发人抗鼠抗体（HAMA）有关。课题组前期研究发现，自制新单抗3A4（CD45RA）能识别几乎100%复发的AML细胞和近90％的AML-LSC，3A4识别抗原可能是良好的免疫治疗新靶点，但国际上尚无有关该靶点的研究报道，更无CD45RA-CD3 BiTE抗体相关的研究。本项目拟采用一系列细胞及分子生物学方法构建鼠源性和人源化双特异性小分子（55kDa）和高分子(105kDa) 3A4-CD3 BiTE抗体基因并优化结构后在CHO细胞中表达，鉴定其生物学特性及体内外靶向杀伤AML及其LSC的作用及机制，为进一步开发该新颖抗体药物制剂提供实验依据，具有重要的科学意义和潜在的临床应用前景。

复发难治性白血病是临床难题，而白血病干细胞是复发的根源。CD3-CD19双特异性抗体（CD3-CD19 BiTE）治疗B系肿瘤的临床疗效良好。课题组前期研究发现，自制新单抗3A4（CD45亚类）能识别几乎100%复发的AML细胞和近90％的AML-LSC，可能是良好的新靶点。本项目经4年时间的潜心研究，成功设计、优化和构建了鼠源性CD3-3A4 双特异性抗体（MuCD3-3A4 BiTE）和人源化CD3-3A4 BiTE（HuCD3-3A4 BiTE1和HuCD3-3A4 BiTE2）基因，通过构建pCDNA3.1真核表达载体，转染中华仓鼠卵巢（CHO）细胞中，在G418加压下克隆化三次，成功建立了MuCD3-3A4 BiTE-CHO细胞株一株和HuCD3-3A4 BiTE-CHO细胞株二株。三个细胞株细胞培养上清经流式细胞术、免疫组化和RT-PCR等方法检测，证实三个细胞株所分泌的BiTE抗体具有生物学活性，RT-PCR检查发现CHO细胞株中存在CD3-3A4 BiTE抗体基因。培养上清经浓缩50倍后，以效靶比1:1，48小时共培养后的靶细胞杀伤率达到(82.03±5.12)%，明显高于3A4靶点阴性的对照组Nalm-6细胞（1.21±0.24）%，（P < 0.0001）。Western blot研究三个BiTE抗体蛋白的分子量约为55kda，与设计结果相符。由于受新冠肺炎疫情的影响，该项目的动物体内实验尚在进行中，预计2022年6月30日前能全部完成。结论：本项目成功构建了鼠源性和人源化CD3-3A4 BiTE-CHO细胞株，所分泌的BiTE抗体具有良好的生物学活性，为进一步开发治疗性制剂奠定了基础。