靶向AML-LSC的新型嵌合抗原受体3A4-CAR基因的构建及优化

Leukemia stem cells (LSC) are the main cause of relapse due to their resistence to conventional chemotherapy. Recent studies on CD19 modified chimeric antigen receptor (CD19-CAR) T cells have demonstrated the promising results for refractory B-lineage malignancies. However, studies on the use of this technique based on CD45 isotype antibody for targeting AML and LSC have been NOT reported. Our previous findings showed that our new antibody ZCH (Zhejiang Children's Hospital)-6-3A4（3A4, muring IgG1），an anti-human CD45 isotype antibody, have the ability to recognize both newly diagnosed and relapsed leukemia cells. 3A4-NCTD (norcantharidin) immunotoxin studies have been proved to have the significant targeting killing effect on AML cells or therapeutic outcome of leukemia animal models. However, the affinity and the killing effect of this antibody will be significantly reduced after humanization. The aim of this project is to study the preparation of our novel monoclonal antibody based 3A4-CAR T cells consisting of 3A4-CD8-CD137 (4-1BB)-ζ，3A4-CD8-CD137-ζ-IL15，and suicide gene inducible caspase 9 (iCasp9) gene containing iCasp9-CD8-3A4-CD137-ζ and iCasp9-3A4-CD8-CD137-ζ-IL15 CAR genes which are to be inserted into the lentiviral vector pELNS to transfect the normal allogeneic peripheral blood mononuclear cells (MNC) to generate 3A4-CAR T cells. The targeting killing effects on AML cells and their LSCs by those 3A4-CAR modified T cells will be comprehensively studied and explored in both in vitro and in vivo settings using AML leukamia cell coculture and NOD/SCID mouse models for further optimization. It is expected that this study will provide the essential basis for 3A4-CARs to targeting the relapsed or refractory AML and their AML-LSCs, which will be of great important significance and clinical application potentials.

白血病干细胞（LSC）是该病复发的根源，常规化疗多耐药。CD19-嵌合抗原受体（CD19-CAR）T细胞在克服B系肿瘤耐药的临床试验中获成功，但用CD45亚类-CAR T细胞靶向AML及其LSC则国内外未见报道。研究发现，新单抗ZCH-6-3A4（简称3A4，CD45亚类）能识别初诊及复发难治AML及其LSC，有明显的靶向杀伤/治疗作用，但当该抗体经人源化改造后亲和力下降。本项目拟构建3A4-CD137-ζ CAR，插入慢病毒载体pELNS后转染正常人血MNC研制新型CAR-T细胞，通过体外实验和动物模型鉴定其对AML的靶向杀伤作用。并将iCaspase9自杀基因和/或IL-15基因整合到该CAR结构中，通过比较其扩增、杀伤能力及持续存活时间的变化，进一步优化并筛选功能最佳的3A4 CAR。本课题将为3A4-CAR T细胞治疗难治性AML奠定基础，具有十分重要的研究意义和潜在的应用前景。

急性白血病是严重威胁人类生命的血液系统恶性肿瘤，虽然联合化疗已经取得了明显的进步，但仍有20-50%的病例因复发死亡，白血病干细胞（LSC）是该病复发的根源。近年来，CD19嵌合抗原受体（CD19CAR）T细胞在克服难治性B系肿瘤中获得巨大成功，但利用该技术靶向AML及其LSC则少见报道。课题组前期研究发现，新单抗3A4能识别90%以上的初诊及复发难治性AML及其LSC，有明显的靶向杀伤/治疗作用，但利用CAR细胞技术研究该抗体识别靶点的研究国内外未见报道。本项目通过4年的潜心研究，成功构建并筛选到体内外靶向杀伤/治疗人复发难治性AML及其LSC的最佳二代3A4CAR基因，并成功构建了pcDNA3.1真核表达载体（3A4CAR-pcDNA3.1）及慢病毒表达载体(3A4CAR-pLenti)，两者均能成功转（感）染人外周血T细胞，其瞬时转（感）染效率分别为19%和88%，慢病毒载体的转染效率明显高于pcDNA3.1载体。慢病毒稳定感染效率为50%~62%。体外实验证明，3A4CAR-T细胞共孵育5小时对AML细胞株KG1a和病人原代复发难治性白血病细胞的靶向杀伤效率为38%-63%，其杀伤机制是3A4CAR-T细胞通过上调颗粒酶B和释放CD107a阳性颗粒而发挥靶向杀伤作用。白血病动物模型体内实验证明，3A4CAR-T细胞能发挥明显的靶向治疗作用，100天AML模型有效率为50%，明显高于模型鼠和普通T细胞治疗组，后两组小鼠在63天内全部死亡，差异具有显著意义（p = 0.000)。结论：本项目成功研制优化筛选到体内外最佳二代3A4CAR基因；3A4CAR-T细胞体内外均能发挥明显的靶向杀伤和治疗作用。该研究为进一步开发基于3A4的免疫治疗奠定了重要的工作基础，具有重要意义和潜在的临床应用前景。