靶向肿瘤干细胞人源Dll4抗体抗肿瘤活性及机制研究

Although the conventional treatment of cancer has the ability to inhibit tumor growth, it cannot eliminate all of tumor cells completely. For patients, clinicians, and cancer biologist, the most frustrating feature of malignancies is their adaptability which translate into drug-resistant cancer recurrence and metastasis, often after clinical and even pathological complete respones...One possible explanation for the biological plasticity of cancer is their cellular heterogeneity. In recent year, a distinct cellular hierarchy has been identified in several hematopoietic and solid tumor. Many cancers seem to contain a small population of pluripotent "tumor initiating cells" or "cancer stem cells" (CSC). The CSC hypothesis states that CSC possess some of the biological properties of normal stem cells, including indefinite selfreplication, asymmetric cell division, and resistance to toxic agents...In recent days, accumulation evidence to date for a role of Delta like 4 (Dll4), a ligand of Notch signaling pathway, contributes to cancer stem cell self-renewal in several hematopoietic and solid tumor and involves in tumor angiogenesis. ..In our research, combination with in vitro antibody affinity maturation technology, anti-hDll4 scFV will be generated from phage antibody library. Then the scFV gene was expressed in mammalian cell by fusion with human IgG1 constant region for full-length antibody. When the Notch was blocked by anti-hDll4 antibody, the anti- proliferation effect of antibody to cancer stem cell was performed by colony formation and in vivo limiting dilution assay. And the anti- angiogenesis activity of the antibody to cancer cell was carried out by cell migration assay and thoracic aorta rings. The mechanisms of inhibiting cancer stem cell self-renewal and tumor angiogenesis was dissected by RT-PCR and Western blot. Finally, the inhibition of tumor growth degree of anti-hDll4 antibody was determined by NOD/SCID mice xenograft models. In our research aims to provide a novel strategy to overcome cancer stem cell and tumor angiogenesis.

目前，针对肿瘤的传统疗法虽能杀灭大部分肿瘤细胞，但无法根除肿瘤。肿瘤干细胞学说认为，肿瘤组织中存在一群与正常干细胞类似，具有自我更新和增殖能力的肿瘤干细胞。Notch信号通路的一个配体Dll4，通过与Notch受体相互作用参与肿瘤干细胞的自我更新、增殖和肿瘤血管生成。.在本研究中，以人Dll4为抗原，结合体外亲和力成熟技术，从天然全人源噬菌体抗体库中筛选出高亲和力单链抗体，连接人IgG1恒定区，通过电转化法转入哺乳动物细胞融合表达全人源全长抗Dll4抗体；通过阻断Dll4与受体相互作用，利用克隆形成法、体内有限稀释法等方法测定抗体抗肿瘤干细胞增殖效果，以划痕损伤修复和主动脉环等方法测定抗体抗血管生成能力；借助RT-PCR、Western blot技术研究抗体抗肿瘤干细胞增殖及抗血管生成机制，并用无胸腺小鼠嫁接瘤模型研究抗Dll4抗体体内抗肿瘤活性，为有效防治肿瘤提供新理论和药物新靶点。

Dll4（ Delta like 4） 是 Notch 信号通路中 Notch 受体的一个重要配体，通过与相邻细胞表面 Notch 受体的结合，参与调控毗邻细胞的增殖、凋亡、 分化以及干细胞增殖和更新等等生物学过程。我们的研究对象是本实验室利用杂交瘤技术筛选得到的能够特异性靶向人 DLL4 抗原、阻断 DLL4/Notch 信号通路并有效抑制肿瘤生长的抗人 Dll4 抗体 MMGZ01，并应用了一种基于计算机结构模拟的方法，对 MMGZ01 进行人源化改造，设计得到了包括互补决定区 (complementarily determining regions, CDR)移植抗体在内的 7 种人源化抗体， 并从中筛选出亲和力最为理想的抗体，为了方便检测，我们设计合成了 MMGZ01 的嵌合抗体作为人源化抗体的参照。本研究通过细胞水平、 组织水平以及整体动物水平等多层次研究，综合评价了抗人 Dll4 人源化抗体体内外抗血管生成以及抗肿瘤活性；初步探索了抗人 Dll4 人源化抗体促进无功能血管生成的机制；探究了抗人 Dll4 人源化抗体对肿瘤干细胞的形成与自我更新能力的抑制作用，并进一步研究了抗人 Dll4 人源化抗体对人乳腺癌的体内抑瘤活性。因此，本研究对象抗人 Dll4 人源化抗体具有高亲和性和高生物学活性，有希望成为靶向肿瘤治疗的候选抑制剂，为肿瘤治疗提供了新的思路， 具有潜在的临床应用前景，同时也为后续 Notch 信号通路的研究奠定了良好基础。本项目发表SCI论文9篇，其中5分以上论文2篇，申请专利2项，取得了很好的预期效果。