Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, 200240, Shanghai, People's Republic of China.
Institute of Translational Medicine, Shanghai Jiao Tong University, 800 Dongchuan Road, 200240, Shanghai, People's Republic of China.
BMC Cancer. 2022 Oct 25;22(1):1092. doi: 10.1186/s12885-022-10165-7.
Antibodies and derivative drugs targeting immune checkpoints have been approved for the treatment of several malignancies, but there are fewer responses in patients with pancreatic cancer. Here, we designed a nanobody molecule with bi-targeting on PD-L1 and CXCR4, as both targets are overexpressed in many cancer cells and play important roles in tumorigenesis. We characterized the biochemical and anti-tumour activities of the bispecific nanobodies in vitro and in vivo.
A nanobody molecule was designed and constructed. The nanobody sequences targeting PD-L1 and CXCR4 were linked by the (GS) flexible peptide to construct the anti-PD-L1/CXCR4 bispecific nanobody. The bispecific nanobody was expressed in E. coli cells and purified by affinity chromatography. The purified nanobody was biochemically characterized by mass spectrometry, Western blotting and flow cytometry to confirm the molecule and its association with both PD-L1 and CXCR4. The biological function of the nanobody and its anti-tumour effects were examined by an in vitro tumour cell-killing assay and in vivo tumour inhibition in mouse xenograft models.
A novel anti-PD-L1/CXCR4 bispecific nanobody was designed, constructed and characterized. The molecule specifically bound to two targets on the surface of human cancer cells and inhibited CXCL12-induced Jurkat cell migration. The bispecific nanobody increased the level of IFN-γ secreted by T-cell activation. The cytotoxicity of human peripheral blood mononuclear cells (hPBMCs) against pancreatic cancer cells was enhanced by the molecule in combination with IL-2. In a human pancreatic cancer xenograft model, the anti-PD-L1/CXCR4 nanobody markedly inhibited tumour growth and was superior to the combo-treatment by anti-PD-L1 nanobody and anti-CXCR4 nanobody or treatment with atezolizumab as a positive control. Immunofluorescence and immunohistochemical staining of xenograft tumours showed that the anti-tumour effects were associated with the inhibition of angiogenesis and the infiltration of immune cells.
These results clearly revealed that the anti-PD-L1/CXCR4 bispecific nanobody exerted anti-tumour efficacy in vitro and inhibited tumour growth in vivo. This agent can be further developed as a therapeutic reagent to treat human pancreatic cancer by simultaneously blocking two critical targets.
针对免疫检查点的抗体和衍生药物已被批准用于治疗多种恶性肿瘤，但在胰腺癌患者中的反应较少。在这里，我们设计了一种针对 PD-L1 和 CXCR4 的双靶向纳米抗体分子，因为这两个靶点在许多癌细胞中过度表达，并在肿瘤发生中发挥重要作用。我们在体外和体内表征了双特异性纳米抗体的生化和抗肿瘤活性。
设计并构建了一种纳米抗体分子。靶向 PD-L1 和 CXCR4 的纳米抗体序列通过（GS）柔性肽连接，构建了抗 PD-L1/CXCR4 双特异性纳米抗体。双特异性纳米抗体在大肠杆菌细胞中表达，并通过亲和层析纯化。通过质谱、Western blot 和流式细胞术对纯化的纳米抗体进行生化表征，以确认分子及其与 PD-L1 和 CXCR4 的结合。通过体外肿瘤细胞杀伤试验和小鼠异种移植模型中肿瘤抑制来研究纳米抗体的生物学功能及其抗肿瘤作用。
设计、构建和表征了一种新型抗 PD-L1/CXCR4 双特异性纳米抗体。该分子特异性结合人癌细胞表面的两个靶标，并抑制 CXCL12 诱导的 Jurkat 细胞迁移。双特异性纳米抗体增加了 T 细胞激活后 IFN-γ 的分泌水平。该分子与 IL-2 联合增强了人外周血单核细胞（hPBMC）对胰腺癌细胞的细胞毒性。在人胰腺癌细胞异种移植模型中，抗 PD-L1/CXCR4 纳米抗体显著抑制肿瘤生长，优于抗 PD-L1 纳米抗体和抗 CXCR4 纳米抗体联合治疗或阳性对照药物阿特珠单抗的治疗效果。异种移植瘤的免疫荧光和免疫组织化学染色显示，抗肿瘤作用与抑制血管生成和免疫细胞浸润有关。
这些结果清楚地表明，抗 PD-L1/CXCR4 双特异性纳米抗体在体外发挥抗肿瘤疗效，并在体内抑制肿瘤生长。该药物可进一步开发为通过同时阻断两个关键靶点治疗人类胰腺癌的治疗试剂。
