CRIg纳米抗体双功能分子探针对非小细胞肺癌靶向诊疗作用的研究

Tumor-associated macrophages (TAMs) are a prominent component of lung cancer stroma and contribute to tumor progression in non-small-cell lung cancer (NSCLC). These cells and biomarker represent as interesting vivo sensors for monitoring tumor microenvironment and progress. However, the methods for specific monitoring and treatment of TAMs during tumor development are still poorly defined. Complement receptor of the lg superfamily (CRIg) is expressed on rodent and human resident macrophages, mainly restricted to liver resident macrophage. CRIg is not only a receptor for complement C4, which mediates the clearance of pathogens, but also shows a suppress pattern on T cell activation and proliferation in vitro. The recent studies have shown that CRIg+ cells are found in the TAMs from patients with NSCLC. Consequently, CRIg seems to be a promising marker for target TAMs and molecular imaging of TAMs, allowing non-invasive localization of lung tumor in vivo. We aim to investigate the relationship of immune suppression mechanism mediated by CRIg in TAMs during tumor progressing and to develop a radiotracer for the visualization of CRIg for monitoring TAMs during NSCLC mice model using radiolabeled single-domain antigen-binding fragments against CRIg (Nanobodies CRIg，NbCRIg). We have reported that NbCRIg specifically bind to CRIg was excellent probes for in vivo single-photon-emission computed tomography (SPECT)/µCT imaging. In the current study, the specificity of NbCRIg targeted TAMs is going to assess via ex vivo flow cytometry and immunohistochemistry analysis in a murine lewis lung carcinoma (LLC) cell line induced NSCLC mouse model and in the tumor tissue of NSCLC patients. Second, 99mTc labeled NbCRIg is going to evaluate as radiolabeled SPECT/µCT tracer for in vivo monitoring of TAMs in untreated mice and tumor bear mice. We will gauge whether CRIg monitoring TAMs could indicate cancer progressing. Furthermore, we will further characterize the CRIg+ macrophage and fully investigate the interaction between CRIg+ macrophage and T cells in vitro. Finally, the novel treatment base on CRIg nanobody will be developed and evaluated in NSCLC tumor bear-mice. This would be of great benefit for the future theranostics of NSCLC patients.

肿瘤相关巨噬细胞（TAMs）是非小细胞肺癌（NSCLC）的固有组成部分，在免疫耐受和诱发免疫反应等过程中起关键作用。现针对TAMs准确标记和干预均无特异手段，因此TAMs特异性影像与靶向性治疗策略的建立是NSCLC治疗领域重要的科学问题。本课题组已率先构建CRIg特异性纳米抗体探针（NbCRIg）并成功用于体内外SPECT/CT成像，本课题拟通过荧光标记的NbCRIg验证其结合NSCLC患者肿瘤和荷瘤小鼠模型中TAMs的特异性；使用CRIg胞外段稳定表达细胞系和T细胞的共培养，研究CRIg抑制T细胞活性和增殖的分子机制；应用99mTcNbCRIg对荷瘤小鼠TAMs进行在体实时、可重复SPECT分子成像；最终注射NbCRIg对荷瘤小鼠进行治疗，对比其与常用抗肿瘤药物的疗效。为影像学在分子与功能水平对NSCLC中TAMs的追踪和干预提供无创、靶向治疗和实时监测同步一体化奠定全新的技术方法基础。

肿瘤相关巨噬细胞（TAMs）是非小细胞肺癌（NSCLC）的固有组成部分，在免疫耐受和诱发免疫反应等过程中起关键作用。本项目针对TAMs准确标记和干预均无特异手段的问题，以期建立TAMs特异性影像与靶向性治疗策略。本课题组利用已经构建CRIg特异性纳米抗体探针（NbCRIg），结合非小细胞肺癌荷瘤小鼠和NSCLC病人样本进行研究。首先对荷瘤动物模型生长曲线绘制和鉴定，对模型和病人样本中的肿瘤相关蛋白的mRNA表达和炎性分子表达进行了检测，利用FACS方法和组织学鉴定NbCRIg结合TAMs的特异性，并对CRIg+ TAMs亚群的极性鉴定，确定了新型分子探针NbCRIg与荷瘤小鼠TAMs结合的特异性，并在患者标本中确定新型分子探针与TAMs结合的特异性。其次对NbCRIg进行荧光影像标记的，对NbCRIg在体肿瘤成像的时间进行选择，对探针在体影像的特异性，肿瘤分布、数量及定位的准确性进行评估，并使用组织化学验证影像学结果。利用在体影像学确定了NbCRIg能够对非小细胞肺癌小鼠模型的肿瘤进行标记。最后，针对CRIg对细胞活化抑制的分子机制进行探索，结合蛋白质晶体结构解析，明确了CRIg和其受体的分子结合机制研究，揭示NbCRIg和CRIg的结合位点，确定NbCRIg为CRIg的中和抗体。此外，还首次证实NbCRIg能对多种类风湿性动物模型在体进行早期诊断，病程监控和疗效评价。最后在本项目的基础上，结合鉴于CRIg在肝脏中特异性大量表达的特点，对NbCRIg在急性肝炎的应用进行了推广，开拓了的新研究方向。