基于PD-L1肿瘤非侵入性精准检测的Affibody构建以及体内评价研究

Tumor immunotherapy represented by PD-1/PD-L1 antibody has shown great advantages and development potential as an innovative treatment. However, PD-1/PD-L1 antibody treatment is only effective in approximately 20-30% of tumor patients, and the effectiveness of treatment in PD-L1-positive patients in some tumors can be greatly improved. The existing PD-L1 detection relies on tumor biopsy, and since PD-L1 is only distributed in part of the tumor, the accuracy of detection is generally low, and it is urgent to develop a new detection strategy. We have previously demonstrated specific imaging of PD-L1 positive tumors in a mouse model by PD-L1 single-chain antibody-coupled isotopes. However, the molecular weight of the single-chain antibody is relatively large, and the retention time in the body is long, and the coupled isotope has certain damage to the human body. As a high affinity and high stability peptide, Affibody has a fast metabolism and unique advantages in molecular detection. We intend to construct an Affibody screening platform to screen small peptide molecules that recognize transmembrane proteins such as PD-L1, which are used for non-invasive detection of tumors. It is expected to replace biopsy to improve the accuracy of detection, screening clinical patients for patients in the future. Accurate treatment lays the foundation for theory and experimentation.

以PD-1/PD-L1抗体为代表的肿瘤免疫治疗作为一种创新的治疗方式已显示出了巨大的优势和发展潜力。但是，PD-1/PD-L1抗体治疗仅对大约20-30%的肿瘤病人有效，而在部分肿瘤中PD-L1阳性的病人治疗的有效率可以大幅度提高。现有的PD-L1检测依赖于肿瘤穿刺活检，而由于PD-L1仅分布在肿瘤部分区域，检测的准确性普遍偏低，亟需研发新的检测策略。我们前期通过PD-L1单链抗体偶联同位素显示可在小鼠模型上对PD-L1阳性肿瘤特异性成像。但是单链抗体分子量还比较大，在体内滞留时间长，偶联的同位素对人体有一定的伤害。Affibody作为高亲和力高稳定性的多肽，代谢较快，在分子检测方面有独特的优势。我们拟构建Affibody筛选平台，筛选能识别PD-L1等跨膜蛋白的多肽小分子，用于肿瘤非侵入性检测，有望代替穿刺活检，提高检测的准确性，为未来筛选临床病人，对病人进行精准治疗奠定理论和实验基础。

PD-1/PD-L1抗体治疗仅对大约20-30%的肿瘤病人有效，而在部分肿瘤中PD-L1阳性的病人治疗的有效率可以大幅度提高。现有的PD-L1检测依赖于肿瘤穿刺活检，而由于PD-L1仅分布在肿瘤部分区域，检测的准确性普遍偏低，亟需研发新的检测策略。Affibody作为高亲和力高稳定性的多肽，代谢较快，在分子检测方面有独特的优势。.本研究我们成功构建了Affibody噬菌体文库，通过噬菌体表面筛选技术，能够得到与PD-L1特异性结合的亲和配体。将Affibody中的13个可变氨基酸位点设计为NNK的形式并合成引物，形成双链后插入到pCom3bXSS噬菌体质粒中。并且我们比较了两种建库的方式，通过PCR形成双链建库的效率是通过退火形成双链的5倍。而且我们发现生长克隆数目也会随着转化DNA的量有梯度增长关系，并且存在转化上限。转化重组质粒到TG1中。我们得到了库容为6.25×109的Affibody文库。在建库的基础上我们开展固相淘选，筛选到与PD-L1特异性结合的亲和配体，并评估了Affibody分子在荷瘤小鼠体内的肿瘤靶向能力和动态分布。我们构建的Affibody筛选平台，筛选能识别PD-L1等跨膜蛋白的多肽小分子，用于肿瘤非侵入性检测，有望代替穿刺活检，提高检测的准确性，为临床检测肿瘤环境中PD-L1的表达提供了科学依据和实验基础。因此本研究具有重要的临床现实意义和良好的应用前景。.本研究成果将为解决现有的PD-L1检测困境提供一种新的思路。