Petroková Hana, Mierzwicka Joanna Maria, Chakraborty Prosenjit, Rašková Kafková Leona, Vaculová Jana, Škarda Jozef, Fischer Ondřej, Kosztyu Petr, Kuchař Milan, Raška Milan, Malý Petr
Laboratory of Ligand Engineering, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV Research Center, Průmyslová 595, Vestec, 252 50, Czech Republic.
Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hněvotínská 3, Olomouc, 779 00, Czech Republic.
J Transl Med. 2025 Jun 13;23(1):655. doi: 10.1186/s12967-025-06699-6.
The treatment of non-small cell lung cancer (NSCLC) patients is correlated with the efficacy of immune checkpoint blockade therapy (ICB) targeting programmed cell death ligand 1 (PD-L1) or its cognate receptor (PD-1) on cancer cells or infiltrating immune cells. Analysis of PD-L1/PD-1 expression in tumor tissue represents a crucial step before PD-L1/PD-1 blocker usage.
We used directed evolution of protein variants derived from a 13 kDa Myomedin loop-type combinatorial library with 12 randomized amino acid residues to select high-affinity binders of human PD-L1 (hPD-L1). After the ribosome display, individual clones were screened by ELISA. Detailed analysis of binding affinity and kinetics was performed using LigandTracer. The specificity of Myomedins was assessed using fluorescent microscopy on HEK293T-transfected cells and cultured cancer cells in vitro, formalin-fixed paraffin-embedded (FFPE) sections of human tonsils, and FFPE tumor samples of NSCLC patients.
Seven identified PD-L1 binders, called MLE, showed positive staining for hPD-L1 on transfected HEK293T cells and cultured MCF-7 cells. MLE031, MLE105, MLE249, and MLE309 exhibited high affinity to both human and mouse PD-L1-transfected HEK293T cells measured with LigandTracer. The diagnostic potential of MLE variants was tested on human tonsillitis tissue and compared with diagnostic anti-PD-L1 antibody DAKO 28-8 and PD-L1 IHC 22C3 pharmDx antibody. MLE249 and MLE309 exhibited an excellent overlap with diagnostic DAKO 28-8 (Pearson´s coefficient (r) = 0.836 and 0.731, respectively) on human tonsils on which MLE309 exhibited also excellent overlap with diagnostic 22C3 antibody (r = 0.876). Using three NSCLC tissues, MLE249 staining overlaps with 28-8 antibody (r = 0.455-0.883), and MLE309 exhibited overlap with 22C3 antibody (r = 0.534-0.619). Three MLE proteins fused with Fc fragments of rabbit IgG, MLE249-rFc, MLE309-rFc and MLE031-rFc, exhibited very good overlap with anti-PD-L1 antibody 28-8 on tonsil tissue (r = 0.691, 0.610, and 0.667, respectively). Finally, MLE249-rFc, MLE309-rFc and MLE031-rFc exhibited higher sensitivity in comparison to IHC 22C3 antibody using routine immunohistochemistry staining system Ventana, which is one of gold standards for PD-L1 diagnosis.
We demonstrated the development of MLE Myomedins specifically recognizing hPD-L1 that may serve as a refinement tool for clinical PD-L1 detection.
非小细胞肺癌（NSCLC）患者的治疗与针对癌细胞或浸润免疫细胞上程序性细胞死亡配体1（PD-L1）或其同源受体（PD-1）的免疫检查点阻断疗法（ICB）的疗效相关。在使用PD-L1/PD-1阻断剂之前，分析肿瘤组织中PD-L1/PD-1的表达是关键步骤。
我们利用来自一个具有12个随机氨基酸残基的13 kDa肌动蛋白环型组合文库的蛋白质变体的定向进化来筛选人PD-L1（hPD-L1）的高亲和力结合物。核糖体展示后，通过酶联免疫吸附测定（ELISA）筛选各个克隆。使用配体追踪仪对结合亲和力和动力学进行详细分析。通过荧光显微镜在体外转染人胚肾293T（HEK293T）细胞和培养的癌细胞、人扁桃体的福尔马林固定石蜡包埋（FFPE）切片以及NSCLC患者的FFPE肿瘤样本上评估肌动蛋白的特异性。
七个鉴定出的PD-L1结合物，称为MLE，在转染的HEK293T细胞和培养的MCF-7细胞上对hPD-L1呈阳性染色。使用配体追踪仪测量，MLE031、MLE105、MLE249和MLE309对人和小鼠PD-L1转染的HEK293T细胞均表现出高亲和力。在人扁桃体炎组织上测试了MLE变体的诊断潜力，并与诊断性抗PD-L1抗体DAKO 28-8和PD-L1免疫组化（IHC）22C3药物诊断抗体进行比较。MLE249和MLE309在人扁桃体上与诊断性DAKO 28-8表现出极好的重叠（皮尔逊系数（r）分别为0.836和0.731），其中MLE309与诊断性抗体22C3也表现出极好的重叠（r = 0.876）。使用三个NSCLC组织，MLE249染色与28-8抗体重叠（r = 0.455 - 0.883），MLE309与22C3抗体重叠（r = 0.534 - 0.619）。三种与兔免疫球蛋白G（IgG）的Fc片段融合的MLE蛋白，即MLE249-rFc、MLE309-rFc和MLE031-rFc，在扁桃体组织上与抗PD-L1抗体28-8表现出非常好的重叠（r分别为0.691、0.610和0.667）。最后，使用作为PD-L1诊断金标准之一的常规免疫组化染色系统Ventana，与IHC 22C3抗体相比，MLE249-rFc、MLE309-rFc和MLE031-rFc表现出更高的敏感性。
我们展示了特异性识别hPD-L1的MLE肌动蛋白的开发，其可作为临床PD-L1检测的优化工具。
