Laboratory of Ligand Engineering, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV Research Center, Průmyslová 595, 252 50, Vestec, Czech Republic.
Department of Immunology, University Hospital Olomouc, Zdravotníků 248/7, 77900, Olomouc, Czech Republic.
J Transl Med. 2024 May 6;22(1):426. doi: 10.1186/s12967-024-05210-x.
Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1 lymphocytes. Due to solid tumor heterogeneity of PD-1 populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1 tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.
We designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.
Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1 populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with Galium isotope. Radiochemical purity of Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1 populations in solid tumors.
Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.
程序性细胞死亡蛋白 1（PD-1）属于免疫检查点蛋白，可确保免疫反应的负调控。在非小细胞肺癌（NSCLC）中，抗 PD-1 治疗的敏感性及其疗效，主要与肿瘤浸润 PD-1 淋巴细胞的增加有关。由于 PD-1 群体的实体瘤异质性，新型低分子量抗 PD-1 高亲和力诊断探针可提高肿瘤组织活检中 PD-1 肿瘤浸润淋巴细胞（TIL）表达谱的可靠性，并通过免疫 PET 成像提高体内定位效率。
我们通过随机化 12 个可变残基设计了一个 13 kDa 的β-折叠 Myomedin 支架组合文库，并与核糖体展示相结合，鉴定出抗 PD-1 Myomedin 变体（MBA 配体），这些变体可特异性结合人源和鼠源 PD-1 转染的 HEK293T 细胞，以及自发过表达细胞表面 PD-1 的人 SUP-T1 细胞。
通过荧光 LigandTracer 测量与转染 HEK293T 细胞表面表达的人源和鼠源 PD-1 的结合亲和力，选择最有前途的变体 MBA066（hPD-1 KD=6.9 nM；mPD-1 KD=40.5 nM）、MBA197（hPD-1 KD=29.7 nM；mPD-1 KD=21.4 nM）和 MBA414（hPD-1 KD=8.6 nM；mPD-1 KD=2.4 nM）。通过用镓同位素标记的去铁胺缀合的 MBA 进行体内成像，证明了 MBA 蛋白在体内成像 PD-1 群体的潜力。Ga-MBA 蛋白的放射化学纯度达到 94.7-99.3%，在人血清中 120 分钟后的体外稳定性在 94.6-98.2%之间。使用全身正电子发射断层扫描与计算机断层扫描（PET/CT）成像监测 Ga-MBA 蛋白在小鼠体内的分布，直至注射后 90 分钟，并在 12 个小鼠器官中进行死后检查。通过与抗 PD-1 抗体对人扁桃体和 NSCLC 组织活检的冷冻切片进行共染色，证明了 MBA 蛋白的特异性，并证明了它们在实体瘤中定位 PD-1 群体的潜力。
通过定向进化，我们开发了一组独特的小结合蛋白，可提高体外 PD-1 诊断水平，并可通过 PET/CT 成像进行体内检测。
