Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a K_d of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant Z_CD69:4 had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule Z_CD69:4 is suitable for further development for imaging of activated immune cells.

由于不同类型疾病的炎症过程范围广泛，因此需要更敏感和更特异的生物标志物来改善预防和治疗。分化簇 69 (CD69) 是最早由活化白细胞表达的细胞表面蛋白之一。在这里，我们表征并优化了潜在的新成像探针，即靶向 CD69 的 Affibody 分子，用于对激活的免疫细胞进行成像。分析先前从 Z 变体大肠杆菌中进行的选择中分离出的候选物人 CD69 的重组胞外域的文库，鉴定了一种对鼠和人 CD69 具有亲和力的交叉反应性 Z 变体。通过随机化主要 Z 变体，然后从库中进行选择来进行亲和力成熟。评估了所得 Z 变体对人和鼠 CD69 的亲和力和热稳定性。在通过 DOTA 螯合剂结合 Z 变体并用 Indium-111 进行放射性标记后，通过 SPECT/CT 在大鼠中评估体内生物分布。具有 K _{d 的}主要 Z 变体鉴定出对人和鼠 CD69 的亲和力约为 50 nM。亲和力成熟产生了 5 个额外的 Z 变体，它们具有改进的或相似的亲和力。所有克隆都表现出合适的稳定性。大鼠的放射性标记和体内生物分布表明，所有变体的肾清除速度都很快，而背景吸收和清除各不相同。变体 Z _CD69:4对人和鼠 CD69 (34 nM) 的亲和力最高，体内背景结合最低。总之，我们描述了对 CD69 具有亲和力的新型 Affibody 分子的发现、优化和评估。Affibody 分子 Z _CD69:4适用于进一步开发活化免疫细胞的成像。