Thayer School of Engineering, Dartmouth College, 14 Engineering Dr, Hanover, NH 03755, USA.
Department of Microbiology and Immunology, Geisel School of Medicine, Dartmouth College, 1 Medical Center Dr, Lebanon, NH 03756, USA.
Protein Eng Des Sel. 2017 Oct 1;30(10):713-721. doi: 10.1093/protein/gzx051.
As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated. A representative clone re-formatted as an scFv-CH1-Fc molecule demonstrated specific binding to both B7H6-Ig and native membrane-bound B7H6 on tumor cell lines with a binding avidity comparable to the previously characterized B7H6-targeting antibody, TZ47. Furthermore, these clones recognized an epitope distinct from that of TZ47 and the natural NK cell ligand NKp30, and demonstrated specific activity against B7H6-expressing tumor cells when expressed as a chimeric antigen receptor (CAR) in T cells.
作为一种应激诱导的自然杀伤 (NK) 细胞配体，B7H6 在先天肿瘤免疫监视中发挥作用，是一种在多种实体瘤和血液系统恶性肿瘤细胞上表达的相当肿瘤选择性标志物。在这里，我们描述了一种针对人 B7H6 配体的新型单链片段可变 (scFv) 分子的分离和鉴定。通过对酵母表面展示的非免疫人源 scFv 文库进行定向进化，分离到了 8 个候选 scFv 克隆，它们属于一个家族，彼此之间的差异最多可达 4 个氨基酸突变，对可溶性 B7H6-Ig 的亲和力达到纳摩尔级。代表性的 scFv-CH1-Fc 分子重构成 scFv-CH1-Fc 分子，可特异性结合肿瘤细胞系上的 B7H6-Ig 和天然膜结合的 B7H6，其结合亲和力与先前鉴定的 B7H6 靶向抗体 TZ47 相当。此外，这些克隆识别与 TZ47 和天然 NK 细胞配体 NKp30 不同的表位，当在 T 细胞中作为嵌合抗原受体 (CAR) 表达时，对表达 B7H6 的肿瘤细胞具有特异性活性。
