Production of a novel bispecific protein ULBP1×CD19-scFv targeting the NKG2D receptor and CD19 to promote the activation of NK cells

• Published in: Protein expression and purification Vol. 178; p. 105783
• Main Authors: Zhao, Qing, Pang, Jie, Yan, Fushan, Jiang, Yi, Cui, Dongxu, Liu, Juanjuan, Jing, Lei, Li, Yuyin, Liu, Zhenxing, Tao, Li, Zhao, Xiaocui, Diao, Aipo
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.02.2021
• Subjects: Bispecific protein; CD19; Nature killer cell; Pichia pastoris; ULBP1; Bispecific protein; Nature killer cell; Pichia pastoris; ULBP1; CD19
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2020.105783

Natural killer (NK) cells are potent cytotoxic effector cells of the innate immune system and play an important role in tumor immunosurveillance and control. NKG2D is an activating receptor of NK cells. The NKG2D receptor-ligand system has contributed to immune cells recognizing tumor cells and the tumor microenvironment. In order to stretch the application of NK cells on adoptive immunotherapy for B-cell malignancies, we designed and produced a novel bispecific ULBP1×CD19-scFv fusion protein, in which the extracellular domain of NKG2D ligand ULBP1 was fused to a single chain variable fragment (scFv) of anti-CD19. The vector expressing ULBP1×CD19-scFv protein was constructed and expressed in Pichia pastoris. Effects of medium composition, concentration of methanol as the inducer, induction time and broth content in shake flask on the expression of the recombinant protein were investigated. The results showed that the optimized conditions for ULBP1×CD19-scFv expression were 1% methanol induction for 96 h with 15% broth content. The secreted recombinant protein was purified using ammonium sulfate fractionation and Ni-NTA affinity chromatography and the purity is about 93%. The cytotoxicity of NK92-MI cells against CD19+ Raji cells was enhanced in the presence of purified ULBP1×CD19-scFv protein. These results indicated that ULBP1 could be used as an activating element of bispecific killer engagers (BiKEs) and Pichia pastoris yeast might be an alternative expression host for BiKEs production. •A novel bispecific ULBP1 × CD19-scFv fusion protein was constructed.•ULBP1 × CD19-scFv fusion protein was expressed in P. pastoris.•Expression condition of ULBP1 × CD19-scFv fusion protein was optimized.•ULBP1 × CD19-scFv fusion protein was purified by ammonium sulfate precipitation and His-tag affinity chromatography.•Presence of ULBP × CD19-scFv increased cytotoxic activity of NK-92 MI cells against Raji cells in vitro.