Rong Liang, Lim Rebecca M, Yin Xiaosuo, Tan Liyao, Yang Jae H, Xie Jianming
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA; Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089, USA.
J Mol Biol. 2022 Apr 30;434(8):167513. doi: 10.1016/j.jmb.2022.167513. Epub 2022 Feb 24.
We have previously developed a universal chimeric antigen receptor (CAR), which recognizes dinitrophenyl (DNP) and can redirect T and NK cells to target cancer and HIV antigens using DNP-conjugated antibodies as adaptor molecules. However, the DNP-antibody conjugates are generated by random modification, which may not be optimal for this modular system. Here, we report the development of enhanced adaptor molecules by site-specific DNP modification. We use the genetic code expansion technology to generate single-chain fragment variable (scFv) antibodies with site-specific DNP. We compare four anti-CD19 scFv mutants and find that the one with DNP at the flexible peptide linker between V and V is the most effective in redirecting anti-DNP CAR-T cells against CD19 cells. The other three mutants are ineffective in doing so due to reduced DNP exposure or abrogated CD19 binding. We also use the anti-CD22 scFv as another model adaptor molecule and again find that the peptide linker is ideal for DNP derivatization. Our approach can potentially be used to design enhanced adaptor molecules to redirect the DNP-mediated universal CAR against other tumor antigens.
我们之前开发了一种通用嵌合抗原受体（CAR），它能识别二硝基苯基（DNP），并可利用与DNP偶联的抗体作为衔接分子，将T细胞和自然杀伤（NK）细胞重定向至靶向癌症和HIV抗原。然而，DNP-抗体偶联物是通过随机修饰产生的，这对于该模块化系统可能并非最佳选择。在此，我们报告了通过位点特异性DNP修饰开发增强型衔接分子的情况。我们利用遗传密码扩展技术生成具有位点特异性DNP的单链可变片段（scFv）抗体。我们比较了四种抗CD19 scFv突变体，发现V与V之间柔性肽接头处带有DNP的突变体在将抗DNP CAR-T细胞重定向至靶向CD19细胞方面最为有效。其他三种突变体由于DNP暴露减少或CD19结合被消除而无效。我们还使用抗CD22 scFv作为另一种模型衔接分子，再次发现该肽接头对于DNP衍生化是理想的。我们的方法有可能用于设计增强型衔接分子，以将DNP介导的通用CAR重定向至靶向其他肿瘤抗原。
