Lam Norris, Finney Richard, Yang Shicheng, Choi Stephanie, Wu Xiaolin, Cutmore Lauren, Andrade Jorge, Huang Lei, Amatya Christina, Cam Margaret, Kochenderfer James N
National Institutes of Health, National Cancer Institute, Center for Cancer Research, Surgery Branch, Bethesda, MD, USA.
National Institutes of Health, National Cancer Institute, Center for Cancer Research, Office of the Director, Bethesda, MD, USA.
Mol Ther Oncolytics. 2023 Jul 19;30:132-149. doi: 10.1016/j.omto.2023.07.001. eCollection 2023 Sep 21.
To address CD19 loss from lymphoma after anti-CD19 chimeric antigen receptor (CAR) T cell therapy, we designed a bicistronic construct encoding an anti-CD19 CAR and an anti-CD20 CAR. We detected deletions from the expected bicistronic construct sequence in a minority of transcripts by mRNA sequencing. Loss of bicistronic construct transgene DNA was also detected. Deletions of sequence were present at much higher frequencies in transduced T cell mRNA versus gamma-retroviral vector RNA. We concluded that these deletions were caused by intramolecular template switching of the reverse transcriptase enzyme during reverse transcription of gamma-retroviral vector RNA into transgene DNA of transduced T cells. Intramolecular template switching was driven by repeated regions of highly similar nucleic acid sequence within CAR sequences. We optimized the sequence of the bicistronic CAR construct to reduce repeated regions of highly similar sequences. This optimization nearly eliminated sequence deletions. This work shows that repeated regions of highly similar nucleic acid sequence must be avoided in complex CAR constructs. We further optimized the bicistronic construct by lengthening the linker of the anti-CD20 single-chain variable fragment. This modification increased CD20-specific interleukin-2 release and reduced CD20-specific activation-induced cell death. We selected an optimized anti-CD19/CD20 bicistronic construct for clinical development.
为解决抗CD19嵌合抗原受体（CAR）T细胞治疗后淋巴瘤中CD19缺失的问题，我们设计了一种双顺反子构建体，其编码抗CD19 CAR和抗CD20 CAR。通过mRNA测序，我们在少数转录本中检测到预期双顺反子构建体序列的缺失。还检测到双顺反子构建体转基因DNA的缺失。与γ-逆转录病毒载体RNA相比，转导的T细胞mRNA中序列缺失的频率要高得多。我们得出结论，这些缺失是由γ-逆转录病毒载体RNA逆转录为转导T细胞的转基因DNA过程中逆转录酶的分子内模板转换引起的。分子内模板转换由CAR序列内高度相似核酸序列的重复区域驱动。我们优化了双顺反子CAR构建体的序列，以减少高度相似序列的重复区域。这种优化几乎消除了序列缺失。这项工作表明，在复杂的CAR构建体中必须避免高度相似核酸序列的重复区域。我们通过延长抗CD20单链可变片段的接头进一步优化了双顺反子构建体。这种修饰增加了CD20特异性白细胞介素-2的释放，并减少了CD20特异性活化诱导的细胞死亡。我们选择了一种优化的抗CD19/CD20双顺反子构建体用于临床开发。
