INSERM UMR1098, Right, EFSBFC, UFC, Laboratoire de Thérapeutique Immuno-Moléculaire Et Cellulaire Des Cancers, 8 rue du Dr Jean François Xavier Girod, 25020, Besançon, France.
Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris (AP-HP), Université de Paris, Service d'Immunologie, Paris, France.
J Transl Med. 2021 Jun 21;19(1):265. doi: 10.1186/s12967-021-02925-z.
Genetically engineered chimeric antigen receptor (CAR) T lymphocytes are promising therapeutic tools for cancer. Four CAR T cell drugs, including tisagenlecleucel (tisa-cel) and axicabtagene-ciloleucel (axi-cel), all targeting CD19, are currently approved for treating B cell malignancies. Flow cytometry (FC) remains the standard for monitoring CAR T cells using a recombinant biotinylated target protein. Nevertheless, there is a need for additional tools, and the challenge is to develop an easy, relevant, highly sensitive, reproducible, and inexpensive detection method. Molecular tools can meet this need to specifically monitor long-term persistent CAR T cells.
Based on 2 experimental CAR T cell constructs, IL-1RAP and CS1, we designed 2 quantitative digital droplet (ddPCR) PCR assays. By targeting the 4.1BB/CD3z (28BBz) or 28/CD3z (28z) junction area, we demonstrated that PCR assays can be applied to approved CD19 CAR T drugs. Both 28z and 28BBz ddPCR assays allow determination of the average vector copy number (VCN) per cell. We confirmed that the VCN is dependent on the multiplicity of infection and verified that the VCN of our experimental or GMP-like IL-1RAP CAR T cells met the requirement (< 5 VCN/cell) for delivery to the clinical department, similar to approved axi-cel or tisa-cel drugs.
28BBz and 28z ddPCR assays applied to 2 tumoral (acute myeloid leukemia (AML) or multiple myeloma (MM) xenograft humanized NSG mouse models allowed us to quantify the early expansion (up to day 30) of CAR T cells after injection. Interestingly, following initial expansion, when circulating CAR T cells were challenged with the tumor, we noted a second expansion phase. Investigation of the bone marrow, spleen and lung showed that CAR T cells disseminated more within these tissues in mice previously injected with leukemic cell lines. Finally, circulating CAR T cell ddPCR monitoring of R/R acute lymphoid leukemia or diffuse large B cell lymphoma (n = 10 for tisa-cel and n = 7 for axi-cel) patients treated with both approved CAR T cells allowed detection of early expansion, which was highly correlated with FC, as well as long-term persistence (up to 450 days), while FC failed to detect these events.
Overall, we designed and validated 2 ddPCR assays allowing routine or preclinical monitoring of early- and long-term circulating approved or experimental CAR T cells, including our own IL-1RAP CAR T cells, which will be evaluated in an upcoming phase I clinical trial.
基因工程嵌合抗原受体（CAR）T 淋巴细胞是癌症治疗有前途的治疗工具。四种 CAR T 细胞药物，包括 tisagenlecleucel（tisa-cel）和 axicabtagene-ciloleucel（axi-cel），均针对 CD19，目前均获准用于治疗 B 细胞恶性肿瘤。流式细胞术（FC）仍然是使用重组生物素化靶蛋白监测 CAR T 细胞的标准。然而，需要额外的工具，挑战是开发一种简单、相关、高度敏感、可重复且廉价的检测方法。分子工具可以满足这种需要，专门监测长期持续存在的 CAR T 细胞。
基于 2 种实验性 CAR T 细胞构建体，IL-1RAP 和 CS1，我们设计了 2 种定量数字液滴（ddPCR）PCR 检测方法。通过针对 4.1BB/CD3z（28BBz）或 28/CD3z（28z）连接区，我们证明 PCR 检测方法可应用于批准的 CD19 CAR T 药物。28z 和 28BBz ddPCR 检测均可确定每个细胞的平均载体拷贝数（VCN）。我们证实 VCN 取决于感染复数，并验证了我们的实验性或类似 GMP 的 IL-1RAP CAR T 细胞的 VCN 符合交付给临床部门的要求（<5 VCN/细胞），类似于批准的 axi-cel 或 tisa-cel 药物。
28BBz 和 28z ddPCR 检测应用于 2 种肿瘤（急性髓系白血病（AML）或多发性骨髓瘤（MM）异种移植人源化 NSG 小鼠模型），使我们能够在注射后定量检测 CAR T 细胞的早期扩增（高达第 30 天）。有趣的是，在最初的扩增之后，当循环 CAR T 细胞受到肿瘤的挑战时，我们注意到第二个扩增阶段。对骨髓、脾脏和肺部的研究表明，在先前注射白血病细胞系的小鼠中，CAR T 细胞在这些组织中的扩散更多。最后，对接受两种批准的 CAR T 细胞治疗的 R/R 急性淋巴细胞白血病或弥漫性大 B 细胞淋巴瘤患者（tisa-cel 为 10 例，axi-cel 为 7 例）的循环 CAR T 细胞 ddPCR 监测可检测到早期扩增，这与 FC 高度相关，并且长期持续存在（长达 450 天），而 FC 未能检测到这些事件。
总的来说，我们设计并验证了 2 种 ddPCR 检测方法，可常规或临床前监测早期和长期循环的批准或实验性 CAR T 细胞，包括我们自己的 IL-1RAP CAR T 细胞，这些细胞将在即将进行的 I 期临床试验中进行评估。
