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318 Development of CD70 armored Delta-like ligand 3 CAR-T cells for the treatment of small cell lung cancer
  1. Chao Wang,
  2. Yu Yang,
  3. Ling Chen,
  4. Fuwei Jiang,
  5. Fanxiang Gao,
  6. Cuiqing Yang,
  7. Qingyang Wang and
  8. Zhuoxiao Cao
  1. Simnova Biotherapeutics, Shanghai, China
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Small cell lung cancer (SCLC) is an aggressively lethal malignancy with a high unmet medical need. Delta-like ligand 3 (DLL3) has been identified as a promising cancer antigen for target immunotherapy in treating SCLC. DLL3-targeting therapies such as T cell engagers and chimeric antigen receptor T cells (CAR-T), so far have shown favorable safety profiles and encouraging therapeutic results.

Methods A diverse panel of single-chain variable fragment (scFv) with high binding affinity for human DLL3 were generated and was compared in a second-generation CAR-T format for target-dependent T cell activation and cytotoxicity. A construct encoding the selected scFv CAR and an armored CD70 was generated and the effector functions of the engineered CAR-T cells were evaluated in vitro. The antitumor efficacies were evaluated against multiple xenograft models in immunodeficient mice. The potential toxicity was characterized in a mouse tumor model with mouse cross-reactive DLL3 CAR-T cells.

Results It was notable to find that a class of membrane-proximal (MPR) epitope targeted DLL3 CARs revealed potent cytotoxicity in treating low DLL3 density tumor cells, in contrast to membrane-distal region (DSL) targeted DLL3 CARs. Consistently, the MPR targeted CAR-T cells was superior in mediating cytokine secretion in response to tumor recognitions. Using transgenic mouse models, we observed that MPR targeted CAR-T cells achieved better tumor control than DSL targeted CAR-T cells. The long-lasting antitumor activities of MPR targeted CAR-T therapy was further evidenced by tumor re-challenge study, where 100% of the cured mice showed tumor rejection. No potential organ toxicity was found in normal tissues and CAR-T infiltration was only observed in tumors and spleens, indicating that MPR targeted CAR-T cells are of safety with no serious off-target toxicity. To further enhance the expansions and persistence, we armored the CAR with a CD70 construct. Using serial killing assays, we demonstrated that CD70 armored CAR-T promoted durable cytotoxicity following serial rounds engagement of low DLL3-expressed tumor cells. Furthermore, the tumor lysing capacity of CD70 armored CAR-T cells was not compromised to TGF-β (transforming growth factor β) inhibition compared to the conventional CAR-T cells.

Conclusions Collectively, our study indicates a combine of a proximal targeting CAR and an armored CD70 construct improve CAR-T persistence and antitumor activity in vitro and in vivo, and result in a potential DLL3 CAR candidate for further preclinical and clinical studies.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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