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287 Novel, boosted, fully human ROR1-targeting CAR T cells effectively eliminate hematologic and solid tumors, and resist tumor microenvironment
  1. Tri Tran,
  2. Bal Krishna Chand Thakuri,
  3. Saule Nurmukhambetova,
  4. Brittany Steimle,
  5. Ngoc Tran,
  6. Darong Wu,
  7. Peirong Hu,
  8. Pradyot Dash and
  9. Dina Schneider
  1. Lentigen, Gaithersburg, MD, USA


Background Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is widely expressed in various hematologic and solid tumors. A clinical trial employing ROR1-CAR T-cells with scFv-R12 binder showed poor tumor infiltration and dysfunctionality of CAR T-cells in NSCLC & TNBC patients,1 suggesting that further CAR-binder/construct optimization may be required to improve CAR T-cell persistence and control of T-cell inhibitory factors in the tumor microenvironment.

Methods We designed new ROR1-CAR constructs, either non-boosted (CAR-1), or boosted with TGFbRII Dominant Negative fragment (TGFbRIIDN), or membrane-bound IL-7 (mbIL7), to overcome the TGFb1 inhibitory effect in the tumor microenvironment, and to enhance T-cell persistence, respectively. We also designed ROR1-CAR with R12 binder1 (CAR-2) as control. The ROR1-CARs were transduced into human primary T-cells using lentiviral vectors. In vitro cytotoxicity was assessed by co-culture with hematologic mantle cell lymphoma (MCL, Jeko-1), or solid ovarian (OVCAR-3), pancreatic (CAPAN-2) or lung (NCI-H226) tumor cell lines; TNF-a, IFN-γ and IL-2 cytokine release was assessed by ELISA. To simulate tumor microenvironment, CAR T-cells were challenged with TGFb1 or cultured without exogenous IL-2. In vivo xenograft studies were performed in NSG mice bearing Jeko-1 or OVCAR-3 xenografts; tumor progression was monitored, and CAR T cells expansion in mouse peripheral blood was analyzed by flow cytometry.

Results CAR-1-transduced T-cells showed enhanced activation and cytotoxicity against Jeko-1 MCL as compared to CAR-2 in vitro, and eliminated tumors in the Jeko-1 model in vivo. CAR-1 also mediated potent killing and enhanced cytokine release as compared to CAR-2 upon incubation with OVCAR-3, CAPAN-2, and NCI-H226 solid tumor cell lines in vitro. Surprisingly, in in vivo ovarian OVCAR-3 xenograft model, CAR-2 failed to reject tumors, whereas CAR-1 mediated remissions; analysis of CAR T-cells in peripheral blood revealed rapid expansion of CAR+T-cell population and enrichment for the central memory phenotype in CAR-1 as compared to CAR-2. In addition, we successfully boosted CAR-1 with TGFßRIIDN, which attenuated the inhibitory effect of TGFß1 on CAR T-cell cytotoxic activity in vitro. We also equipped CAR-1 with mbIL7, which enhanced cytotoxic activity and mediated extended functionality of the CAR T-cells without exogeneous IL-2 for up to 18 days.

Conclusions The novel fully human ROR1-CAR1 T-cells effectively eliminated both hematologic and solid tumors in vivo, and were superior to ROR1-CAR-2 T cells. Furthermore, the boosting elements TGFßRIIDN and mbIL7 are promising tools to overcome the inhibitory effects of tumor microenvironment and sustain CAR T-cell persistence.


  1. Srivastava S et al., Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade. Cancer Cell, 2021, 39, 193–208.

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