The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is critical during embryogenesis regulating cell migration, differentiation and growth. It is demonstrated that ROR1 expression is low in adult tissues, and unfortunately the expression increases in various malignancies such as Mantle Cell Lymphoma (MCL), Multiple Myeloma (MM), Triple Negative Breast Cancer (TNBC), Non-Small Cell Lung Carcinoma (NSCLC), colorectal cancer (CRC) or Melanoma (MEL), all being highly aggressive and their management is challenging. ROR1 targeted therapies are continuously developing, with remarkable achievements in terms of novel mAbs, ADCs or small molecules. Our study aims to highlight that the use of CAR T-cells could improve the outcomes and offer new perspectives by targeting ROR1.

The full length of CAR was synthesized and subcloned into lentivirus vector by Creative Biolabs. The insert was confirmed by Sanger seq. The iCasp9-pMD2.G-psPAX2 virus was generated by transfecting HEK293FT packaging cells. The target pHR-iCasp9 plasmid, the packaging vector, psPAX2 and the envelope, pMD2.G were acquired from AddGene. FuGENE® was used as transfection reagent. Lentiviral supernatants were harvested at 24h and 48h. CAR ROR1 Jurkat cells were spinoculated in 8 µg/mL polybrene and viral supernatant. On day 3 after transduction, fluorescence microscopy confirmed the presence of mCherry+ cells. CAR ROR1 Jurkat cells were expanded for 14 days, followed by mCherry/GFP-highly positive cells sorting, on a FACS ARIA III platform. The co-culture efficacy of different effector-target (E:T) ratios was evaluated by flow cytometry. Target cells were identified by staining with an APC anti-human ROR1 antibody. The supernatant was stored and further used for LDH titration by an ELISA assay.

The presence of eGFP and mCherry red signal confirmed the successful generation of anti-ROR1 CAR T-cells. The analysis at different E:T ratios showed that the CAR T cells successfully inhibited the targets, with a significant cytokine release measured by ELISA assays, all tests being done in comparison with Mock - Jurkat cells. The highest inhibitory rate was observed in the MCL cell line, accompanied by a significantly higher cytokine release compared to the controls. As the CAR T-cells are immortalized due to the use of Jurkat cells, we evaluated the suicidal capacity using various concentrations of AP1903. AP1903 induced iCasp9 dimerization, and the flow cytometry analysis revealed that apoptosis was initiated in most of the CAR T-cells. Apoptotic rate was determined by a 7-AAD/PO-PRO1 flow cytometry assay on an LSRII cytometer. This remained evident at a very low concentration (6.25 nM), which is still well below the clinical concentration for AP1903.

Anti-ROR1 CAR T-cells showed a significant inhibitory rate against multiple targets, indicating that the new approach could be considered for further in vivo evaluations.