P-07.04 Mantle Cell Lymphoma in sight: development of anti-ROR1 CAR T-cell therapy, andenhancement of its safety profile by iCasp9 suicidal gene expression

<h3>Background</h3> Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a key member of the Wnt protein family that activates the MAPK and JAK/STAT pathways and promotes tumoral cell proliferation. Its overexpression in hematological cancers like mantle cell lymphoma (MCL) has made it a prime target for therapies like monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor (CAR) T-cells. MCL is an aggressive cancer type with urgent need for novel therapeutic approaches. This study aimed to develop a novel CAR T-cell therapy targeting ROR1 using in vitro MCL models. Also, iCasp9 suicide gene was incorporated, enabling selective elimination of CAR T-cells to mitigate toxicity risks. <h3>Materials and Methods</h3> The CAR construct was synthesized into lentivirus vector, by Creative Biolabs. The plasmid was introduced into Jurkat T cells via viral transfection. The screening for enhanced green fluorescent protein (eGFP) expression confirmed successful transfection. eGFP-positive cells were sorted using fluorescence-activated cell sorting (FACS). CAR T-cells were pre-activated using Dynabeads targeting CD3/CD28. Cytotoxic efficacy was evaluated at different effector-to-target (E:T) ratios (1:4 and 1:9), using mock Jurkat cells as controls. Next, the iCasp9-pMD2.G-psPAX2 virus was generated by transfecting HEK293FT packaging cells. Lentiviral supernatants were collected, and Jurkat cells were transduced with polybrene. Fluorescence microscopy confirmed successful transduction of mCherry-positive cells, and FACS sorted mCherry/GFP-bright populations. AP1903 was used to activate the iCasp9 system, and fluorescence microscopy was used for apoptosis markers investigation. <h3>Results</h3> Anti-ROR1 CAR T-cells showed limited cytotoxicity against MCL, mostly at lower E:T ratios, while the cytokine release was increased across all CAR T-treated groups. The iCasp9 suicide gene was successfully activated by AP1903, effectively inducing apoptosis in most of the mCherry/GFP-positive CAR T-cells population, within one hour. Nonetheless, a decrease in mCherry expression during cell passages raises concerns about its long-term utility. <h3>Conclusions</h3> Anti-ROR1 CAR T-cells present a promising therapeutic approach for MCL. While the engineered CAR T-cells showed limited efficacy against MCL in terms of cytotoxicity, the successful integration and activation of the iCasp9 suicide gene highlight its utility in mitigating side effects by enabling CAR T-cell depletion. Future efforts will focus on perfecting the CAR design and conducting more in vitro and in vivo studies. Acknowledgement: Grant no 760278/26.03.2024,’Creating a Research Group of Excellence to Develop Cell and Immune Therapy Technology to Target the Tumor Microenvironment, CF106/31.07.2024,’ funded by the Ministry of Education of Romania and the European Union’s Next Generation EU program and National Recovery and Resilience Plan. D. Kegyes: None. C. Chinde: None. A.B. Tigu: None. D. Cenariu: None. M.L. Nistor: None. A. Bancos: None. G. Ghiaur: None. C. Tomuleasa: None.