American Association for Cancer Research
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Supplementary data (Figures S1 to S12) from CML Hematopoietic Stem Cells Expressing IL1RAP Can Be Targeted by Chimeric Antigen Receptor–Engineered T Cells

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posted on 2023-03-31, 02:41 authored by Walid Warda, Fabrice Larosa, Mathieu Neto Da Rocha, Rim Trad, Eric Deconinck, Ziad Fajloun, Cyril Faure, Denis Caillot, Marius Moldovan, Severine Valmary-Degano, Sabeha Biichle, Etienne Daguindau, Francine Garnache-Ottou, Sebastien Tabruyn, Olivier Adotevi, Marina Deschamps, Christophe Ferrand

Figure S1. In vitro detection (using #A3C3) of recombinant IL-1RAP protein by ELISA Figure S2. Procedure for generating IL-1RAP CART cells and CD4/CD8 ratio at the end of the process Figure S3. Staining of IL-1RAP CAR at the cell surface of T cell line or primary T -lymphocytes Figure S4. Impact of the IL-1RAP soluble form on IL-1RAP CART-cells toxicity Figure S5. Tissue macroarray (TMA) using #A3C3 monoclonal antibody Figure S6. Experimental immunosafety human CD34+ engrafted NOG murine model Figure S7. Colony Forming Unit (CFU-GM) experiment after coculture of autologous CART-cells with CD34+ HSC. Figure S8. In-vitro and in-vivo iCASP/AP1903 suicide gene safety switch Figure S9. Production and characterization of IL-1RAP (soluble and membrane) transfected cell line Figure S10. Cytokine secretion profiling of IL-1RAP CART-cells after co-culturing with targets Figure S11. Tumoral xenograft murine model Figure S12. Autologous cytotoxicity of IL-1RAP CART-cells directed against initial CML disease

Funding

Ligue contre le cancer

Programme de Recherche Translationnelle INCa

BPI France

European Regional Development Fund of the Region BFC

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ARTICLE ABSTRACT

Chronic myeloid leukemia (CML) is a chronic disease resulting in myeloid cell expansion through expression of the BCR-ABL1 fusion transcript. Tyrosine kinase inhibitors (TKI) have significantly increased survival of patients with CML, and deep responders may consider stopping the treatment. However, more than 50% of patients relapse and restart TKI, subsequently suffering unknown toxicity. Because CML is a model immune system–sensitive disease, we hypothesize that chimeric antigen receptor (CAR) T cells targeting IL1 receptor-associated protein (IL1RAP) in quiescent CML stem cells may offer an opportunity for a permanent cure. In this study, we produced and molecularly characterized a specific monoclonal anti-IL1RAP antibody from which fragment antigen-binding nucleotide coding sequences were cloned as a single chain into a lentiviral backbone and secured with the suicide gene iCASP9/rimiducid system. Our CAR T-cell therapy exhibited cytotoxicity against both leukemic stem cells and, to a lesser extent, monocytes expressing IL1RAP, with no apparent effect on the hematopoietic system, including CD34+ stem cells. This suggests IL1RAP as a tumor-associated antigen for immunotherapy cell targeting. IL1RAP CAR T cells were activated in the presence of IL1RAP+ cell lines or primary CML cells, resulting in secretion of proinflammatory cytokines and specifically killing in vitro and in a xenograft murine model. Overall, we demonstrate the proof of concept of a CAR T-cell immunotherapy approach in the context of CML that is applicable for young patients and primary TKI-resistant, intolerant, or allograft candidate patients. These findings present the first characterization and proof of concept of a chimeric antigen receptor directed against IL1RAP expressed by leukemic stem cells in the context of CML.

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