Inclusion of the Inducible Caspase 9 Suicide Gene in CAR Construct Increases Safety of CAR.CD19 T Cell Therapy in B-Cell Malignancies.

T cells engineered with chimeric antigen receptor (CAR-T cells) are an effective treatment in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia or B-cell non-Hodgkin lymphoma. Despite the reported exciting clinical results, the CAR-T cell approach needs efforts to improve the safety profile, limiting the occurrence of adverse events in patients given this treatment. Besides the most common side effects, such as cytokine release syndrome and CAR-T cell-related encephalopathy syndrome, another potential issue involves the inadvertent transduction of leukemia B cells with the CAR construct during the manufacturing process, thus leading to the possibility of a peculiar mechanism of antigen masking and treatment resistance. In this study, we investigated whether the inclusion of the inducible caspase 9 (iC9) suicide gene in the CAR construct design could be an effective safety switch to control malignant CAR+ B cells, ultimately counteracting this serious adverse event. iC9 is a suicide gene able to be activated through binding with an otherwise inert small biomolecule, known as AP1903. The exposure of iC9.CAR.CD19-DAUDI lymphoma and iC9.CAR.CD19-NALM-6 leukemia cells in vitro to 20 nM of AP1903 resulted into the prompt elimination of CAR+ B-leukemia/lymphoma cell lines. The results obtained in the animal model corroborate in vitro data, since iC9.CAR.CD19+ tumor cells were controlled in vivo by the activation of the suicide gene through administration of AP1903. Altogether, our data indicate that the inclusion of the iC9 suicide gene may result in a safe CAR-T cell product, even when manufacturing starts from biological materials characterized by heavy leukemia blast contamination.

Guercio M ，Manni S ，Boffa I ，Caruso S ，Di Cecca S ，Sinibaldi M ，Abbaszadeh Z ，Camera A ，Ciccone R ，Polito VA ，Ferrandino F ，Reddel S ，Catanoso ML ，Bocceri E ，Del Bufalo F ，Algeri M ，De Angelis B ，Quintarelli C ，Locatelli F ... -  《Frontiers in Immunology》

Generation of Suicide Gene-Modified Chimeric Antigen Receptor-Redirected T-Cells for Cancer Immunotherapy.

Minagawa K ，Al-Obaidi M ，Di Stasi A 《-》

Strategy to prevent epitope masking in CAR.CD19+ B-cell leukemia blasts.

Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. METHODS: We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.

Quintarelli C ，Guercio M ，Manni S ，Boffa I ，Sinibaldi M ，Di Cecca S ，Caruso S ，Abbaszadeh Z ，Camera A ，Cembrola B ，Ciccone R ，Orfao A ，Martin-Martin L ，Gutierrez-Herrero S ，Herrero-Garcia M ，Cazzaniga G ，Nunes V ，Songia S ，Marcatili P ，Marin FI ，Ruella M ，Bertaina V ，Vinti L ，Del Bufalo F ，Algeri M ，Merli P ，De Angelis B ，Locatelli F ... -  《-》

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety.

Hoyos V ，Savoldo B ，Quintarelli C ，Mahendravada A ，Zhang M ，Vera J ，Heslop HE ，Rooney CM ，Brenner MK ，Dotti G ... -  《-》

Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

Budde LE ，Berger C ，Lin Y ，Wang J ，Lin X ，Frayo SE ，Brouns SA ，Spencer DM ，Till BG ，Jensen MC ，Riddell SR ，Press OW ... -  《-》