CXCR4-modified CD33.CAR-CIK with enhanced bone marrow homing in Acute Myeloid Leukemia

Chimeric Antigen Receptor (CAR) Cytokine-Induced Killer (CIK) cell therapy is an emerging treatment for acute myeloid leukemia (AML) although some implementations are required. Specifically, it appears crucial to improve CAR-CIK infiltration ability into the bone marrow (BM) niche to eradicate leukemia stem cells (LSCs) at their location. CAR-CIK ex vivo manipulation influences the expression of several chemokine receptors and may dampen the capacity of infused cells to migrate to the BM. The chemokine ligand 12 (CXCL12), produced by mesenchymal stromal cells (MSCs) within the niche, and its chemokine receptor 4 (CXCR4) modulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 overexpressed on blasts, enhancing their homing in the niche. CXCR4 expression is consistently downregulated during the culture of CIKs. Therefore, combining the expression of CD33.CAR and CXCR4 might promote CAR-CIK homing to the BM and subsequent leukemia eradication, specifically of LSCs. Two bicistronic Sleeping Beauty transposon vectors were designed: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. Phenotype and CAR-related in vitro effector functions were compared. Migration in vitro toward rhCXCL12 or MSC supernatants was tested using a transwell migration assay. CAR-CIKs in vivo BM homing ability was evaluated in sub-lethally irradiated NSG mice. CAR-CIKs engraftment was assessed in BM, blood, spleen, and lungs. We noticed that both CD33.CAR(2A)CXCR4-CIKs (n=22, P