Combining the expression of CD33.CAR and CXCR4 to augment CAR-CIKs homing to bone marrow niche and leukemic stem cell eradication in acute myeloid leukemia

Chimeric Antigen Receptor (CAR) cytokine-induced killer (CIK) cell therapy is a promising treatment for acute myeloid leukemia (AML). Specifically, it is crucial to improve CAR CIK-cells infiltration ability into the bone marrow (BM) niche to eradicate leukemia stem cells (LSCs) at their location. Actually, BM mesenchymal stromal cells (MSCs) interact with LSCs, residing in the niche, releasing different chemokines and soluble factors. The chemokine ligand 12 (CXCL12), produced by MSCs, and its chemokine receptor 4 (CXCR4) regulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 overexpressed on blasts, promoting their homing in the niche. On the contrary, CXCR4 expression is drastically downregulated on CIKs during culture. Combining the expression of CD33.CAR and CXCR4 might facilitate CAR-CIKs homing to the BM and subsequent leukemia eradication. We designed two bicistronic Sleeping Beauty transposon vectors: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. We observed both CD33.CAR(2A)CXCR4-CIKs (n=22, P<0.0001) and CXCR4(IRES)CD33.CAR-CIKs (n=9, P<0.0001) maintained CXCR4 overexpression during culture, whereas in CD33.CAR-CIKs was drastically downregulated (n=22). However, CD33.CAR expression was lower in CXCR4(IRES)CD33.CAR-CIKs (n=8, P<0.0001) compared with CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=11) exhibit a significant co-expression of both proteins against control (P=0.001). Chemotaxis assays toward recombinant CXCL12 confirmed both CXCR4(IRES)CD33.CAR-CIKs (n=7, P=0.01) and CD33.CAR(2A)CXCR4-CIKs (n=8, P=0.0006) displayed a migration advantage over CD33.CAR-CIKs (n=12) with a mean percentage of migration of 58.5% and 67.2% respectively, compared to 40.1%. Interestingly, CD33.CAR(2A)CXCR4-CIKs (n=2) showed an increased specific chemotactic response toward HD- (n=3) and AML-MSCs (n=2) supernatants, as demonstrated by the use of CXCR4 antagonist Plerixafor. Moreover, CXCR4(IRES)CD33.CAR-CIKs and CD33.CAR(2A)CXCR4-CIKs retained killing of CD33+ KG1 target cell line, maintained their capacity to produce IL-2 and IFN-y and to proliferate after CD33 antigen exposure. However, CXCR4(IRES)CD33.CAR-CIKs exhibited lower effector responses against control, due to inferior CAR expression. Taking together, these data demonstrating enhanced migration while maintaining CAR functionalities, make CD33.CAR(2A)CXCR4 the best candidate for further in vivo homing and anti-leukemic evaluation.