Constructing a Second-Gen CD33-Targeting CAR

A second-generation CD33-targeting CAR which incorporates the My96 scFv sequence, which has been used in the immunoconjugate AVE9633, was constructed as described earlier [26 (link)]. In short, the My96 scFv was combined in frame with CD8 hinge and transmembrane domain, 4-1BB/CD137 co-stimulatory domain, and CD3ζ activation domain. A leader peptide derived from granulocyte-macrophage colony-stimulating factor receptor unit α (GM-CSFRα) was included to facilitate CAR cell surface expression. Third-generation self-inactivating baboon envelope-pseudotyped lentiviral vectors (BaEV-LVs) were produced by transient transfection into HEK293T cells using MACSfectin™ (Miltenyi Biotec, Bergisch Gladbach, Germany) [18 (link), 27 (link)].