Construction of the lentiviral vector

The HuScL-2 Human Single-Chain Antibody Library (Creative Biolabs) was screened; after four rounds, 40 clones from the fourth eluate were selected for analysis in ELISA using monoclonal phages. The highest affinity clone was selected and its single-chain variable fragment (scFv) was used to build the anti-HLA-G CAR construct by synthesizing DNA corresponding to the leader peptide sequence 5′-ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC-3′. This was fused to the anti-HLA-G scFv sequence, followed by the DNA sequence encoding the transmembrane and cytosolic domains of KIR2DS4 (amino acids 246–304), the P2A (porcine teschovirus-1 2A) sequence (amino acids 1–19), the full-length DAP-12 (DNAX-activating protein of molecular mass 12 kD) sequence (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_003332.3","term_id":"291045269","term_text":"NM_003332.3"}}NM_003332.3), and inducible caspase-9 (iC9) (FK506-binding protein 12 (amino acids 1–108) fused to caspase-9 (amino acids 135–416)). The insert was cloned into the pCAR-(puroless) lentiviral vector (Creative Biolabs) via the EcoRI/XbaI restriction sites.