How to isolate useful human antibodies for the construction of CARs

To construct cancer cell-recognizing CARs, we selected a cancer-specific peptide-MHC complex as our target antigen, and tried to isolate human antibodies. For this purpose, we needed a human antibody library, the target peptide-MHC complex, and a screening procedure.
To construct a human antibody library, the immunoglobulin VH and VL genes were amplified by PCR, reconstructed on a phagemid vector, and displayed on M13 phage.

WT1/HLA-A2402 and MAGE-A4/HLA-A0201 were reconstructed by gentle refolding of HLA, beta-2 micro-globulin, and the epitope peptide. The antibodies were screened with pMHC-conjugated streptavidin magnet beads, a pMHC-immobilized plate, and peptide-pulsed cells.

After screening numerous clones, the obtained antibodies were characterized according to their epitope specificity and affinity. Then, a CAR was constructed, and its expression, recognition specificity, and cell toxicity against endogenous antigen-expressing cells were assessed.