In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

Figure 1

CD16 and CAR vector design and expression in NK-92 cells. (a) Schematic representation of the chimeric human FcγRIII-human FcεRIγ molecule and the trastuzumab- (4D5-) based CAR against HER2: the CD16/γ chimeric cDNA comprised the leader (S) and the two extracellular domains (EC1 and EC2) of human , two amino acids (aa) of the extracellular domain of human FcεRIγ, and the intact transmembrane (TM) and intracellular (IC) domains. The trastuzumab- (4D5-) based CAR contains the VL: hum Ab 4D5-8 light chain; a linker; VH: hum Ab 4D5-8 heavy chain and two amino acids (aa) of the extracellular domain of the human FcεRIγ; and the intact transmembrane (TM) and intracellular (IC) domains. (b) Transgene expression of NK-92 cell line transduced with CD16-γ chimeric receptor or trastuzumab-γ CAR. Anti-CD16 (clone 3G8) was used to determine CD16 expression on CD16-γ transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line). Anti-human IgG2a-Fc (clone HP6002) was used to determine trastuzumab-γ CAR expression on trastuzumab-γ CAR transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line).

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