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. 2020 Jan 17;135(10):713–723. doi: 10.1182/blood.2019002779

Figure 4.

Figure 4.

Identification of CD13 as a target to kill AML cells by CAR T cells. (A) Experimental schema. About 3000 cell membrane protein cDNAs were purified and transfected into HEK293T cells separately, followed by flow analysis with Nbs expressing phage and FITC-labeled secondary antibody against phage M13 protein. (B) Flow analysis of Nbs binding to HEK293T cells with CD13 overexpression. (C) Confirmation of CD13 cDNA expression in HEK293T cells by western blot. (D) Western blot was performed to confirm the gRNA/CRISPR-guided CD13-knockout effect in THP-1 cells. Three independent gRNAs were transduced into THP-1 separately, followed by puromycin selection and single individual clone expansion. (E) Flow analysis of Nb157- or Nb163-binding CD13-knockout THP-1 cells. (F) Cytotoxicity assay of CAR/UTD T cells to wild-type (wt) THP-1 or 2 CD13-knockout THP-1 cell lines (CD13 KO) (n = 4). *P < .05, **P <.01, d***P < .001, Student t test. ns. not significant.