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. Author manuscript; available in PMC: 2021 Apr 15.
Published in final edited form as: Cancer Res. 2020 Aug 31;80(20):4552–4564. doi: 10.1158/0008-5472.CAN-20-1418

Figure 2.

Figure 2.

Specificity of M25 IgG binding to tumor cells and to different members of the alkaline phosphatase family. (A) M25 IgG1 binds specifically to mesothelioma cell lines M28 and VAMT-1 with no binding to control normal human cells (HS27, a normal foreskin fibroblast cell line; HK-2, a normal kidney epithelial cell line; and HS775Li, a normal primary liver cell line). Apparent KD was estimated by curve-fitting (GraphPad). (B) M25 IgG1 binds specifically to ALPPL2 and ALPP, but not ALPI or ALPL. Flow cytometry analysis of M25 IgG1 binding to CHO-K1 cells transiently expressing each of the four members of the human alkaline phosphate family. ALPPL2, placental like-2 alkaline phosphatase; ALPP, placental alkaline phosphatase; ALPI, intestinal alkaline phosphatase; ALPL, tissue-nonspecific alkaline phosphatase. (C) Upper panel: Binding region determination using hybrid ALPI/ALPP fusion constructs. Sequence information is shown in Supplemental Figure 2S. Lower panel: flow cytometry analysis of binding to HEK293A cells expressing hybrid constructs. Red, binding to HEK293A cells expressing full-length ALPP. Grey, binding to HEK293A cells expressing full-length ALPI. Orange, binding to HEK293A cells expressing ALPI/ALPP hybrid_1. Blue, binding to HEK293A cells expressing ALPI/ALPP hybrid_2. (D) M25 IgG1 IHC staining of FDA standard panel of human frozen normal tissues. Other than placenta trophoblasts, none of the normal tissues studied showed positive staining. Scale bar: 50 μm. Enlarged windows are close-up views; scale bar (blue): 30 μm.