Fig. 1: Characterization of binding and internalization properties of the humanized FLT3 monoclonal antibody 20D9h3. | Leukemia

Fig. 1: Characterization of binding and internalization properties of the humanized FLT3 monoclonal antibody 20D9h3.

From: Effective eradication of acute myeloid leukemia stem cells with FLT3-directed antibody-drug conjugates

Fig. 1

A, B 20D9h3 monoclonal antibody (mAb) was complexed with a pHrodo Deep Red-labelled secondary antibody and incubated with Ba/F3 cells stably expressing empty MSCV-IRES-YFP vector (pMIY ev) or human wildtype FLT3 at low (hFLT3low) or high (hFLT3high) surface levels. Internalization was measured by flow cytometry after 1 h, 5 h and 24 h depicted as mean fluorescence intensity (MFI) ratio to unstained control (A) or by microscopy after 24 h (B). Green = stably expressed YFP, red = 20D9h3-mAb visualized by pHrodo-labelled secondary antibody. Scale bar = 10 µm; Nikon CFI Apochromat TIRF 60x NA 1.49 oil immersion objective. mean±s.d.; n = 3. Binding of 20D9h3-mAb to Ba/F3-pMIY cells expressing ev, hFLT3high, FLT3 paralogues (VEGFR, PDGFRα, CSF-1R and c-KIT) (C) or FLT3 orthologues (to cynomolgus monkey (cynoFLT3), rat (rFLT3) and mouse (mFLT3) FLT3) (D) measured by flow cytometry after 1 h incubation. MFI was normalized to binding of IgG1 isotype control. mean±s.d.; n = 3. E Binding of 20D9h3-mAb and IgG1 isotype control with wildtype (wt) or Leu234Ala/Leu235Ala (LALA)-mutated Fc domain to Ba/F3-pMIY cells expressing ev, immunoglobulin gamma Fc receptor (FcγRI, FcγRII, FcγRIII) or hFLT3high measured by flow cytometry after 1 h incubation and depicted as MFI. mean±s.d.; n = 4.

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