Figure 1.
Evaluation of epitope specificity and internalization capability of anti-FLT3 monoclonal antibodies. (A) Affinity of 7 anti-FLT3 mAb clones to recombinant human FLT3 measured in enzyme-linked immunosorbent assay normalized to the binding of the antibodies to bovine serum albumin control. KD (mean ± SD; n = 3) is depicted. (B) Schematic FLT3 receptor. Black arrows indicate common mutations, and blue arrows indicate the 3 identified epitopes of 7 anti-FLT3 antibodies analyzed in linear epitope mapping by PEPperPRINT. Figure was created with BioRender.com. (C) Binding and temperature-induced internalization of anti-FLT3 mAb in Ba/F3 cells expressing human wildtype FLT3 (hFLT3wt). Internalization was induced by incubation for 30 minutes at 37°C (gray) compared with 4°C (black). Remaining surface-bound antibody was detected in flow cytometry. Mean fluorescence intensity (MFI) was normalized to control human IgG1 binding. Mean ± SD of n = 3 is depicted. (D) Cell surface binding of 20D9-mAb or control human IgG1 antibody to Ba/F3 cells stably expressing pMIY (Ba/F3-pMIY) empty vector (ev), hFLT3wt, or ITD mutant human FLT3 (hFLT3ITD) was measured in flow cytometry. Mean ± SD of n = 3. (E) Temperature-induced internalization of 4B12-mAb in MOLM-13 (FLT3+) cells after 30-minutes incubation at 37°C compared with 4°C and HL-60 (FLT3−) after 37°C incubation assessed in immunofluorescence staining. Red = FLT3 staining by 4B12, green = membrane staining by Vybrant DiO, blue = nuclear staining by DAPI. Scale bar, 5 μm. Representative pictures are shown. (F) Cell surface binding of 20D9-mAb or control hIgG1 antibody to Ba/F3-pMIY ev, hFLT3wt, murine (mFLT3wt), or cynomolgues monkey FLT3 (cynoFLT3wt) was measured in flow cytometry. Mean ± SD of n = 3. (G) Cell surface binding of 20D9-mAb or control hIgG1 antibody to Ba/F3-pMIY ev, hFLT3wt, or epitope mutant FLT3 (hFLT3S50P/P54R) measured in flow cytometry. Mean ± SD of n = 3. (H) Cell surface binding of 20D9-mAb or control hIgG1 antibody to Ba/F3-pMIY ev, hFLT3wt, or human CD64 (hCD64) measured in flow cytometry. Mean ± SD of n = 3. ∗P < .05 (unpaired, 2-tailed t test). ns, not significant.

Evaluation of epitope specificity and internalization capability of anti-FLT3 monoclonal antibodies. (A) Affinity of 7 anti-FLT3 mAb clones to recombinant human FLT3 measured in enzyme-linked immunosorbent assay normalized to the binding of the antibodies to bovine serum albumin control. KD (mean ± SD; n = 3) is depicted. (B) Schematic FLT3 receptor. Black arrows indicate common mutations, and blue arrows indicate the 3 identified epitopes of 7 anti-FLT3 antibodies analyzed in linear epitope mapping by PEPperPRINT. Figure was created with BioRender.com. (C) Binding and temperature-induced internalization of anti-FLT3 mAb in Ba/F3 cells expressing human wildtype FLT3 (hFLT3wt). Internalization was induced by incubation for 30 minutes at 37°C (gray) compared with 4°C (black). Remaining surface-bound antibody was detected in flow cytometry. Mean fluorescence intensity (MFI) was normalized to control human IgG1 binding. Mean ± SD of n = 3 is depicted. (D) Cell surface binding of 20D9-mAb or control human IgG1 antibody to Ba/F3 cells stably expressing pMIY (Ba/F3-pMIY) empty vector (ev), hFLT3wt, or ITD mutant human FLT3 (hFLT3ITD) was measured in flow cytometry. Mean ± SD of n = 3. (E) Temperature-induced internalization of 4B12-mAb in MOLM-13 (FLT3+) cells after 30-minutes incubation at 37°C compared with 4°C and HL-60 (FLT3−) after 37°C incubation assessed in immunofluorescence staining. Red = FLT3 staining by 4B12, green = membrane staining by Vybrant DiO, blue = nuclear staining by DAPI. Scale bar, 5 μm. Representative pictures are shown. (F) Cell surface binding of 20D9-mAb or control hIgG1 antibody to Ba/F3-pMIY ev, hFLT3wt, murine (mFLT3wt), or cynomolgues monkey FLT3 (cynoFLT3wt) was measured in flow cytometry. Mean ± SD of n = 3. (G) Cell surface binding of 20D9-mAb or control hIgG1 antibody to Ba/F3-pMIY ev, hFLT3wt, or epitope mutant FLT3 (hFLT3S50P/P54R) measured in flow cytometry. Mean ± SD of n = 3. (H) Cell surface binding of 20D9-mAb or control hIgG1 antibody to Ba/F3-pMIY ev, hFLT3wt, or human CD64 (hCD64) measured in flow cytometry. Mean ± SD of n = 3. ∗P < .05 (unpaired, 2-tailed t test). ns, not significant.

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