Journal: eBioMedicine
Article Title: Effectorless Fc-fusion improves FLT3L drug-like properties for cancer immunotherapy combinations
doi: 10.1016/j.ebiom.2025.105822
Figure Lengend Snippet: In vitro characterisation of FLT3L-Fc NG2LH activity . Representative sensorgrams of FLT3L-Fc NG2LH binding to recombinant human monomeric FLT3-monoFc (a) and dimeric FLT3-Fc (b). Black solid lines are curves fit to the data using a 1:1 binding model, and coloured lines are raw data. The tested recombinant human FLT3-monoFc concentrations (from bottom to top) are 62.5, 125, 250, 500, 1000, and 2000 nM; recombinant human dimeric FLT3-Fc concentrations (from bottom to top) are 3.75, 7.5, 15, 30, 60, and 120 nM. The sensorgrams were generated after in-line reference cell correction followed by buffer sample subtraction. The experiments were conducted at 37 °C. (c) Binding affinities of recombinant human monomeric FLT3-monoFc and dimeric FLT3-Fc proteins to captured FLT3L-Fc NG2LH. k a association rate constant; k d dissociation rate constant; K D equilibrium dissociation constant. Data for FLT3L-Fc NG2LH binding to recombinant human FLT3-monoFc were averaged from three independent experimental Biacore runs. Data for FLT3L-Fc NG2LH binding to recombinant human dimeric FLT3-Fc were averaged from two independent experimental Biacore runs. (d) ADCP for FLT3L-Fc NG2LH and its variants. Data shown are a representative set taken from one of three independent experiments. Average (SD) of duplicate values are shown. (e) Proliferation of OCI-AML5 cells in vitro. A representative dose response curve is shown, Average (SD) of triplicate values. Data were normalised to 10 μg/mL FLT3L-Fc NG2LH as the maximum (100%) response and the assay media alone control as the minimum (0%) response. (f) Human cDC1 differentiation from cord blood stem cells. Frequency of cDC1 at the end of culture is shown (percent of live cells). Results were obtained from 2 donors. (g) In vitro proliferation of mouse bone marrow cells in response to mFLT3L or mFLT3L-Fc. Average (SD) of duplicate values are shown.
Article Snippet: CD34+ human cord blood stem cells (StemCell Technologies) were seeded at 5000 cells per well in 96-well U-bottom plates (Costar) in 100 μL expansion media (StemSpan media–StemCell Technologies, 10% FBS, 40 ng/mL IL-3, 200 ng/mL SCF, 100 ng/mL TPO–cytokines from Peprotech).
Techniques: In Vitro, Activity Assay, Binding Assay, Recombinant, Generated, Control
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