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. 2020 Sep 3;12(1):1801230. doi: 10.1080/19420862.2020.1801230

Figure 2.

Figure 2.

A lead panel of purified scFv specifically bind to and inhibit the enzymatic function of recombinant human trimeric ARG2. Representative data further characterizing clones C0020185 (Δ), C0020186 (▲) and C0020187 (□) originally identified in the parallel in vitro ARG2 biochemical and inhibition high throughput screens. Each clone binds (a) recombinant biotinylated trimeric human ARG2, but not (b) recombinant biotinylated trimeric human ARG1 and (c) effectively neutralizes recombinant trimeric human ARG2 in a scFv concentration-dependent manner. Direct binding was measured using HTRF®, titrating in purified scFv and using recombinant biotinylated human ARG2 or ARG1 trimer at a fixed concentration of 12 nM or 24 nM, respectively. A hook effect was observed at highest concentrations of scFv used.23 ARG2 enzyme inhibition assay plotted as percentage. IC50 values ± standard deviation were determined for C0020185 (145.8 nM ± 9.1 nM), C0020186 (807.4 nM ± 312 nM) and C0020187 (446.8 nM ± 84.5 nM). The small molecule Arginase inhibitor NG-hydroxy-L-arginine (NHLA; ○) was used as a positive control (IC50 20640.0 nM ± 3933.8 nM). HTRF® and enzyme inhibition assay data points represent the mean of duplicate wells ± standard deviation across independent experiments (n = 2 and n = 4, respectively).