Journal: American Journal of Hematology

Article Title: A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation

doi: 10.1002/ajh.27578

Figure Lengend Snippet: RKER‐216 epitope in ALK2 overlaps with BMP6 binding site, and the presence of RKER‐216 competitively inhibits BMP6 from binding to ALK2. (A, E) Binding affinity ( K D ) between ALK2 and RKER‐216 or ALK2 and BMP6 was determined by SPR. RKER‐216 diluted in HBS‐EP+ buffer was injected through a CM4 chip immobilized with ALK2‐Fc at 25 RU (A) while BMP6 diluted in HBS‐EP+ buffer with 30 nM arginine was injected through a CM4 chip immobilized with ALK2‐Fc at 350 RU (E). The results were analyzed by Biacore Insight Evaluation software using 1:1 binding model for RKER‐216 and steady‐state affinity for BMP6. The mean K D ± SEM from 3 separate experiments is reported and a representative sensogram is shown. (B) RKER‐216 binds to regions in ALK2 covering the F2 loop and β4 sheet (blue) as determined by HDX‐MS. The H/D exchange protected regions (blue) were mapped onto a model structure of ALK2 (PDB ID: 7YRU) shown in ribbon (left) and in surface (right) representation in complex with BMP6 structure (PBD ID: 2R52). The model of ALK2:BMP6 complex was generated by aligning individual structure of ALK2 and BMP6 into the structure of ALK1:BMP9 (PDB ID: 4FAO). The sequence alignment between ALK2 and ALK3 are shown (bottom), highlighting the binding region (in blue) of RKER‐216 in ALK2. (C‐D) BLI competition assays were performed in two ways. (C) ALK2 binding site was saturated with 5 nM of RKER‐216 prior to the competition with BMP6 at 0 to 100 nM. In addition to the vehicle control, BMP6 at 100 nM was added to unsaturated ALK2 to confirm binding ability of BMP6 in the competition. No binding activity was observed in the competition, indicating that BMP6 at 100 nM was unable to complete RKER‐216 away for binding ALK2. (D) ALK2 binding site was saturated with 100 nM of BMP6 prior to the competition with RKER‐216 at 0 to 50 nM. RKER‐216 at 50 nM was added to unsaturated ALK2 to confirm binding ability of RKER‐216. This group also served as a control for examining whether RKER‐216 binds to ALK2 or the ALK2‐BMP6 complex, as changes in the wavelength reflects both the size and affinity of the interactor. In the zoomed plot of RKER‐216 competition, data were aligned to zero on the Y ‐axis prior to the competition step for visual comparison, and 50 nM RKER‐216 without saturation had the greatest wavelength shift, indicating that RKER‐216 dislodged BMP6 from ALK2 instead of binding to the ALK2‐BMP6 complex. Experiments were repeated 3 times and a representative sensogram is shown.

Article Snippet: Briefly, the library was panned for antibodies recognizing the extracellular domain of human ALK2 (637‐AR; R&D Systems) with three rounds of enrichment and counter‐selected against the Fc control for depletion.

Techniques: Binding Assay, Injection, Software, Generated, Sequencing, Control, Activity Assay, Comparison

Journal: American Journal of Hematology

Article Title: A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation

doi: 10.1002/ajh.27578

Figure Lengend Snippet: RKER‐216 decreases hepcidin transcription in vitro and controls iron availability by lowering hepcidin secretion in vivo. (A) Hep3B cells were serum starved with 1% FBS overnight and incubated with an ascending dose of RKER‐216 (0.02–1 μg/mL) in the absence or presence of 5 ng/mL of BMP6, BMP2/6, or BMP2 for 6 h ( n = 4–5 per group). (B) Characterization of human ACVR1 knockout in HepG2 and Huh7 cells by quantifying ACVR1 and BMPR1A transcript copy number ( n = 3 per group). (C) ACVR1 KO and the negative control cells were treated with RKER‐216 at 1–30 μg/mL for 6 h ( n = 3 per group). Relative HAMP mRNA levels were determined by qRT‐PCR and transcripts were normalized to an internal control RPL19 . The average of PBS control without ligand stimulation or ACVR1 wildtype without RKER‐216 was set to 1. For ligand preference, hepcidin stimulated by each ligand was set to 100%. Values represent mean ± SEM. Results were compared across RKER‐216 and BMP ligands by one‐ or two‐way ANOVA with Tukey's post hoc test. Means without a common superscript differ significantly ( p < 0.05) or * p < 0.05, *** p < 0.001 relative to untreated cells of the same genotype. (D‐G) B6N male mice at 8 weeks were treated with a single SC dose of isotype control or RKER‐216 at 3 mg/kg for times as indicated ( n = 5 per group). Serum was collected to quantify (D) RKER‐216 exposure and (E) serum hepcidin by ELISA, and (F) serum iron and (G) serum transferrin saturation (TSAT) by colorimetric assays. Values represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 relative to the isotype control mice of the same time point by Student's t ‐test.

Article Snippet: Briefly, the library was panned for antibodies recognizing the extracellular domain of human ALK2 (637‐AR; R&D Systems) with three rounds of enrichment and counter‐selected against the Fc control for depletion.

Techniques: In Vitro, In Vivo, Incubation, Knock-Out, Negative Control, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay