Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: Localization of laminin-related integrins and the effect of functional blocking of integrin α7 in THP-1 macrophages cultured on laminin-211/221/221E8 or laminin-511 for 5 days . A , morphology and multiple immunofluorescent staining for laminin-related integrins (α7, α6, and α3) and phalloidin in THP-1 macrophages cultured on laminin (LM)-211/-511. Nuclei were counterstained with DAPI. Immunostaining was performed four times independently, imaging a total of 300 to 350 cells per antibody. B , morphology of THP-1 macrophages cultured on laminin-211/-221/-221E8 in the presence of integrin α7 function-blocking antibodies (anti-α7) or isotype control IgG. In the presence of integrin α7 function-blocking antibodies, dendritic-like branching and extended cellular processes are observed. These experiments were repeated at least five times independently. C , morphology of THP-1 macrophages cultured on laminin-511 in the presence of integrin α7 function-blocking antibodies. D , western blotting of integrin α7 under conditions of integrin α7-targeted small interfering RNA (siITGA7) on day 5. siITGA7 #9 and #1 reduced integrin α7 levels on laminin-211. Nonspecific siRNA was used as control (siCtrl); β-actin was used as a loading control. E , gene expression levels of integrin α7. Data are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 as determined by Tukey’s post hoc test. F , morphology of THP-1 macrophages treated with integrin α7-targeted small interfering RNA #9, #1 or nonspecific siRNA on day 5. The knockdown experiment was repeated three times independently. Integrin α7 knockdown induced dendritic processes in THP-1 macrophages cultured on laminin-211 ( arrows ). Nonspecific siRNA did not induce obvious morphological alterations. The scale bars represent 10 μm. DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G.

Article Snippet: An isotype control IgG was obtained from Bio X Cell (BE0366).

Techniques: Functional Assay, Blocking Assay, Cell Culture, Staining, Immunostaining, Imaging, Control, Western Blot, Small Interfering RNA, Gene Expression, Knockdown

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Choice of cell line for sACE2 2 .v2.4-IgG1 production impacts pharmacokinetics and glycosylation (A and B) sACE2 2 .v2.4-IgG1 was expressed and purified from human Expi293F (gray) or nonhuman ExpiCHO-S (black) cultures that were both transiently transfected. A 10 mg/kg amount of sACE2 2 .v2.4-IgG1 was injected into the tail vein of human FcRn transgenic mice. (A) ACE2 catalytic activity and (B) protein concentrations based on ELISA were measured in plasma. Data are mean ± SEM, n = 3 mice per time point. (C) N-glycan types from glycomics analysis of sACE2 2 .v2.4-IgG1 produced in ExpiCHO-S vs. Expi293F cells. (D) Abundance of sialylated (Neu5Ac) and fucosylated N-glycan structures. (E and F) O-Glycan analysis of sACE2 2 .v2.4-IgG1 produced in (E) ExpiCHO-S and (F) Expi293F cells. After PNGaseF treatment of protein, O-glycans were released, purified, permethylated, and analyzed by MALDI-TOF-MS. O-glycan structures were assigned using Glycoworkbench software based on precursor masses and the common mammalian biosynthetic pathway. Glycan structures are indicated on the x axis by their m/z ratio. GlcNAc, blue squares; GalNAc, yellow squares; Gal, yellow circles; Neu5Ac, purple diamonds.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Drug discovery, Glycoproteomics, Purification, Transfection, Injection, Transgenic Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Produced, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: An engineered derivative of sACE2 2 .v2.4-IgG1 and its glycosylation (A) Left, the structure (PDB: 6M17 ) of dimeric ACE2 (chains “A” and “B” in dark and light green) bound to RBD (gray ribbons). Glycans are shown as orange sticks. PD, protease domain; CLD, collectrin-like dimerization domain. Center and right, residues mutated to fill cavities (blue spheres), introduce disulfides (yellow spheres), or add N-glycosylation motifs (purple spheres) are shown on a single ACE2 subunit. Lead candidate sACE2 2 .S19-IgG1 has mutations V491I, M662T, N720S. (B) N-glycan types on sACE2 2 .S19-IgG1 produced in Expi293F cells. (C) Abundance of sialylated and fucosylated N-glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells. (D) O-Glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells following O-glycan release and MALDI-TOF-MS analysis. (E) Occupancy of the N-glycosylation sites based on glycopeptidomics analysis of sACE2 2 .S19-IgG1 from Expi293F (green), sACE2 2 .v2.4-IgG1 from Expi293F (pale gray), and sACE2 2 .v2.4-IgG1 from ExpiCHO-S (dark gray). sACE2 2 .S19-IgG1 has added glycosylation sites at positions 660 and 718. (F) Percent of the glycoforms at each N-glycosylation site that have at least one sialic acid.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Glycoproteomics, Introduce, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: BLI kinetics for monovalent binding of decoy receptors to Spike RBD

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Binding Assay, Mutagenesis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: High sialylation and YTE mutations in the Fc region enhance the pharmacokinetics of sACE2 2 .S19-IgG1 (A) Intravenous administration of proteins at 10 mg/kg into human FcRn mice. Blood was collected into heparin via retroorbital route at the plotted time points. Plasma levels of the indicated proteins were measured by ELISA. (B and C) Proteins were injected s.c. in the flank of human FcRn mice at a dose of 10 mg/kg (solid lines) or 100 mg/kg (broken line). (B) ACE2 catalytic activity in plasma and (C) protein concentrations based on ELISA. (D) sACE2 2 .S19-IgG1(YTE) was treated with PNGase F or neuraminidase. Proteins (20 μg) were analyzed without further purification by SDS-PAGE under non-reducing conditions and stained with Coomassie. The calculated molecular weight (MW) of the mature polypeptide (excluding glycans) is 218 kD for the dimer. PNGase F has an MW of 36 kD. A. ureafaciens neuraminidase is a mixture of isoenzymes. (E) Proteins (10 μg) were analyzed by IEF gel electrophoresis. Glycosidase-treated proteins were analyzed without (−) and with (+) purification by gel filtration following treatment. (F) Purified proteins were injected i.v. at 10 mg/kg into human FcRN mice and concentrations in plasma were measured by ELISA for 5 days. For PK studies in this figure, data are mean ± SEM for n = 3 per group and proteins were purified from transiently transfected Expi293F.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Drug discovery, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Injection, Activity Assay, Purification, SDS Page, Staining, Molecular Weight, Nucleic Acid Electrophoresis, Filtration, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Pharmacokinetic properties of optimized decoy receptors in Tg32 mice

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Produced, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: A single dose of sACE2 2 .S19-IgG1(YTE) sourced from Expi293F culture protects K18-hACE2 mice from lethal SARS-CoV-2 challenge (A and B) K18-hACE2 mice were inoculated intranasally with 1 × 10 4 PFU 2019n-CoV/USA_WA1/2020 virus. Mice were administered a single i.v. dose (10 mg/kg) of sACE2 2 .S19-IgG1(YTE) (red; purified from transiently transfected Expi293F culture) or IgG1 control (gray) 24 h post-inoculation. Survival (A) and weights (B) for N = 10 mice per treatment group. p value determined by Gehan-Breslow-Wilcoxon test. (C and D) Lungs of inoculated mice were harvested at day 7 and relative viral loads were determined by qPCR for mRNA expression levels of SARS-CoV-2 Spike (C) and Nsp (D). Mean ± SEM, N = 4 per treatment group. p values determined by unpaired t test.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Virus, Purification, Transfection, Control, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Sialylation of decoy receptors purified from different sources

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Purification

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: The v2.4 and S19 mutations in ACE2 are predicted to have low immunogenicity (A) Computational analysis of calculated affinity of peptides to HLA-II allotypes. The wild-type sACE2 2 -IgG1 sequence (top row) is scanned for peptides predicted to be displayed on a common set of 14 HLA-II allotypes. In this analysis, the minimum threshold for a peptide to be considered an antigen is predicted affinity for four HLA-II allotypes (Nhits ≥4) from a set of 14 common alleles. For sACE2 2 -IgG1 derivatives, the sequence is grayed out except for the regions where mutations are introduced to highlight whether a mutation is within a predicted epitope and/or changes the probability of presentation. Peptides that were analyzed experimentally by yeast display are indicated below with dark red bars. (B) Peptides were displayed on yeast and binding to HLA-II following an HLA-DM-dependent peptide loading reaction was measured by flow cytometry. The correlation plot shows the agreement between two independent replicates measuring mean fluorescence units (MFU) for bound HLA-II. Polyserine negative control reactions are blue, positive control peptide/HLA-II pairs are orange, and ACE2 peptides are gray. (C) Yeast display measurements of HLA-II binding to ACE2 peptides is plotted from no signal (dark blue) to high binding signal (orange).

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Immunopeptidomics, Sequencing, Mutagenesis, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Positive Control