biotinylated human cd16a v158  (Sino Biological)

Journal: Cell reports

Article Title: Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

doi: 10.1016/j.celrep.2025.116593

Figure Lengend Snippet: (A) BW-CD16A-ζ reporter cells were incubated with SM5–1 antibody, 100 μg/mL isotype, or 1:100 dilution Cytotect and AD169- or Δ3-infected MRC5 fibroblasts (MOI = 5 and 72 hpi), with CD16A activation quantified by mouse IL-2. (B) Fluorescent images of AD169-infected MRC5 cells (MOI = 2 and 96 hpi) incubated with AF647-labeled SM5–1 or SM5–1-mFc (20 μg/mL) for 2 h at 37°C. Cells fixed with 4% paraformaldehyde and scanned using Zeiss LSM 710/Elyra S.1 at 63×. Scale bars, 21 μm. (C and D) Flow cytometry measured the percent of cells positive for (C) extracellular or (D) internalized antibody after incubating 67 nM antibody with infected cells (as above) or mock-infected cells for 2 h at 37°C. Extracellular antibody detected with goat anti-human-Fcy AF647; intracellular with pHrodo-Red-labeled primary antibody. Data are mean ± SD, for n = 2 with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns: non-significant as determined by two-way ANOVA followed by Tukey’s multiple comparisons test in GraphPad. Data are representative of one experiment; each experiment was repeated twice.

Article Snippet: Biotinylated human CD16A V158 , Sino Biological , Cat# 10389-H27H1B.

Techniques: Incubation, Infection, Activation Assay, Labeling, Flow Cytometry

Journal: Cell reports

Article Title: Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

doi: 10.1016/j.celrep.2025.116593

Figure Lengend Snippet: (A) Binding of host Fc receptors (FLAG-tagged CD16A-GST, FcRn-GST, or gp34-M) to immobilized human Fc was measured in the presence of competitor (gp34-M, t-gp68, and hu-Fc) by ELISA. Assay area under the curve (AUC) was normalized to controls lacking a competitor and presented as a heatmap. (B and C) Binding of antibody Fc variants to gp34-M, t-gp68, and (B) FcRn or (C) CD16A assessed by ELISA with normalized inverse 50% effective concentration (EC 50 ) values shown as a heatmap with more color indicating better binding. (D and E) The nsEM 3D reconstruction and 2D class averages for particles containing (D) one Fc with one gp34-M bound near the CH 2 tip and (E) one Fc with one t-gp68 bound to the CH 2 -CH 3 interface. RoseTTAFold-based gp34 (green) and t-gp68 (blue) models fit the 3D reconstruction with the Fc crystal structure (PDB: 2GJ7). Scale bars, 5 nm. (F and G) SPR measured (F) t-gp68 binding to immobilized Fc and (G) binding of human IgG1 antibody to immobilized gp34-M with K D values determined using BIAevaluation X100 software. (H and I) Flow cytometry measured (H) binding to and (I) internalization of pHrodo-Red-labeled antibody (67 nM) by AD169-infected MRC5 cells (MOI = 2 and 96 hpi) in the presence of t-gp68 (2 μM) and/or gp34-M (0.1 μM). Extracellular antibody detected with goat-anti-human-Fcy AF647; in (I), the dashed line represents the threshold for detection. Data shown are mean ± SD, n = 2, with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns, non-significant. All analyses are performed in GraphPad using two-way ANOVA with Tukey’s multiple comparisons test. Representative data of one experiment is shown; each experiment is repeated at least twice.

Article Snippet: Biotinylated human CD16A V158 , Sino Biological , Cat# 10389-H27H1B.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Software, Flow Cytometry, Labeling, Infection

Journal: Cell reports

Article Title: Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

doi: 10.1016/j.celrep.2025.116593

Figure Lengend Snippet: (A) Schematic of the yeast display and competitive staining strategy used for Fc selection. (B) To select for gp34-resistance, an Fc library (Δgp34 R0) was incubated with AF647-CD16A (V158) tetramers with 1 μM unlabeled gp34-M, followed by four rounds of FACS (Δgp34 R4). (C) To select for clones that also exhibit gp68-resistance, a library (Δgp68 R0) was constructed from R47 and incubated with AF647-CD16A (V158) and PE-FcRn tetramers at pH 6.0 with 5 μM unlabeled t-gp68, followed by four rounds of FACS (Δgp68 R4). (D) Binding affinities of Fc variant hu4D5 antibodies determined by BLI for FcRn, CD16A V158, or F158 or SPR for gp34-M and t-gp68, with the K D mean ± SEM ( n = 2) are shown. (E) Fc binding to different vFcγRs ectodomains expressed on CHO cells evaluated by staining with Fc variant hu4D5 antibodies and goat-anti-Fcγ-AF647 detection. Mean AUC shown as a heatmap, with darker color indicating more binding; repeated twice with n = 2. For (D) and (E) significance versus WT was assessed by one-way ANOVA with Tukey test for multiple comparisons. (F–H) ADCC was performed using hu4D5-Fc variant antibodies, HER2-positive SKOV3 target cells, and NK-92 V158 or NK-92 F158 effector cells, with cell lysis, was measured by calcein release. ADCP performed by incubating SM5–1 Fc variants with (G) gB-coated pHrodo-Green/APC-polystyrene beads or (H) pHrodogreen-labeled AD169 virions and then THP1 monocytes (50 beads or 2 PFU per cell). Phagocytosis scores were calculated as the percent antigen-presenting cell/fluorescein isothiocyanate (APC/FITC)-positive cells multiplied by the APC geometric mean fluorescence intensity (GMFI) and compared to WT. Representative data (mean ± SD) are shown, with each experiment repeated at least twice. Statistical analyses performed using two-way ANOVA followed by Tukey’s multiple comparison test, with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns, non-significant.

Article Snippet: Biotinylated human CD16A V158 , Sino Biological , Cat# 10389-H27H1B.

Techniques: Staining, Selection, Incubation, Clone Assay, Construct, Binding Assay, Variant Assay, Lysis, Labeling, Fluorescence, Comparison

Journal: Cell reports

Article Title: Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

doi: 10.1016/j.celrep.2025.116593

Figure Lengend Snippet: (A and B) (A) Cell staining by and (B) internalization of Fc variant SM5–1 antibodies (20 μg/mL) with AD169- or mock-infected MRC5 cells. Comparisons performed using one-way ANOVA with Tukey’s multiple comparisons; data are presented as mean ± SD ( n = 2). (C) CD16A activation measured as mouse IL-2 production after incubation of SM5–1 antibodies with BW-CD16A-ζ reporter cells and AD169-or Δ3-infected MRC5 cells (MOI = 5 and 72 hpi). Data normalized to results for SM5–1 WT with Δ3; curves from n = 3 replicates were fit to 4PL or AUC using GraphPad. Mean EC 50 values listed unless no activation was detected at 100 μg/mL; p values depicting EC 50 differences for AD169 versus Δ3; * represents significance versus SM5–1 WT. (D) CD16A activation AUC normalized to SM5–1 WT with Δ3; data shown are the mean ± SEM of three experiments. (E) NK cell activation measured as percent CD107a-positive NK-92 (V158) cells after incubation with AD169-infected MRC5 cells (MOI = 2 and 96 hpi) and Fc-variant SM5–1 antibodies or polyclonal antibodies from Cytogam (CMV+) or CMV-negative serum (CMV−; 30 μg/mL). Data shown are mean ± SD ( n = 2) and average of n = 3 experiments, with comparison using two-way ANOVA with Tukey’s multiple comparisons test and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns, non-significant.

Article Snippet: Biotinylated human CD16A V158 , Sino Biological , Cat# 10389-H27H1B.

Techniques: Staining, Variant Assay, Infection, Activation Assay, Incubation, Comparison

Journal: Cell reports

Article Title: Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

doi: 10.1016/j.celrep.2025.116593

Figure Lengend Snippet: (A–C) CD16A activation measured as mouse IL-2 production after incubation of neutralizing SM5–1 or non-neutralizing antibodies with BW-CD16A-ζ reporter cells in the presence of HFF cells infected (MOI = 3 and 96 hpi) with (A) AD169 or Δ3, (B) Merlin or its vFcγR-deficient strain Merlin-Δ4, and (C) TB40E. (D) CD16A activation after incubation of Cytotect (500 μg/mL) with AD169, Merlin, or mock-infected cells. Data normalized to the maximum response per viral strain and presented as mean ± SD ( n = 3) for one representative experiment, with significance determined by one-way ANOVA with Tukey test for multiple comparisons. (E) Ex vivo purified human donor PBMC cells and 27–287 antibodies (20 μg/mL) combined with Merlin- or Merlin-Δ4-infected HFF cells (10:1 E:T ratio or 1:1 NK:T ratio, 96 h hpi). NK cell degranulation (CD107a-positive) measured after 4 h; data shown are mean ± SD ( n = 3) of one representative experiment with isotype control subtracted. (F) Immortalized skin fibroblasts (SFi-hTert) infected at low MOI, with 27–287 antibodies (5 μg/mL) and autologous NK cells added at indicated E:T ratios 72 hpi. After 7 days of culture in an Incucyte, the integrated GFP fluorescence was measured per antibody with NK cells relative to no NK cells. Each condition was assessed in technical quadruplicate, with curves fit to 4PL using GraphPad. Statistics compare G2 (black stars) or G2E (pink stars) to WT over different E/T ratios. Two-way ANOVA with Tukey’s multiple comparisons test was performed on all datasets in (A)–(E), except (C), with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns, non-significant.

Article Snippet: Biotinylated human CD16A V158 , Sino Biological , Cat# 10389-H27H1B.

Techniques: Activation Assay, Incubation, Infection, Ex Vivo, Purification, Control, Fluorescence