human fcγriib  (Sino Biological)

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Schematic overview of the IgE elimination experiment. Humanized FcγRIIb mice ( b – e ; n = 5 mice per group) or FcγRIIb -/- mice ( f , g ; n = 4 mice per group) were injected with 5 mg/kg FcRTACs,followed by injection of 2.5 mg/kg IgE 30 min later. Blood samples were collected at the indicated time points (red arrows) to measure the serum levels of total IgE ( b , d , f , g ) and free IgE ( c , e ). Data are presented as means ± SEM. Statistical significance was determined using two-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ). The curves for “Only IgE”, “Omalizumab”, and “NK-2-12” are shared between panels b and d , as well as c and e . Panel ( a ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/53ug670 .

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Injection

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Alignment of SK3 and SK4 Sequences. HCDRs and LCDRs are indicated with red. b SPR-based competition assay for SK3 and SK4. NK-2-12-SK3 was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Subsequently, SK4 Fab and hFcγRIIb were injected. c Structure of the SK4 Fab in complex with FcγRIIb (PDB ID: 9M5B ). The heavy and light chains of the SK4 Fab are shown in orange and light blue, respectively. The ribbon diagram of FcγRIIb is colored green, with its surface illustrated in white and the contact area with SK4 Fab highlighted in brown. d Comparison of the calculated SK3 scFv-FcγRIIb complex structure versus the SK4 Fab-FcγRIIb complex structure. e SPR-based affinity measurements of SK3 and SK3hi binding to hFcγRIIb at pH 7.4 and pH 6.0. SK3 or SK3hi was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Solid curves represent raw data, while dashed curves indicate fitted curves. f Live-cell imaging of NK-2-12-SK3hi-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence or absence of Cy5-labeled NK-2-12-SK3hi (white) for 2 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a super-resolution confocal microscope. Scale bar: 5 μm. Experiments were performed in triplicate and repeated three times with similar results. g , h The rechallenge experiment. FcγRIIb humanized mice ( n = 4 mice per group) were i.p. injected with 2.5 mg/kg IgE following by injection of 2.5 mg/kg NK-2-12-SK3 or NK-2-12-SK3hi. One day later, mice were rechallenged with 2.5 mg/kg IgE without FcRTACs. Blood samples were collected to measure serum IgE levels. Data are presented as means ± SEM. Statistical comparisons were performed using Mixed-effects analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ).

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Competitive Binding Assay, Injection, Comparison, Binding Assay, Live Cell Imaging, Labeling, Incubation, Staining, Imaging, Microscopy

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Schematic diagram of the structure of NK-2-12-v12. b Internalization assay of NK-2-12-V12. Antibodies were pre-mixed with a pH-sensitive dye-labeled anti-human Fc and incubated with Huvec-2b cells. Fluorescence changes were monitored hourly over 24 h. c , Internalization assay of NK-2-12-V12 in presence of endocytosis inhibitors. d , e Live-cell imaging of bispecific antibodies-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence of Cy5-labeled designated antibodies (white) for 1 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a 40× objective ( d , scale bar: 40 μm) or a 60× objective confocal microscope ( e left panel, scale bar: 20 μm) and a super-resolution microscope ( e middle and right panel; scale bar: 10 μm). Experiments were performed in triplicate and repeated three times with similar results. f FcγRIIb humanized mice were injected with 5 mg/kg NK-2-12-SK3 or NK-2-12-V12, followed by administration of 2.5 mg/kg IgE after 30 min. Blood samples were collected to measure the serum IgE levels ( n = 4 mice per group). Data are presented as means ± SEM. Statistical significance was determined using two-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ). The curves for “Only IgE” is shared between Supplementary Fig. . g FcγRIIb humanized mice were injected with 5 mg/kg of NK-2-12-SK3 and NK-2-12-V12. Blood was collected from the tail vein of mice at indicated time points and serum antibody levels were quantitatively detected by ELISA ( n = 5 mice per group). Panel ( a ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/g5wqarw .

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Labeling, Incubation, Fluorescence, Live Cell Imaging, Staining, Imaging, Microscopy, Super-Resolution Microscopy, Injection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Schematic overview of the IgE elimination experiment. Humanized FcγRIIb mice ( b – e ; n = 5 mice per group) or FcγRIIb -/- mice ( f , g ; n = 4 mice per group) were injected with 5 mg/kg FcRTACs,followed by injection of 2.5 mg/kg IgE 30 min later. Blood samples were collected at the indicated time points (red arrows) to measure the serum levels of total IgE ( b , d , f , g ) and free IgE ( c , e ). Data are presented as means ± SEM. Statistical significance was determined using two-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ). The curves for “Only IgE”, “Omalizumab”, and “NK-2-12” are shared between panels b and d , as well as c and e . Panel ( a ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/53ug670 .

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Injection

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Alignment of SK3 and SK4 Sequences. HCDRs and LCDRs are indicated with red. b SPR-based competition assay for SK3 and SK4. NK-2-12-SK3 was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Subsequently, SK4 Fab and hFcγRIIb were injected. c Structure of the SK4 Fab in complex with FcγRIIb (PDB ID: 9M5B ). The heavy and light chains of the SK4 Fab are shown in orange and light blue, respectively. The ribbon diagram of FcγRIIb is colored green, with its surface illustrated in white and the contact area with SK4 Fab highlighted in brown. d Comparison of the calculated SK3 scFv-FcγRIIb complex structure versus the SK4 Fab-FcγRIIb complex structure. e SPR-based affinity measurements of SK3 and SK3hi binding to hFcγRIIb at pH 7.4 and pH 6.0. SK3 or SK3hi was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Solid curves represent raw data, while dashed curves indicate fitted curves. f Live-cell imaging of NK-2-12-SK3hi-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence or absence of Cy5-labeled NK-2-12-SK3hi (white) for 2 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a super-resolution confocal microscope. Scale bar: 5 μm. Experiments were performed in triplicate and repeated three times with similar results. g , h The rechallenge experiment. FcγRIIb humanized mice ( n = 4 mice per group) were i.p. injected with 2.5 mg/kg IgE following by injection of 2.5 mg/kg NK-2-12-SK3 or NK-2-12-SK3hi. One day later, mice were rechallenged with 2.5 mg/kg IgE without FcRTACs. Blood samples were collected to measure serum IgE levels. Data are presented as means ± SEM. Statistical comparisons were performed using Mixed-effects analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ).

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Competitive Binding Assay, Injection, Comparison, Binding Assay, Live Cell Imaging, Labeling, Incubation, Staining, Imaging, Microscopy

Journal: Nature Communications

Article Title: Chimeras co-targeting antigens and FcγRIIb trigger degradation of extracellular soluble proteins and pathological aggregates

doi: 10.1038/s41467-025-67207-4

Figure Lengend Snippet: a Schematic diagram of the structure of NK-2-12-v12. b Internalization assay of NK-2-12-V12. Antibodies were pre-mixed with a pH-sensitive dye-labeled anti-human Fc and incubated with Huvec-2b cells. Fluorescence changes were monitored hourly over 24 h. c , Internalization assay of NK-2-12-V12 in presence of endocytosis inhibitors. d , e Live-cell imaging of bispecific antibodies-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence of Cy5-labeled designated antibodies (white) for 1 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a 40× objective ( d , scale bar: 40 μm) or a 60× objective confocal microscope ( e left panel, scale bar: 20 μm) and a super-resolution microscope ( e middle and right panel; scale bar: 10 μm). Experiments were performed in triplicate and repeated three times with similar results. f FcγRIIb humanized mice were injected with 5 mg/kg NK-2-12-SK3 or NK-2-12-V12, followed by administration of 2.5 mg/kg IgE after 30 min. Blood samples were collected to measure the serum IgE levels ( n = 4 mice per group). Data are presented as means ± SEM. Statistical significance was determined using two-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the exact P value were provided in Supplementary Table ). The curves for “Only IgE” is shared between Supplementary Fig. . g FcγRIIb humanized mice were injected with 5 mg/kg of NK-2-12-SK3 and NK-2-12-V12. Blood was collected from the tail vein of mice at indicated time points and serum antibody levels were quantitatively detected by ELISA ( n = 5 mice per group). Panel ( a ) was created in BioRender. Zhao, J. (2025) https://BioRender.com/g5wqarw .

Article Snippet: C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Humanized FcγRIIb mice (NM-HU-2000010), FcγRIIb-/- mice (NM-KO-00130), Fcer1g-KO mice (NM-KO-190187), humanized FcRn mice (NM-HU-190070) and humanized PCSK9 mice (NM-HU-00075) were purchased from Shanghai Model Organisms Center, Inc. Humanized FcγRIIb mice were generated by CRISPR/Cas9-mediated homologous recombination to exclusively replace the mouse Fcgr2b extracellular domain (exons 3-4) with human Fcgr2b sequences while retaining native transmembrane and intracellular regions.

Techniques: Labeling, Incubation, Fluorescence, Live Cell Imaging, Staining, Imaging, Microscopy, Super-Resolution Microscopy, Injection, Enzyme-linked Immunosorbent Assay