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(A) Structure of Fc-ARM containing a target-binding terminus (TBT) and antibody-binding terminus (ABT). (B) Schematic illustration showing the activation of an NK cell by Fc-ARM via the crosslinking of FcγRIIIa and antigen. A complex model of <t>HER2/Fc-ARM/IgG/FcγRIIIa</t> is shown in Fig. S3. (C) 3D structure predictions of two types of Fc-ARMs. Z HER2:342 and Z domain were used as the TBT (red) and ABT (orange), respectively.
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(A) Structure of Fc-ARM containing a target-binding terminus (TBT) and antibody-binding terminus (ABT). (B) Schematic illustration showing the activation of an NK cell by Fc-ARM via the crosslinking of FcγRIIIa and antigen. A complex model of <t>HER2/Fc-ARM/IgG/FcγRIIIa</t> is shown in Fig. S3. (C) 3D structure predictions of two types of Fc-ARMs. Z HER2:342 and Z domain were used as the TBT (red) and ABT (orange), respectively.
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(A) Structure of Fc-ARM containing a target-binding terminus (TBT) and antibody-binding terminus (ABT). (B) Schematic illustration showing the activation of an NK cell by Fc-ARM via the crosslinking of FcγRIIIa and antigen. A complex model of HER2/Fc-ARM/IgG/FcγRIIIa is shown in Fig. S3. (C) 3D structure predictions of two types of Fc-ARMs. Z HER2:342 and Z domain were used as the TBT (red) and ABT (orange), respectively.

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: (A) Structure of Fc-ARM containing a target-binding terminus (TBT) and antibody-binding terminus (ABT). (B) Schematic illustration showing the activation of an NK cell by Fc-ARM via the crosslinking of FcγRIIIa and antigen. A complex model of HER2/Fc-ARM/IgG/FcγRIIIa is shown in Fig. S3. (C) 3D structure predictions of two types of Fc-ARMs. Z HER2:342 and Z domain were used as the TBT (red) and ABT (orange), respectively.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Binding Assay, Activation Assay

Recruitment of fluorescence-labeled IgG (IgG-Fluor) via A-Z or A-ZZ to SK-BR-3 and MDA-MB-231 cells, which are HER2 positive and negative, respectively. Cells were seeded and incubated overnight. Fluorescence-labeled IgG (1.0 μM) and each Fc-ARM (100 nM) were added. Nuclei were stained with Hoechst 33 342 (blue). Scale bar = 20 μm.

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: Recruitment of fluorescence-labeled IgG (IgG-Fluor) via A-Z or A-ZZ to SK-BR-3 and MDA-MB-231 cells, which are HER2 positive and negative, respectively. Cells were seeded and incubated overnight. Fluorescence-labeled IgG (1.0 μM) and each Fc-ARM (100 nM) were added. Nuclei were stained with Hoechst 33 342 (blue). Scale bar = 20 μm.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Fluorescence, Labeling, Incubation, Staining

The effect of the concentration of Fc-ARMs and fluorescence-labeled IgG on ternary complex formation on SK-BR-3. (A) and (B) SK-BR-3 cells were treated with increasing concentrations of Fc-ARM with an excess amount of fluorescence-labeled IgG (1.0 μM). (C) and (D) SK-BR-3 cells were treated with increasing concentrations of IgG with Fc-ARM at a concentration of 10 nM. The y -axis indicates the median fluorescence intensity and the x -axis indicates the concentration of each sample. Two experimental repeats were performed.

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: The effect of the concentration of Fc-ARMs and fluorescence-labeled IgG on ternary complex formation on SK-BR-3. (A) and (B) SK-BR-3 cells were treated with increasing concentrations of Fc-ARM with an excess amount of fluorescence-labeled IgG (1.0 μM). (C) and (D) SK-BR-3 cells were treated with increasing concentrations of IgG with Fc-ARM at a concentration of 10 nM. The y -axis indicates the median fluorescence intensity and the x -axis indicates the concentration of each sample. Two experimental repeats were performed.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Concentration Assay, Fluorescence, Labeling

ADCC induced by the combination of Fc-ARM and non-target IgG. SK-BR-3 cells were treated with Fc-ARM (A) A-Z and (B) A-ZZ, and IgG. Fc-ARM (10 nM) was added to the cells and incubated for 30 min. After unbound molecules were washed away, IgG (100 nM) was added. The y -axis indicates the cellular cytotoxicity ratio and the x -axis indicates the ratio of the number of NK cells to cancer cells.

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: ADCC induced by the combination of Fc-ARM and non-target IgG. SK-BR-3 cells were treated with Fc-ARM (A) A-Z and (B) A-ZZ, and IgG. Fc-ARM (10 nM) was added to the cells and incubated for 30 min. After unbound molecules were washed away, IgG (100 nM) was added. The y -axis indicates the cellular cytotoxicity ratio and the x -axis indicates the ratio of the number of NK cells to cancer cells.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Incubation