Journal: The Journal of Biological Chemistry

Article Title: Production of site-specific antibody conjugates using metabolic glycoengineering and novel Fc glycovariants

doi: 10.1016/j.jbc.2024.108005

Figure Lengend Snippet: Fc glycovariant design and manufacturing process. A , crystallographic structure of human immunoglobulin G1 (IgG1) fragment crystallizable (Fc) domain (PDB ID: 5JII ), with glycovariant sites and canonical N297 glycan site indicated. B , process of Fc glycovariant manufacturing through supplementation with 1,3,4-O-Bu3ManNAz, an azido-functionalized ManNAc analog. C and D , Nonreducing ( C ) and reducing ( D ) SDS-PAGE analysis of the F5111 antibody in wild type (WT), N297A, and engineered Fc glycovariant formats. HC, heavy chain; LC, light chain.

Article Snippet: The heavy chain (HC) sequences and light chain (LC) sequences for the hIgG1 lambda F5111 antibody were separately cloned into the gWiz vector (Genlantis) ( ).

Techniques: SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Production of site-specific antibody conjugates using metabolic glycoengineering and novel Fc glycovariants

doi: 10.1016/j.jbc.2024.108005

Figure Lengend Snippet: Fc glycovariants express robustly and incorporate azides to allow fluorescent labeling while retaining antigen and Fc receptor binding. A , yield of antibodies from human embryonic kidney (HEK) 293F cells following purification with protein G. B , average number of dye molecules per antibody molecule, determined by dye/protein ratio of azide-functionalized wild type (WT) F5111 antibody and glycovariants thereof labeled with dibenzocyclooctyne (DBCO)-linked fluorescent dye, as measured by UV/Vis spectroscopy. C , biolayer interferometry studies of the equilibrium binding between immobilized IL-2 and soluble F5111 glycovariants. D , biolayer interferometry studies of the equilibrium binding between immobilized FcRn and soluble F5111 glycovariants. E and F , biolayer interferometry studies of the equilibrium binding between immobilized FcγRI ( E ) or FcγRIIa ( F ) and soluble F5111 glycovariants with re-introduced N297 glycosylation site.

Article Snippet: The heavy chain (HC) sequences and light chain (LC) sequences for the hIgG1 lambda F5111 antibody were separately cloned into the gWiz vector (Genlantis) ( ).

Techniques: Labeling, Binding Assay, Purification, UV-Vis Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Production of site-specific antibody conjugates using metabolic glycoengineering and novel Fc glycovariants

doi: 10.1016/j.jbc.2024.108005

Figure Lengend Snippet: Azide incorporation in Fc glycovariants can be optimized through use of different analogs, production in alternative cell lines, or incorporation of multiple glycosylation sites. A , average number of dye molecules per antibody molecule, determined by dye/protein ratio of azide-functionalized and DBCO-linked fluorescent dye-labeled S4 F5111 glycovariant antibody produced with varying amounts of 1,3,4-O-Bu 3 ManNAz (ManNAz) or Bu 4 GalNAz (GalNAz) in HEK 293F or Chinese hamster ovary (CHO)-S cells, as measured by UV/Vis spectroscopy. B , reducing SDS-PAGE analysis of wild-type (WT) F5111 antibody and glycovariants thereof, including double and triple glycomutant antibodies. C , the average number of dye molecules per antibody molecule upon expression of antibody glycovariants with supplemented azide-functionalized analogs. Either WT F5111 antibody and double or triple mutant glycovariants thereof transiently expressed in HEK 293F cells or the S146 trastuzumab glycovariant stably expressed in ExpiCHO cells in the presence of the sialyltransferase ST6GAL1 were labeled with DBCO-linked fluorescent dye, and dye/protein ratio was measured by UV/Vis spectroscopy. HC, heavy chain; LC, light chain.

Article Snippet: The heavy chain (HC) sequences and light chain (LC) sequences for the hIgG1 lambda F5111 antibody were separately cloned into the gWiz vector (Genlantis) ( ).

Techniques: Labeling, Produced, UV-Vis Spectroscopy, SDS Page, Expressing, Mutagenesis, Stable Transfection

Journal: Science Advances

Article Title: Bioengineered particles expand myelin-specific regulatory T cells and reverse autoreactivity in a mouse model of multiple sclerosis

doi: 10.1126/sciadv.add8693

Figure Lengend Snippet: ( A ) Rapamycin-loaded MPs are fabricated by single emulsion and conjugated with tetrameric MHC class II/ peptide complexes and single-chain fusion IL-2/ anti–IL-2 (F5111) immunocytokines to generate Tol-MPs. ( B ) Tol-MPs are designed to preferentially engage antigen-specific T regs and promote their expansion. The immunocytokine blocks IL-2 signaling on non-T regs that lack CD25, and the local release of rapamycin is intended to suppress pathogenic effector T cells that engage the Tol-MP. The expansion of myelin-specific T regs can halt and reverse damage to the myelin sheath of nerves in the CNS to treat MS. PVA, poly(vinyl alcohol); PLGA, poly(lactic co -glycolic acid); PBAE, poly(beta-amino ester).

Article Snippet: DiD-labeled particles were conjugated with different combinations of OVA tetramer, F5111 IC, control tetramer, and isotype control IgG (Bio X Cell, Lebanon, NH).

Techniques: Emulsion

Journal: Science Advances

Article Title: Bioengineered particles expand myelin-specific regulatory T cells and reverse autoreactivity in a mouse model of multiple sclerosis

doi: 10.1126/sciadv.add8693

Figure Lengend Snippet: ( A ) Scanning electron micrograph images of PLGA/PBAE MPs loaded with rapamycin during particle synthesis. ( B ) Frequency distribution of dry particle diameters (bin size = 0.5 μm). ( C ) Average dry diameter and Z -average hydrodynamic diameter of MPs measured using scanning electron microscopy and dynamic light scattering (DLS), respectively. Error bars for scanning electron microscopy represent the SEM of 100 particles. Error bars for DLS represent the SEM of three replicates. ( D ) Rapamycin release from MPs. Error bars represent the SEM of three replicates. ( E ) Protein density and ( F ) conjugation efficiency of MHC class II tetramers containing ovalbumin (OVA) peptide and F5111 IC to the surface of MPs. Error bars in (E) and (F) represent the SEM of four replicates.

Article Snippet: DiD-labeled particles were conjugated with different combinations of OVA tetramer, F5111 IC, control tetramer, and isotype control IgG (Bio X Cell, Lebanon, NH).

Techniques: Electron Microscopy, Conjugation Assay

Journal: Science Advances

Article Title: Bioengineered particles expand myelin-specific regulatory T cells and reverse autoreactivity in a mouse model of multiple sclerosis

doi: 10.1126/sciadv.add8693

Figure Lengend Snippet: ( A ) MPs or free proteins were incubated with CD4 + T cells, and flow cytometry was used to measure pSTAT5, a key downstream indicator of IL-2 signaling. ( B ) pSTAT5 expression in CD25 + Foxp3 + T regs and CD25 − Foxp3 − T cells. Soluble human IL-2 (hIL-2) (blue) induces pSTAT5 in both CD25 + and CD25 − T cells. pSTAT5 expression in non-T regs is indicated with the black arrow. F5111 IC MPs (green) and soluble F5111 IC (orange) exclusively stimulate CD25 + T regs . ( C ) Soluble F5111 IC and F5111 IC MPs induce similar dose-dependent pSTAT5 expression in T regs and non-T regs . Error bars represent the SEM. JAK, Janus kinase.

Article Snippet: DiD-labeled particles were conjugated with different combinations of OVA tetramer, F5111 IC, control tetramer, and isotype control IgG (Bio X Cell, Lebanon, NH).

Techniques: Incubation, Flow Cytometry, Expressing

Journal: Science Advances

Article Title: Bioengineered particles expand myelin-specific regulatory T cells and reverse autoreactivity in a mouse model of multiple sclerosis

doi: 10.1126/sciadv.add8693

Figure Lengend Snippet: ( A ) MHC class II tetramers loaded with OVA or a control peptide were conjugated to fluorescent MPs for binding studies. MPs were incubated with naïve CD4 + T cells harvested from OT-II mice or CD4 + CD25 + T cells harvested and activated in vitro for 4 days. ( B and C ) OT-II (OVA-specific) CD4 + T cells showed significantly higher levels of binding to OVA-specific MPs compared to control and blank MPs. Antigen-specific binding was observed at all MP doses tested (per 100,000 cells). ( D and E ) OVA specificity dominates MP binding to cells. MPs conjugated with OVA/isotype control antibody or OVA/F5111 IC bound to OVA-specific CD4 + CD25 + T cells to a greater extent than did MPs conjugated with control tetramer and either isotype control antibody or F5111 IC (** P < 0.01 and **** P < 0.0001). Error bars represent the SEM. MFI, mean fluorescence intensity; Ab, antibody.

Article Snippet: DiD-labeled particles were conjugated with different combinations of OVA tetramer, F5111 IC, control tetramer, and isotype control IgG (Bio X Cell, Lebanon, NH).

Techniques: Control, Binding Assay, Incubation, In Vitro, Fluorescence

Journal: Science Advances

Article Title: Bioengineered particles expand myelin-specific regulatory T cells and reverse autoreactivity in a mouse model of multiple sclerosis

doi: 10.1126/sciadv.add8693

Figure Lengend Snippet: Summary of EAE studies. N/A, not applicable.

Article Snippet: DiD-labeled particles were conjugated with different combinations of OVA tetramer, F5111 IC, control tetramer, and isotype control IgG (Bio X Cell, Lebanon, NH).

Techniques: