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: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: Blood was collected from mothers just before or after birth and from neonates birth. A) Transport rates for all IgG subclasses expressed as cord/maternal ratios found at birth. The transport rates differed significantly from each other (P<0.0001), except for IgG2 and IgG3 (not significant), as tested by one-way Anova and Tukey's multiple comparison test. (B–E)IgG subclass 1–4 serum levels were quantified by nephelometry and each pair was plotted on a X axis displaying days of each pregnancy against IgG concentration. Average neonate concentration was significantly higher than in the mother for IgG1 and IgG4 as tested by a paired-T test as shown (child/mother ratio = 1.55 and 1.38, respectively) while averge concentrations for IgG2 and IgG3 were not significantly different in mothers and their children (child/mother ratios not significantly different from 1). One pre-term baby was identified displaying low transport of all IgG (square symbol). (F) Child/mother transport ratio of subclasses IgG2-4 for each pair was plotted relative to the IgG1 transport ratios.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Comparison, Concentration Assay

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: In general, IgG1 is transported better than IgG4, both of which are transported better than IgG2 and IgG3, which have similar transport rates. Two-tailed Pearson correlation revealed a significant correlation for all subclasses, IgG1 R 2 = 0.379, P = 0.0018; IgG2 R 2 = 0.2910, P = 0.0096; IgG3 R 2 = 0.2415, P = 0.0202; IgG4 R 2 = 0.2881, P = 0.0121. Thus, for all subclasses, relatively more IgG was transported at lower maternal IgG.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Two Tailed Test

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: Binding of titrated amounts of soluble human FcRn (62.5–8000 nM) at pH 6.0 to human IgG variants immobilized onto CM5 sensor flow cells. The relative affinity constants derived (KD) are indicated.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Binding Assay, Derivative Assay

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: Results from IgG1 total (A) and IgG2 total (B) were plotted against the sum of IgG1κ and IgG1λ, or IgG2κ and IgG2λ, respectively. The results of regression analysis are indicated in each panel, along with Pearson's correlation.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques:

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: IgG1κ (A), IgG1λ(B) and IgG2κ (C) IgG2λ (D) light chain isotype from sera in were quantified by subclass- and light chain specific ELISA and each mother-child pair was plotted on the x- and y-axis, respectively. A paired t-test revealed no significant difference between the light chains isotypes within each antibody subclass. Average neonate concentration was significantly higher than in the mother for IgG1κ (A) and IgG1λ (B) as indicated in each graph by P values (child/mother ratio = 1.60 and 1.56, respectively) while average concentrations for IgG2 κ and IgG2 λ (C and D) were not significantly different in mothers and their children. (E) Child/mother transport ratio of IgG1λ, IgG2κ and IgG2λ for each pair was plotted relative to IgG1κ transport ratios. While both IgG2 isotypes perform worse than IgG1 when concentration increases, no difference is visible between the IgG2-light chain isotypes.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: (A) Recombinant human IgG2κ and λ in Balb/C mice was injected and measured by total IgG ELISA for a two week period following injection of 200 µg IgG. Calculated half-lives were 7.2±1.48 and 6.4±0.84 days for IgG2κ and IgG2λ. (B) Enrichment of IgG2 κ isoforms was performed as described in Dillon et al 2008. HPLC elution profiles of IgG2 κA and κB structural isomeres on a Dionex ProPac WCX-10 (4.0_250 mm) column are depicted. IgG2κB isoform was generated by incubation of 3 mg/ml IgG2κ in 200 mM Tris buffer at pH 8 with 6 and 1 mM of cysteine and cystamine, respectively. For IgG2κA synthesis 0.9M guanidine hydrochloride (GuHCl) was also added. The samples were kept in the dark and placed at 4°C for 48–72 h. Following incubation the antibody was run on a Zeba spin desalting column (Pierce) for buffer exchange into PBS. (C) The clearance of IgG2λ, IgG2κA, and IgG2κB in Balb/C mice. Calculated half-lives were 4.0±0.58, 5.39±0.85, and 3.7±1.04 days for IgG2κA, IgG2κB and IgG2λ, respectively. (D) Clearance of IgG2κA and IgG2κB in WT and FcγR −/− C57Bl/6 mice. Calculated half-lives were 6.2±2.62, 6.43±1.69, and 7.5±1.89 days for IgG2κA, IgG2κB and IgG2λ, respectively, in WT mice but 1.08±0.28, 1.19±0.23, and 0.7±0.92 days for IgG2κA, IgG2κB and IgG2λ, respectively, in FcRn −/− mice. Graphs in (A, C–D) depict mean and standard deviations of results obtained for 4 mice per group. Half-lives were calculated assuming exponential decay and reported in days ± standard error of means. No significant difference in half-life was detected between the two isotypes.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Recombinant, Injection, Enzyme-linked Immunosorbent Assay, Generated, Incubation, Buffer Exchange

Journal: PLoS ONE

Article Title: On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

doi: 10.1371/journal.pone.0108319

Figure Lengend Snippet: (A) The average IgG1 and IgG2 placental transport (maternal/child) ratios were compared according to their light chain isotype. (B) Clearence of IgG1 and IgG2 κ and λ was investigated in humans by collecting blood from hypogammaglobulinemia patients four weeks after an IVIg transfusion. IgG1 and IgG2 light chain isotypes κ and λ were quantified in serum by subclass- and light chain specific ELISA and subclass composition was compared to that found in the IVIg used. No preferential clearance of one light chain isotype was detectable in either IgG subclass.

Article Snippet: Similarly, mouse anti-human IgG2 (HP6002, Southern Biotech, 1/100 for lambda assay, 1/50 for kappa assay) was used for coating, and HRP-labelled mouse anti-human λ JDC12 (Southern Biotech, 1/1600) or k (HP6062, 1/1300) for detection.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines

doi: 10.1038/s42003-021-02881-w

Figure Lengend Snippet: a A basic panel of FP-barcoded reporter cell lines. K530 cells were transduced with different combinations of 4 FPs to produce 16 uniquely FP-barcoded reporter cell lines. The absence/presence of fluorescence from FPs EBFP2, mTurquoise2 (mTq2), mNeonGreen (mNG), and mCardinal (mCar) are designated as four digits of binary barcodes as shown on the right of histograms for each individual cell line. b Demultiplexing of pooled FP-barcoded reporter cell lines by flow cytometry. c The 16 barcoded reporter cell lines were transduced to express human CD4, CD8a, CD86, and CD154 molecules in a shifted pattern relative to FP expression. These cells were pooled and stained with corresponding mouse monoclonal antibodies (as indicated on the top of each histogram) followed by a PE-conjugated anti-mouse IgG antibody. Signals from individual reporter cell lines were demultiplexed as shown in b and the binding by corresponding antibodies were plotted as half-offset histograms. In all cases, the detected expression patterns were consistent with antigen expression by barcoded cells before multiplexing as shown on the right of histograms for each individual cell line. Isotype Ctrl, mouse IgG1, κ-isotype control antibody. Data from one experiment are shown. Data from another independent repeat experiment are shown in Supplementary Fig. .

Article Snippet: Isotype control antibodies included the following: Mouse IgG1 κ-isotype control (Rockland 010-001-330), Human IgG1κ (hIgG1K, Southern Biotech 0151K-01), and Human IgG1 Lambda (hIgG1L, Southern Biotech 0151L-01).

Techniques: Transduction, Fluorescence, Flow Cytometry, Expressing, Staining, Bioprocessing, Binding Assay, Multiplexing, Control

Journal: iScience

Article Title: Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation

doi: 10.1016/j.isci.2023.106385

Figure Lengend Snippet: GAL1+ mesenchymal stromal cells favor the development of murine Pre-BCR+ B-ALL

Article Snippet: The expression of GAL1 was controlled with an anti mouse GAL1 antibody (R&D Systems) conjugated with the Alexa-Fluor 488 antibody labeling kit (Thermo Fisher).Patient samples and human cell lines.

Techniques:

Journal: iScience

Article Title: Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation

doi: 10.1016/j.isci.2023.106385

Figure Lengend Snippet: Pre-BCR signaling and human pre-B ALL growth are impaired in the absence of GAL1

Article Snippet: The expression of GAL1 was controlled with an anti mouse GAL1 antibody (R&D Systems) conjugated with the Alexa-Fluor 488 antibody labeling kit (Thermo Fisher).Patient samples and human cell lines.

Techniques: