Journal: iScience

Article Title: JNJ-78306358, a first-in-class bispecific T cell engaging antibody targeting CD3 and HLA-G

doi: 10.1016/j.isci.2025.111876

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Article Snippet: Goat anti-hIgG1-AF647 (Fab’) 2 , Jackson ImmunoResearch , 109-606-098; RRID: AB_2337899.

Techniques: Recombinant, Lysis, Staining, Blocking Assay, Saline, Molecular Weight, Marker, Western Blot, Binding Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Polymer, Enzyme-linked Immunosorbent Assay, Activation Assay, Cell Culture, Membrane, Sterility

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 1. Flow cytometry analyses. A, Example of gating strategy to analyze the binding of scFv (single-chain fragment variable)-phages on rabbit atheromatous proteins coupled to beads. The control (mouse anti-pVIII and Alexa Fluor 488–conjugated anti-mouse antibodies) and the P3 clone are illustrated. Binding of scFv-phages (B) and soluble scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) (C) on rabbit atheromatous proteins coupled to beads. Protein binding was detected using a mouse anti-pVIII antibody and an Alexa Fluor 488-conjugated anti-mouse antibody for scFv-phages, and using a rabbit anti-human Fc antibody and an Alexa Fluor 488–conjugated anti-rabbit antibody for scFv-Fc. Mean values±SD were calculated using the geometric mean of fluorescence (P2 geomean) obtained with 3 different antibody batches. The error bars represent the mean values±SD. CN indicates control negative; FSC-A,forward scatter area; VH, variable heavy; VL, variable light domains.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Flow Cytometry, Binding Assay, Control, Protein Binding, Fluorescence

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 2. Immunohistochemical analysis of arterial tissue sections from atheromatous rabbits and Apoe−/− mice and human endarterectomy specimens using the indicated scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G). The different areas in transversal sections are identified: adventitia (A), media (M), and intima (I). Sections were incubated with the indicated scFv-Fc antibodies (P3, C4, D4, and C8), followed by the HRP-conjugated goat anti-human Fcγ antibody, and the DAB substrate kit reagent. The yellow-brown staining indicates the presence of the antigen recognized by the scFv-Fc. No staining was observed in mouse and rabbit sections incubated with the secondary antibody alone, and there is low background noise in the human sections (upper panels). Scale bars, 100 µm. Nuclei were counterstained with hematoxylin. DAB indicates 3,3′-diaminobenzidine; and HRP, horseradish peroxidase.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Immunohistochemical staining, Incubation, Staining

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 3. SDS-PAGE analysis of immune complexes. After immunoprecipitate elution in 2×SDS Laemmli buffer, immune complexes were separated on polyacrylamide gels (4%–10%) and stained with silver nitrate. SDS-PAGE 1 and 2: proteins extracted from human endarterectomy specimens were immunoprecipitated with the P3 and C4 scFv-Fc (single- chain fragment variable fused to the crystallizable fragment of immunoglobulin G) (from mammalian cells), respectively. SDS-PAGE 3: Gal3R (recombinant galectin-3 protein) was immunoprecipitated with a commercial anti-GAL3 Ab (anti-galectin-3 antibody). MW indicates molecular weight; M, marker; NA, non attributed; PH, proteins from human biopsies.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: SDS Page, Staining, Immunoprecipitation, Recombinant, Molecular Weight, Marker

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 4. Binding of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) and control antibodies to recombinant proteins by ELISA assay. A, P3 scFv-Fc showed stronger binding to recombinant galectin-3 than to the galectin-3BP (galectin-3 binding protein) ligand. Galectin-1 and Glycoprotein αIIbβ3 (GPαIIbβ3) were used to assess the specificity of galectin-3 binding. An irrelevant antigen (BSA) served as a negative control. P3 scFv-Fc binding was detected with a horseradish peroxidase (HRP)-conjugated anti-human Fcγ antibody, and absorbance was measured at 405 nm. Data are the means of 3 independent experiments repeated in triplicate. According to the Kruskal-Wallis test followed by the Dunn test, differential analyses provided the following P values: P=0.0222 for galectin-3 vs galectin-3BP, P=0.0020 for galectin-3 vs galectin-1, P=0.0178 for galectin-3 vs BSA, and P=0.1087 for galectin-3 vs GPαIIbβ3. B, The specificity of the commercial antibodies against galectin-3, galectin-1, and galectin-3BP, of the mouse AP2 (AP2 clone) antibody (from Dr Nurden) against GPαIIbβ3 integrin, and of CN (control negative) scFv-Fc (negative control; from Laboratoire Français de Fractionnement et de Biotechnologies) was assesses by ELISA with recombinant galectin-3, galectin-1, galectin-3BP, BSA, and GPαIIbβ3. The anti-galectin-3 and anti-galectin-3BP antibodies were detected with an HRP-conjugated anti-rabbit antibody. The galectin-1 and AP2 antibodies were detected with an HRP-conjugated anti-mouse antibody. CN was detected with a peroxidase- conjugated anti-human FCγ antibody. The error bars reprement the mean values±SD.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Binding Assay, Control, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 5. Colocalization of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) with an anti-galectin-3 antibody and an anti-LOX1 (lectin-type oxidized LDL receptor 1) antibody in the intima of human endarterectomy specimens and coronary sections. A through D, Co-staining of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) and an anti-galectin-3 antibody in human endarterectomy sections by immunofluorescence. An Alexa Fluor 488 anti-mouse antibody (+488 anti-Ms [mouse]) was used to reveal the specific binding of the commercial anti-human galectin-3 antibody (Ms anti-galectin-3) (A and C). The Alexa Fluor 568 anti-human antibody (+568 anti-Hum) was used to reveal the specific binding of P3 scFv-Fc (Hum P3 scFv-Fc) (B and C). Before image merging, the red and green fluorescence signals were adjusted to comparable levels. The yellow color indicates colocalization of the antigens recognized by P3 scFv-Fc and the anti-human galectin-3 antibody (C). Secondary antibodies alone were used as negative controls (D). Size bars: 100 µm. E through H, The Alexa Fluor 488 anti-rabbit antibody (+488 anti-Rb [rabbit]) was used to reveal the specific binding of the commercial anti-LOX1 antibody (Rb anti-LOX1) (E and G). The Alexa Fluor 647 anti-human antibody (+568 anti-Hum) was used to reveal the specific binding of P3-scFv-Fc (Hum P3 scFv-Fc) (F and G). Before image merging, the red and green fluorescence signals were adjusted to comparable levels. The yellow color indicates colocalization of the antigens labeled by P3 scFV-Fc and anti-LOX1 antibody (G). As galectin-3 is also expressed also by other cell types (ie, mast cells, eosinophils, neutrophils, endothelial cells, and activated T and B cells8,27), areas outside the macrophage location also are stained by P3 scFv-Fc (G). Size bars: 100 μm.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Staining, Immunofluorescence, Binding Assay, Fluorescence, Labeling

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 6. In vitro immunofluorescence analysis of Apoe−/− aorta sections with P3 scFv-Fc (single- chain fragment variable fused to the crystallizable fragment of immunoglobulin G) and an anti- LOX1 (lectin-type oxidized LDL receptor 1) antibody. Colocalization analysis by fluorescence microscopy in Apoe−/− mouse aorta sections of the antigens recognized by P3 scFv-Fc and the commercial anti-LOX1 antibody. The Alexa Fluor 488 anti-rabbit antibody (+488 anti-Rb) was used to reveal the specific binding of the anti-LOX1 antibody (Rb anti-LOX1). The Alexa Fluor 647 anti-human antibody (+647 anti-Hum) was used to reveal the specific binding of P3 scFv-Fc (Hum P3 scFv-Fc). Before image merging, the red and green fluorescence signals were adjusted to comparable levels. The yellow color and yellow arrowheads indicate colocalization of the antigens recognized by P3 scFv-Fc and the anti-LOX1 antibody. Size bars: 250 μm.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: In Vitro, Immunofluorescence, Fluorescence, Microscopy, Binding Assay

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 7. Ex vivo imaging of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) in Apoe−/− and wild-type (WT) mice using a fluorescent ultramicroscope. Fluorescence macroscopy analysis of P3 scFv-Fc coupled to Alexa Fluor 568 dye after ex vivo injection in Apoe−/− and WT mice. A human immunoglobulin (Ig)G coupled to Alexa Fluor 568 was used as negative control antibody. P3 scFv-Fc shows specific labeling of the atheroma in the Apoe−/− mouse (yellow arrowheads). Size bars: 500 μm.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Ex Vivo, Imaging, Fluorescence, Injection, Negative Control, Labeling