Journal: Scientific Reports

Article Title: Targeting galectin-3 with a high-affinity antibody for inhibition of high-grade serous ovarian cancer and other MUC16/CA-125-expressing malignancies

doi: 10.1038/s41598-021-82686-3

Figure Lengend Snippet: Expression of LGALS3 and CA125 (MUC16) in human ovarian cancers. Three anonymized human tissue microarrays were constructed by collecting 1–3 cores from paraffin embedded, fixed blocks, as previously described . ( A ) Examples of human tumor cores stained for CA125 (panel i) and Gal3 (panel ii). Examples of core that did not stain for CA125 (panel iv) and tumor that did not stain for Gal3 (panel iii). ( B ) Each core was qualitatively scored for prevalence of CA125 and Gal3 positive cells (0 = < 5%, 1 + = 5–25%; 2 + = 25–50%; 3 + = 50–75% and 4 + = > 75%. The intensity for Gal3 was separately scored as low, medium and high (represented by green, orange and red dots respectively). All cores were independently scored by a blinded reference pathologist (KP).

Article Snippet: The last 2 immunizations used human LGALS3 protein (OriGene; Rockville, MD).

Techniques: Expressing, Construct, Staining

Journal: Scientific Reports

Article Title: Targeting galectin-3 with a high-affinity antibody for inhibition of high-grade serous ovarian cancer and other MUC16/CA-125-expressing malignancies

doi: 10.1038/s41598-021-82686-3

Figure Lengend Snippet: Knockdown sh LGALS3- MDA-MB-231 cells show decreased invasiveness both in vitro and in vivo . ( a ) LGALS3 knockdown cell line, sh LGALS3 -MDA-MB-231 and a similar LGALS1 knockdown, sh LGALS1 -MDA-MB-231 were generated. LGALS3 and LGALS1 silencing was confirmed by Western blot in a manner previously described . β-Actin normalized densitometry quantification values are shown below each Western blot band. ( b ) In a Matrigel assay using triplicate chambers with 1 × 10 4 cells/chamber, invasion of the sh LGALS3 -MDA-MB-231 and sh LGALS1 -MDA-MB-231 were compared to the parental MDA-MB-231 wild-type cell line after 48 h ( p = 0.005, unpaired t test; error bars represent standard error). Three replicates were performed. ( c ) Representative luminescence images of all mice inoculated intravenously (by tail vein) with 5 × 10 6 sh LGALS3 -MDA-MB-231 cells or wild-type MDA-MB-231 control cells as previously described . ( d ) Kaplan–Meier survival curves; median survival for mice (n = 10 female athymic nude mice) implanted with MDA-MB-231 wild-type cells was 60 days (95% CI, 53.8–66.2), while median survival for mice (n = 10 female athymic nude mice) implanted with shLGALS3-MDA-MB-231 cells was not reached ( p = 0.02, log-rank test).

Article Snippet: The last 2 immunizations used human LGALS3 protein (OriGene; Rockville, MD).

Techniques: In Vitro, In Vivo, Generated, Western Blot, Matrigel Assay

Journal: Scientific Reports

Article Title: Targeting galectin-3 with a high-affinity antibody for inhibition of high-grade serous ovarian cancer and other MUC16/CA-125-expressing malignancies

doi: 10.1038/s41598-021-82686-3

Figure Lengend Snippet: Anti-Gal3 monoclonal antibody, 14D11, inhibits MUC16-mediated tumorigenesis in vitro. ( a ) Direct ELISA for Gal3 binding to selected antibodies in the presence or absence of lactose. The presence of increasing lactose concentrations progressively inhibited the binding of Gal3 to 14D11, an isotype control antibody (18C6) and 1F5, an antibody reactive to the N terminal 15 amino acids of Gal3. Each lactose condition was compared to the zero lactose condition for the same antibody by paired t-test. ( b ) Effect of 14D11 on MUC16-mediated activation of ERK/AKT signaling in MUC16-expressing A2780 c344 and SKOV3 c344 cells after 48-h exposure to antibody. MUC16-negative A2780 and SKOV3 cells were used as controls. Densitometry was used to normalize the results against β-actin. ( c ) Effect of 14D11 on Matrigel invasion in MUC16-overexpressing SKOV3 c344 and A2780 c344 cells, and endogenous MUC16-expressing OVCAR3 cells. Anti-MUC16 N-glycosylation site antibody, 18C6, was used as a positive control. The values are the mean of triplicate well from a representative study and the invasion assays were repeated independently ≥ 3times. ( d ) Effect of 14D11 on Matrigel invasion in MDA-MB-231 and sh LGALS3 -MDA-MB-231 cells, performed as in 3C.

Article Snippet: The last 2 immunizations used human LGALS3 protein (OriGene; Rockville, MD).

Techniques: In Vitro, Direct ELISA, Binding Assay, Activation Assay, Expressing, Positive Control

Journal: The Journal of allergy and clinical immunology

Article Title: Identification of Galectin-3 as an Auto-Antigen in IgG4-Related Disease

doi: 10.1016/j.jaci.2018.05.011

Figure Lengend Snippet: Heat map demonstrating differential protein binding of PaCa2 cell lysate by mAb clones #1 and #2. Scale reflects log2-fold change divided by isotype control. Background was determined using an isotype control. Galectin-3 was identified as the antigen with the highest binding affinity for both mAb clones.

Article Snippet: Alternating rows of Nunc Medisorb 96 well plates were coated overnight at 4°C with recombinant galectin-3 (PeproTech, #450-38) at a concentration of 5 μg/mL in PBS.

Techniques: Protein Binding, Clone Assay, Binding Assay

Journal: The Journal of allergy and clinical immunology

Article Title: Identification of Galectin-3 as an Auto-Antigen in IgG4-Related Disease

doi: 10.1016/j.jaci.2018.05.011

Figure Lengend Snippet: IgG4 and IgE isotype-specific anti-galectin-3 antibodies were confirmed by ELISA with 34 and 12 of 121 IgG4-RD subjects demonstrating IgG4 and IgE antibody responses, respectively. Dashed lines represent two standard deviations above the healthy donor mean.

Article Snippet: Alternating rows of Nunc Medisorb 96 well plates were coated overnight at 4°C with recombinant galectin-3 (PeproTech, #450-38) at a concentration of 5 μg/mL in PBS.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of allergy and clinical immunology

Article Title: Identification of Galectin-3 as an Auto-Antigen in IgG4-Related Disease

doi: 10.1016/j.jaci.2018.05.011

Figure Lengend Snippet: A) Circulating Galectin-3 levels were quantified by ELISA and 15.5% of the IgG4-RD cohort were found to have elevated levels compared to age-matched healthy donors. Subjects with elevated galectin-3 levels are indicated by red color. B) Segregating the cohort by those with vs those without elevated galectin-3 levels showed a nearly 3-fold enrichment in IgG4 anti-galectin-3 autoantibody responses among the subset with elevated circulating Gal-3 levels.

Article Snippet: Alternating rows of Nunc Medisorb 96 well plates were coated overnight at 4°C with recombinant galectin-3 (PeproTech, #450-38) at a concentration of 5 μg/mL in PBS.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of allergy and clinical immunology

Article Title: Identification of Galectin-3 as an Auto-Antigen in IgG4-Related Disease

doi: 10.1016/j.jaci.2018.05.011

Figure Lengend Snippet: Three subjects with positive anti-Galectin-3 antibody responses studied longitudinally by ELISA and plotted against IgG4-RD-Responder Index (RI) on the right Y-axis. Yellow symbols indicate dates of B cell depletion therapy with rituximab, after which, anti-galectin-3 antibody titers consistently declined. The black arrow in Subject 336 indicates the initiation of low-dose prednisone that successfully maintained remission in this subject with a subsequent rise in anti-galectin-3 response.

Article Snippet: Alternating rows of Nunc Medisorb 96 well plates were coated overnight at 4°C with recombinant galectin-3 (PeproTech, #450-38) at a concentration of 5 μg/mL in PBS.

Techniques: Enzyme-linked Immunosorbent Assay