Journal: Pharmaceutics

Article Title: Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies

doi: 10.3390/pharmaceutics14071351

Figure Lengend Snippet: Human IgG loading and in vitro release were performed with the ‘injection method’. ( a ) A total of 50 µg IgG in a volume of 25 µL PBS was injected into the center of a BC fleece. To visualize the injection depot, the IgG solution was supplied with Trypan blue. The loaded fleeces were then put in blocking buffer (1% BSA/0.05% Tween-20 in PBS) and IgG release was followed. At several time-points, the fleeces were taken out, and the Trypan blue injection spots were photographed. Graph ( b ) shows the cumulative IgG release in %, with the inset of the first 24 h shown in ( c ). Release data are shown as cumulative release, which are displayed as mean ± SD for a triplicate measurement.

Article Snippet: BC fleeces were loaded with human IgG antibody (50 µg) or anti-CTLA-4 (Syrian hamster IgG, clone 9H10; Bioxcell, Lebanon, NH, USA; 100 µg) according to .

Techniques: In Vitro, Injection, Blocking Assay

Journal: Pharmaceutics

Article Title: Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies

doi: 10.3390/pharmaceutics14071351

Figure Lengend Snippet: Cytotoxicity evaluation of BC extracts in MC38 tumor cells with 7-AAD staining and MTS. BC extracts were prepared by dissolving 1 g of BC in 50 mL culture medium, which yielded an extraction ratio of 20 mg/mL. Empty extracts or extracts supplied with human IgG or anti-CTLA-4 (both starting at a concentration of 50 µg/mL) were tested on MC38 cells. DMSO was used as positive cell killing control. The primary measure was cell viability, which was assessed by 7-AAD staining (live-dead cells exclusion marker; figures a – c ). Higher 7-AAD MFI signal is a hallmark of dying cells with leaky cell membranes. Cytotoxicity was also assessed using a cell metabolism assay (MTS), in which higher cell metabolism is indicative of higher cell viability ( d – f ). Cell metabolism was measured 48 h after incubation. All data are shown as mean ± SD for triplicates. Only the DMSO treatment significantly decreased cell viability, which was assessed with a Student’s t test, with **** denoting p < 0.0001.

Article Snippet: BC fleeces were loaded with human IgG antibody (50 µg) or anti-CTLA-4 (Syrian hamster IgG, clone 9H10; Bioxcell, Lebanon, NH, USA; 100 µg) according to .

Techniques: Staining, Concentration Assay, Marker, Incubation

Journal: Pharmaceutics

Article Title: Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies

doi: 10.3390/pharmaceutics14071351

Figure Lengend Snippet: BC reduces serum antibody levels in vivo. Photographs in ( a ) depict in chronological order the procedures of the BC implantation, starting with loading, placing the BC sample underneath the skin, wound closure, visual inspection of the skin and removing the BC implant at D21 (after the mice were killed). Body weight measurements are depicted in ( b ), for the BC-treated mice, the body weight differences at various time-points were compared with the initial body weight at D0. Body weight differences were assessed with a paired Student’s t -test, with NS denoting no statistical differences compared to the body weights at D0. In ( c ) and ( d ), the serum IgG and anti-CTLA-4 levels are shown for several time-points after treatment, respectively. Statistical differences between the BC and PBS group were assessed with an unpaired Student’s t -test and are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: BC fleeces were loaded with human IgG antibody (50 µg) or anti-CTLA-4 (Syrian hamster IgG, clone 9H10; Bioxcell, Lebanon, NH, USA; 100 µg) according to .

Techniques: In Vivo

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: ICAM-1 IgG autoantibody and sICAM-1 levels across healthy individuals and those with severe COVID-19, high BMI, and autoimmune conditions. (A) Dot plot graph of AUC on the log axis of the optical density at 450 nm measurement by ELISA for determining the levels of IgG antibodies targeting ICAM-1 within plasma from healthy individuals (n = 40), individuals with severe COVID-19 (n = 40), individuals with a high BMI over 30 kg/m 2 (n = 50), and individuals with select autoimmune conditions, SLE or RA (n = 39). (B) Dot plot graph of concentration (pg/mL) on the log axis measured by ELISA for determining the levels of sICAM-1 within plasma from healthy individuals (n = 40), individuals with severe COVID-19 (n = 40), individuals with a high BMI over 30, (n = 50), and individuals with autoimmune conditions SLE or RA (n = 39). Statistical significance was determined using the Mann-Whitney test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Concentration Assay, MANN-WHITNEY

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Correlation of anti-ICAM-1 IgG autoantibody levels with age. (A) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis of IgG antibodies targeting ICAM-1 within plasma compared with age (x-axis) from healthy pediatric individuals ranging in age from 6 mo to 15 yr old (n = 37). (B) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis of IgG antibodies targeting ICAM-1 within plasma compared with age (x-axis) from healthy adult individuals ranging in age from 22 to 75 yr old (n = 40). Statistical significance was determined using the Pearson correlation test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Clinical Proteomics

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: High-resolution IgG epitope mapping of ICAM-1 autoantibodies using ICAM-1 peptide microarrays. (A) Heatmap of z scores for ICAM-1 epitope binding for individual samples (n = 20). ICAM-1 domains 1 through 5 are annotated above the heatmap. (B) Line graphs of median group z scores of ICAM-1 autoantibody binding to individual ICAM-1 peptides in the peptide array. Epitopes of recurrent high binding were qualified as median z scores of ≥0.95 to represent 1 SD ±0.05 above the median, points in red. (C) Table of recurrent high binding peptides, with associated z score and sequence. (D) Table of high binding clusters with associated peptide IDs, z score range, and sequence. Overlapping sequence motifs are bolded, and the common leucine threonine epitope is underlined.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Binding Assay, Peptide Microarray, Sequencing

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Immunoglobulin isotype and subclass determination of anti-ICAM-1 antibodies. (A) Dot plot graph of concentration (µg/mL) on the log y-axis obtained by ELISA for determining the levels of immunoglobulin isotype class within high-autoantibody-presenting plasma, defined as the top 16 positive samples by qualitative ELISA regardless of disease group (n = 16). (B) Dot plot graph of concentration (µg/mL) obtained by ELISA for determining the levels of IgG subclass within high-autoantibody-presenting plasma, defined as the top 16 positive samples by qualitative ELISA regardless of disease group (n = 16). Statistical significance was determined using the Mann-Whitney test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, MANN-WHITNEY

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Anti-ICAM-1 IgG autoantibodies interact with protein glycans. (A) SDS-PAGE separation of ICAM-1 under nonreducing conditions. Lane 1 shows ICAM-1 protein, lane 2 shows ICAM-1 in reaction buffer without PNGase F, and lane 3 shows ICAM-1 with PNGase F. Molecular masses are displayed on the left margin in kDa. (B) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis obtained by ELISA for determining the levels of ICAM-1 IgG autoantibodies targeting ICAM-1 with glycans (control) compared with enzymatically deglycosylated ICAM-1 (PNGASE) within plasma with high levels of detected ICAM-1 autoantibodies, defined as the top 8 positive samples by qualitative ELISA regardless of disease group (n = 8). Statistical significance was determined using the Wilcoxon matched pairs signed rank test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: SDS Page, Enzyme-linked Immunosorbent Assay, Control, Clinical Proteomics

Journal: Oncoimmunology

Article Title: T cell-mediated elimination of cancer cells by blocking CEACAM6–CEACAM1 interaction

doi: 10.1080/2162402X.2021.2008110

Figure Lengend Snippet: Antibody-binding profile of BAY 1834942 as determined by ELISA and flow cytometry. A . Binding of BAY 1834942 to recombinant human and cynomolgus CEACAM6 that were coated on a CM5 sensor chip and analyzed by SPR. B . Selective binding of BAY 1834942 to recombinant human CEACAM6 and cynomolgus monkey CEACAM6 with absence of reactivity to human CEACAM1, −3, or −5 as determined by binding ELISA. C-D . Binding of BAY 1834942 and of a control antibody 9A6 (as huIgG2) to HeLa cells transfected with (b) human and (c) cynomolgus monkey CEACAM6 as determined by flow cytometry. No binding of BAY 1834942 was observed on HeLa wild-type cells. E . The half-maximal binding (EC 50 ) values for BAY 1834942 and the positive control antibody 9A6 as determined by binding to CEACAM6-positive cancer cells by flow cytometry. F . Dose-dependent binding of recombinant CEACAM6-Fc to recombinant human CEACAM1 (coated to plate, 1 µg/mL) by ELISA. Human IgG antibody as Fc-control is shown on the right. G . BAY 1834942 and 9A6, added as precomplex of recombinant CEACAM6-Fc (2 µg/mL) and antibody in concentration series from 0.01–100 nM at 25°C for 1 h, interrupted the interaction between CEACAM1 (coated to plate, 1 µg/mL) and recombinant CEACAM6-Fc as determined in a competition ELISA assay. For detection of bound CEACAM6-Fc in the presence of BAY 1834942, an anti-human IgG horseradish peroxidase (HRP) conjugate was used with Amplex Red (Life Technologies) as substrate. RFU, relative fluorescence unit.

Article Snippet: The anti-CEACAM5/6 antibody h16 C3 (as described in US20130189268, human IgG1, , ), the anti-human CEACAM1 antibody (as described in WO2013054320, human IgG1 (TPP-3006) and human IgG2 (TPP-9145)) and isotype control antibodies TPP-1238 (huIgG2), TPP-1240 (huIgG4-S228P), and TPP-754 (huIgG1) were generated at Bayer AG.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Recombinant, Transfection, Positive Control, Concentration Assay, Fluorescence

Journal: Oncoimmunology

Article Title: T cell-mediated elimination of cancer cells by blocking CEACAM6–CEACAM1 interaction

doi: 10.1080/2162402X.2021.2008110

Figure Lengend Snippet: Immunosuppression and efficacy of BAY 1834942 correlates with receptor density and is dose dependent. A-D . BAY 1834942-induced (30 µg/mL) increase in IFN-γ cytokine release in co-culture assays of 10,000 (a) SNU-C1 colon, (b) NCI-H1993 lung, (c) KS22.24 breast, or (d) CEACAM6-transfected HCT-116 colon cancer cells and 20,000 survivin T cells. Representative examples are shown in the figure. Full data set shown in . IFN-γ levels were measured by ELISA and fold change in the induction of IFN-γ levels over background is indicated with an arrow. The data were normalized to the mean of the isotype control-treated group. E . CEACAM6 expression in SNU-C1, NCI-H1993, KS22.24, and CEACAM6-transfected HCT-116 cells as analyzed by flow cytometry and presented as mean fluorescence intensity (MFI) after binding of CEACAM6 mAb (murine IgG2a variant of BAY 1834942 for use with the Qifi kit (Dako)). Full data set shown in . F.-H . Examples of dose-dependent cytokine secretion in co-culture of 10,000 (f) KS breast cancer, (g) HCC2935 lung cancer, and (h) CEACAM6-transfected HCT116 colon cancer cells with 20,000 survivin T cells. BAY 1834942 and isotype control antibody were used at concentrations ranging from 0.6 ng/mL to 30 µg/mL. IFN-γ levels were measured with ELISA. I.-J . Effect of BAY 1834942 on the killing of HCC2935 lung cancer cells by TILs isolated from pancreatic cancer patients. EpCAM/CD3 bispecific antibody construct (0.25 ng/mL) was used as a T cell-engaging molecule and tumor cell killing was measured by loss of impedance with an Xcelligence instrument. (i) Co-culture of 10,000 HCC2935 tumor cells and 50,000 TILs (TIL-12) with BAY 1834942, 9A6 anti-CEACAM6 mAb (huIgG2 variant), anti-PD-L1 antibody (huIgG2 variant) and isotype control used at 30 µg/mL. (j). Same co-culture as in (i) with BAY 1834942 used at doses 0.02–50 µg/mL and isotype control antibody (50 µg/mL) as a control. Statistical analysis in (a-d) was performed in comparison to the isotype control group using a linear model and corrected for family-wise error rate using Sidak’s method. ***, p < .001.

Article Snippet: The anti-CEACAM5/6 antibody h16 C3 (as described in US20130189268, human IgG1, , ), the anti-human CEACAM1 antibody (as described in WO2013054320, human IgG1 (TPP-3006) and human IgG2 (TPP-9145)) and isotype control antibodies TPP-1238 (huIgG2), TPP-1240 (huIgG4-S228P), and TPP-754 (huIgG1) were generated at Bayer AG.

Techniques: Co-Culture Assay, Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Fluorescence, Binding Assay, Variant Assay, Isolation, Construct