Journal: Vaccines

Article Title: Maternal Immunization with Adjuvanted Recombinant Receptor-Binding Domain Protein Provides Immune Protection against SARS-CoV-2 in Infant Monkeys

doi: 10.3390/vaccines12080929

Figure Lengend Snippet: Primary and secondary antibody responses of 5 adult female rhesus monkeys after immunization with adjuvanted recombinant spike protein-fusion protein (RBD-Fc) prior to conception. Mean (S.E.) antibody titers are shown in relative intensity units (RIUs) for the 1:10,000 serum dilution. Monkeys were screened at baseline to verify they had not been exposed previously to SARS-CoV-2. IgG levels increased significantly over time, crossing the threshold for seropositivity.

Article Snippet: To serve as Positive Controls, our assay protocol included anti-SARS-CoV-2 spike RBD neutralizing antibody (Human IgG1, ACRO Biosystems, SAD-S35, Lot # S35-211VF1-VT run at 1:100 and 1:1000, 1.0 µg/mL and 0.1 µg/mL, respectively), and a pooled human SARS-CoV-2 national IgG positive standard (Frederick National Laboratory Lot # COVID-NS0109, run at 1:10, 1:100 and 1:1000).

Techniques: Recombinant

Journal: Vaccines

Article Title: Maternal Immunization with Adjuvanted Recombinant Receptor-Binding Domain Protein Provides Immune Protection against SARS-CoV-2 in Infant Monkeys

doi: 10.3390/vaccines12080929

Figure Lengend Snippet: Mean (S.E.) spike protein-specific IgG levels in 6 mother and infant rhesus monkey pairs after delivery are shown in ( A ) (dark and light bars, respectively). Antibody titers were quantified at 3 serum dilutions (1:1000, 1:10,000, 1:100,000), and values are expressed in relative intensity units (RIUs). Maternal and neonatal antibody levels were similar at each dilution, resulting in a mean placental transfer rate of 100.3%. The ANOVA indicated only a significant effect of serum dilution ( F {2,15} = 133.1, p < 0.001). Individual data for all infants and the correlation with their mothers’ IgG titer at each dilution are shown in ( B ). Maternal and infant IgG levels were similar at each dilution, positively correlated at each dilution ( r = 0.77, p < 0.08; r = 0.74, p < 0.09, and r = 0.94, p < 0.01, respectively), and significantly correlated across the entire series ( r {18} = 0.98, p < 0.003). Regression lines are plotted to show the mother–infant association at each dilution. Placental transfer rate was calculated to be 100.3% and was markedly higher than the transfer rate of IgG transfer for tetanus IgG1, which was only 78%.

Article Snippet: To serve as Positive Controls, our assay protocol included anti-SARS-CoV-2 spike RBD neutralizing antibody (Human IgG1, ACRO Biosystems, SAD-S35, Lot # S35-211VF1-VT run at 1:100 and 1:1000, 1.0 µg/mL and 0.1 µg/mL, respectively), and a pooled human SARS-CoV-2 national IgG positive standard (Frederick National Laboratory Lot # COVID-NS0109, run at 1:10, 1:100 and 1:1000).

Techniques:

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Median expression of PDL1 gene in different organs in normal and cancer tissue obtained from the TCGA dataset using Gene Expression Profiling Interactive Analysis (GEPIA) . The bar plot shows the lower PDL1 expression in LUAD and LUSC compared with normal tissue. ( B ) Anti-PDL1 (red) and DAPI (blue) staining of lung tissue micro-array (TMA) from patients with NSCLC. Each specimen of cancer and adjacent normal tissue was collected from matched patients with NSCLC. The image shows high expression of PDL1 in normal tissue compared with cancer tissue. However, the density of PDL1-high cells is equal in cancer and normal tissue because of tight cellular packing in the cancer tissue (data in the Supplementary Materials). ( C ) Median expression of CD47 gene in different organs in normal and cancer tissue according to the TCGA dataset obtained using GEPIA. The bar plot shows lower CD47 expression in LUAD and LUSC compared with normal tissue.

Article Snippet: Fifty nanograms of human CD47 protein (Acro BioSystems, catalog no. CD7-H5256, lot no. 646-91TF1-PW) or 100 ng of mouse CD47 protein (Biosystems Acro, catalog no. CD7-M5251) was added in each well of the ELISA plate (Nunc MaxiSorp flat-bottom plate) with 50 μl of PBS and incubated at 4°C for 16 hours.

Techniques: Expressing, Gene Expression, Staining, Microarray

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Immunostaining of lung TMA by anti-CD47 (green), anti-PDL1 (red) antibody, and DAPI (blue). The image shows most of the tumors expressing high CD47 and a limited number of tumors having higher PDL1 expression. Each tissue sample was collected from a different patient having either LUAD or LUSC. The magnified image and the analysis are described in the Supplementary Materials. ( B ) Percentage of cancer cells having PDL1-high and CD47-high in patients with LUAD. Each line represents individual tissue corresponding to each tumor specimen. The data show a poor expression of PDL1 and higher CD47 in all patients with LUAD. An example of the quantification can be found in Supplementary Materials. ( C ) Percentage of cancer cells having PDL1-high and CD47-high in patients with LUSC. The data show the expression of either PDL1 or CD47 in all patients with LUSC. No patient with high CD47 and high PDL1 has been observed. ( D ) Percentage of patients expressing either PDL1-high or CD47-high in more than 5% of cancer cells compared across different stages of LUAD and LUSC. High CD47 expression has been observed in all stages of patients with adenocarcinoma, whereas an almost equal distribution of CD47-high and PDL1-high cell populations has been observed in squamous cell carcinoma.

Article Snippet: Fifty nanograms of human CD47 protein (Acro BioSystems, catalog no. CD7-H5256, lot no. 646-91TF1-PW) or 100 ng of mouse CD47 protein (Biosystems Acro, catalog no. CD7-M5251) was added in each well of the ELISA plate (Nunc MaxiSorp flat-bottom plate) with 50 μl of PBS and incubated at 4°C for 16 hours.

Techniques: Immunostaining, Expressing

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Schematic representation of formulation of ADNs. A single or mixture of antibodies can be used for monospecific or bispecific ADNs. ( B ) The bar graph shows the diameter of the nanoparticles in each stage of the synthesis. Data shown as means ± SEM ( n = 3). ( C ) Change in surface charge in terms of zeta potential of the anti–CD47-PDL1-ADN during synthesis. Data presented as means ± SEM ( n = 3, one-way ANOVA followed by Tukey’s multiple comparison test). ( D ) The stability of the nanoparticle in PBS (pH = 7.2) for 45 days showed no notable change in size and zeta potential. ( E ) Schematic representation of ELISA for detecting the presence of anti-PDL1 and anti-CD47 antibodies on ADNs. ( F ) The presence of the antibody and antigen binding by nanoparticles was measured by ELISA. The data show a concentration-dependent increase in optical density, signifying the presence of both CD47 and PDL1 antibodies in the nanoparticle surface. ( G ) Representative confocal microscopy image showing the internalization of anti–CD47(FITC)-PDL1(APC)-ADN in A549 cell (scale bar, 10 μm). The image shows colocalization of the green and red fluorescence, signifying the presence of both CD47(FITC) and PDL1(APC) antibodies on the nanoparticle. ( H ) Binding of the anti–CD47-PDL1-ADN to CD47 (blue) and PDL1 (black) antigen under competitive assay conditions. ( I ) Dose-dependent association and dissociation between anti–CD47-PDL1-ADN and CD47+PDL1 antigen measured by BLI. The association was monitored for 600 s, followed by dissociation for 600 s. ( J ) Comparison between the bindings of anti–CD47-PDL1-ADNs with dual antigen CD47+PDL1 (1:1) versus single antigen, either CD47 or PDL1.

Article Snippet: Fifty nanograms of human CD47 protein (Acro BioSystems, catalog no. CD7-H5256, lot no. 646-91TF1-PW) or 100 ng of mouse CD47 protein (Biosystems Acro, catalog no. CD7-M5251) was added in each well of the ELISA plate (Nunc MaxiSorp flat-bottom plate) with 50 μl of PBS and incubated at 4°C for 16 hours.

Techniques: Formulation, Zeta Potential Analyzer, Comparison, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Confocal Microscopy, Fluorescence

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Representative fluorescence microscopy image showing the internalization of the anti–CD47-PDL1-ADN (green fluorescence) in A459 cells. The image shows negligible internalization of the DNs with antibody-targeting (scale bars, 10 μm). ( B ) The bar plots show the MFI of fluorescent ADNs in A549 cells. The internalization of anti–CD47-ADN is significantly higher than that of anti–PDL1-ADN or IgG-ADN. Data show means ± SEM ( n = 3, two-way ANOVA followed by Tukey’s multiple comparison test). ( C ) Comparison of time-dependent cellular internalization of monospecific and bispecific ADNs evaluated by flow cytometry. The anti–CD47-ADN have shown higher cellular internalization than anti–PDL1-ADN because of higher cell surface CD47 antigen than PDL1 on A549 cells. Data show means ± SEM ( n = 3, two-way ANOVA followed by Tukey’s multiple comparison tests). ( D ) Cytotoxicity of the untargeted (DNs) and monospecific and bispecific ADNs in LLC cells. ( E ) The bar plot represents the IC 50 values from each ADN in the case of A549, LLC, and KLN-205 cells. The variation of the IC 50 values was observed based on the expression of CD47 and PDL1 on the cancer cells. ( F ) Representative confocal microscopy image of phagocytosis of LLC cells by mouse macrophage (RAW 264.7) in the presence of anti–CD47-PDL1-ADN (scale bar, 10 μm). ( G ) Comparison of the phagocytosis events between cancer cells and macrophages in the presence and absence of anti–CD47-PDL1-ADN. Data show means ± SEM (unpaired t test, two-tailed). ( H ) Schematic for experimental validation of immune checkpoint blocking by anti–CD47-PDL1-ADN in lung cancer cells. ( I ) The data illustrate the dose-dependent blocking of cell surface CD47 antigen in lung cancer cells, resulting in the unavailability of the free antigen for anti-CD47(FITC) mAb. ( J ) Comparison of CD47 antigen blocking by free drug, DNs, and anti–CD47-PDL1-ADN.

Article Snippet: Fifty nanograms of human CD47 protein (Acro BioSystems, catalog no. CD7-H5256, lot no. 646-91TF1-PW) or 100 ng of mouse CD47 protein (Biosystems Acro, catalog no. CD7-M5251) was added in each well of the ELISA plate (Nunc MaxiSorp flat-bottom plate) with 50 μl of PBS and incubated at 4°C for 16 hours.

Techniques: Fluorescence, Microscopy, Comparison, Flow Cytometry, Expressing, Confocal Microscopy, Two Tailed Test, Biomarker Discovery, Blocking Assay

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Schematic representation of the experimental design for the in vivo antitumor efficacy study of ADNs in a syngeneic LLC tumor model. ( B ) Tumor growth curves show the change in tumor volume of each treatment group. The representative images of the tumor are shown. The tumor broke into small pieces because of its friable nature. Data show means ± SEM ( n = 5, two-way ANOVA following Tukey’s multiple comparison test). ( C ) Kaplan-Meier plots show survival response. ( D ) Graph shows normalized RBC levels in each group of mice. Data show means ± SEM ( n = 3, two-way ANOVA following Tukey’s multiple comparison test). ( E ) Representative scatter plot showing intertumoral T cells in KLN-205 tumor. ( F ) The bar graph shows a significant increase in intratumoral cytotoxic T cells in mice treated with anti–CD47-PDL1-ADN. The data show means ± SEM ( n = 3, unpaired t test, two-tailed). ( G ) Immunostaining images of a tumor tissue section from control and anti–CD47-PDL1-ADN-treated groups. The color code of each marker is mentioned on the right side of the images. The images were captured using a TissueFAXS slide scanner.

Article Snippet: Fifty nanograms of human CD47 protein (Acro BioSystems, catalog no. CD7-H5256, lot no. 646-91TF1-PW) or 100 ng of mouse CD47 protein (Biosystems Acro, catalog no. CD7-M5251) was added in each well of the ELISA plate (Nunc MaxiSorp flat-bottom plate) with 50 μl of PBS and incubated at 4°C for 16 hours.

Techniques: In Vivo, Comparison, Two Tailed Test, Immunostaining, Control, Marker