Journal: AAPS Open

Article Title: A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)

doi: 10.1186/s41120-021-00039-w

Figure Lengend Snippet: Fig. 1 NAb detection assay. A Complexes form between the fixed amount of added drug and NAb; the formation of these complexes allows SULFO-TAG–labeled PD-L1 to bind to biotinylated PD-1, which can bind to the streptavidin-ECL plate. B In the absence of NAb, the fixed amount of drug added to the solution binds to biotinylated PD-1 and prevents its interaction with PD-L1. SULFO-TAG–labeled PD-L1 bound to PD-1 is necessary to generate the ECL signal. ECL, electrochemiluminescence; NAb, neutralizing antibody; PD-1, programmed death 1; PD-L1, programmed death ligand 1

Article Snippet: Critical reagents for this assay were as follows: a mouse anti-dostarlimab mAb (Precision Antibodies, Columbia, MD) generated from clone #6G10 at a concentration of 1.15 mg/mL; dostarlimab (WuXi, Philadelphia, PA) at a concentration of 20.7 mg/mL; biotinylated human PD-1 (ACRO Biosystems, Newark, DE) at a concentration of 200 μg/mL; and SULFO-TAG–labeled human PD-L1 (CR0; Meso Scale Diagnostics [MSD], Rockville, MD) at a concentration of 2106 μg/mL.

Techniques: Detection Assay, Labeling, Electrochemiluminescence

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic illustration of US-triggered deep-tissue activation of SPINs to release immunomodulators. b Schematic illustration of sonodynamic activation of SPINs to debulk tumor, enhance tumor immunogenicity, and release immunomodulators in situ as well as synergetic action of IDO inhibition and PD-L1 blocking on enhancing antitumor immunity with alleviated irAEs relative to free-drug administration.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Activation Assay, In Situ, Inhibition, Blocking Assay

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Chemical structures of amphiphilic semiconducting polymeric modulators and schematic illustration of their self-assembly and surface modification to form SPINs. b The molar ratios of each component in different SPINs. c Zeta potentials and hydrodynamic sizes of different SPINs in 1× PBS buffer (pH = 7.4) ( n = 4). d Photographs of erythrocytes after incubation with 1× PBS buffer (negative control), 1% Triton X-100 (positive control), and 1× PBS buffer containing SPINs at the concentration of 100 µg/mL for 2 h, followed by centrifugation. e Hemolysis percentages of erythrocytes after incubation with SPINs at different concentrations for 2 h ( n = 4). f Schematic illustration of US irradiation of SPIN D2 solutions covered with a pork tissue. g ESR spectra of 1 O 2 for SPIN D2 (20 µg/mL) after US irradiation (1.2 W/cm 2 , 3 min) without or with coverage of pork tissues at different thicknesses. h Release profiles of aPD-L1 and NLG919 from SPIN D2 (40 µg/mL) after US irradiation for different time ( n = 4). i PD-L1/PD-1 binding activity assay after treatment with free aPD-L1 or SPIN D2 (40 µg/mL) with or without US irradiation ( n = 4). SPIN D2 – US versus SPIN D2 + US: P < 0.0001. Statistical significance was calculated via a two-tailed Student’s t test. *** P < 0.001. In ( g – i ), the power intensity of US irradiation was 1.2 W/cm 2 (1.0 MHz, 50% duty cycle). Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Modification, Incubation, Negative Control, Positive Control, Concentration Assay, Centrifugation, Irradiation, Binding Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schedule for the establishment of primary and distant tumors, triple systemic injection of SPINs (0.2 mL, 0.6 mg/mL) via tail vein, US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min), and analysis of immune responses. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 6) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). SPIN D2 + US versus drug + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus drug: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) receiving different treatments as indicated. e Schematic illustration of treatment of rechallenged tumor mouse models using SPINs. f Growths of rechallenged tumors in Panc02 tumor-bearing mice after injection of saline or SPIN D2 (0.2 mL, 0.6 mg/mL) with US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min) ( n = 5). Saline versus SPIN D2 + US: P < 0.0001. g The survival curves of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 10). h Flow cytometry analysis of populations of effector memory T cells in the spleen of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 4). Saline versus SPIN D2 + US: P = 0.0059. i Differentially expressed gene numbers in tumor tissues of mice after different treatments. j Relative expression of Carl , Hmgb1-ps1 , Hmgb1-ps2 , Cd80 , Cd86 , Cd40 , Pdcd1 , Cd3e , Cd8a , Ifng , Gzmb , Cxcl1 , Cxcl2 , Cxcl9 , Cxcl10 , Cxcl11 , Ccl4 , Ccl5 , Il1b , Il2 , Il6 , Il7 , Il15 , Ido1 , and Cd274 in tumors of Panc02 tumor-bearing mice after different treatments (the experiment was repeated independently five times with similar results). k Unsupervised hierarchical clustering of relative gene expression in tumors of Panc02 tumor-bearing C57BL/6 mice after different treatments ( n = 5). Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic of sono-immunotherapy of subcutaneous pancreatic mouse tumors covered with 5-cm tissue. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 5) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), SPIN 0 or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). The primary tumors were covered with 5-cm tissue under US irradiation. SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) after different treatments for 60 days. e Schematic of US-mediated deep-tissue sonodynamic therapy of orthotopic pancreatic rabbit tumors. f Radiolabeling stability of 131 I-SPIN 0 after storage in saline or 50% serum at 37 °C for different time ( n = 3). g , h SPECT imaging ( g ) and signal intensity ( h ) of orthotopic pancreatic rabbit tumors after systemic injection of 131 I-SPIN 0 (1.0 mL, 1.5 mg/mL) for different time ( n = 4). The white dotted circle indicated tumors. i Computed tomography (CT) imaging of orthotopic pancreatic rabbit tumors after systemic injection of saline or SPIN 0 (1.0 mL, 1.5 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 30 min). The white dotted circle indicated tumors. j Tumor volume of orthotopic pancreatic rabbit tumors ( n = 3) after treatments as indicated for different days. Saline + US versus SPIN 0 + US: P = 0.0108. k H&E staining images of orthotopic pancreatic rabbit tumors after different treatments. The experiment was repeated independently three times with similar results. l Survival curves of orthotopic pancreatic tumor-bearing rabbits ( n = 4) after different treatments for 20 days. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; * P < 0.05, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Radioactivity, Single Photon Emission Computed Tomography, Imaging, Computed Tomography, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a , b Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( a ), and CD3 + CD8 + CTLs ( b ) in blood of mice ( n = 4) at 30 day after systemic administrations of saline, SPIN 0 , SPIN D2 (0.2 mL, 1.2 mg/mL) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). Saline – US versus drug − US: P = 0.0023; saline − US versus drug + US: P = 0.0006; drug + US versus SPIN D2 + US: P = 0.0071 for CD3 + CD4 + Th cells ( a ); saline − US versus drug − US: P = 0.0004; saline − US versus drug + US: P = 0.0001; drug + US versus SPIN D2 + US: P = 0.0093 for CD3 + CD8 + CTLs ( b ). c , d Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( c ), and CD3 + CD8 + CTLs ( d ) in spleen of mice ( n = 4) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0008; saline − US versus drug + US: P = 0.0005; drug + US versus SPIN D2 + US: P = 0.0015 for CD3 + CD4 + Th cells ( c ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P = 0.0002; drug + US versus SPIN D2 + US: P = 0.0049 for CD3 + CD8 + CTLs ( d ). e Representative H&E staining images of liver after 30 days of treatments in different groups (white arrows indicate the infiltrated lymphocytes). The experiments were repeated independently three times with similar results. f Heatmap to show relative fold of cytokine levels in serum of mice after different treatments for 30 days relative to those in saline control group. g , h Serum levels of ALT ( g ) and AST ( h ) in mice ( n = 5) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0010; saline − US versus drug + US: P = 0.0020; drug + US versus SPIN D2 + US: P = 0.0054 for ALT ( g ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P < 0.0001; drug + US versus SPIN D2 + US: P = 0.0013 for AST ( h ). i Summary comparison of the antitumor immunity and irAEs between SPIN D2 -mediated sono-immunotherapy and free-drug treatment. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Flow Cytometry, Irradiation, Staining, Two Tailed Test

Journal: mAbs

Article Title: Development and pharmacokinetic assessment of a fully canine anti-PD-1 monoclonal antibody for comparative translational research in dogs with spontaneous tumors

doi: 10.1080/19420862.2023.2287250

Figure Lengend Snippet: Enrichment of canine scFv phage display library after panning against cPD-1 antigen. Recombinant cPD-1 was adsorbed to a microtiter plate overnight at 4°C. Wells were washed and blocked with 2% milk in PBS (MPBS) for 1 hour at 37°C. Initial unpanned library (P0) and libraries of phage obtained after each round of selection (P1 through P4) were added to coated plates and incubated for 1 hour at 37°C. Plates were washed with PBS supplemented with 0.1% Tween (PBST) and bound phage was detected using a 1:5000 dilution of HRP-conjugated anti-M13 mAb in MPBS. Bound phage were detected with ABTS. OD was read at 405 nm after 30 min using a Molecular Devices SpectraMax 340 spectrophotometer.

Article Snippet: Briefly, the non-suppressor TOP10F’ Chemically Competent E. coli strain (ThermoFisher Scientific/Invitrogen, C303003) was infected with phage clones that were confirmed to bind to streptavidin immobilized and biotinylated avi-tagged cPD-1 soluble protein (Sino Biological 70,109-D27H-B) by phage ELISA.

Techniques: Recombinant, Selection, Incubation, Spectrophotometry

Journal: mAbs

Article Title: Development and pharmacokinetic assessment of a fully canine anti-PD-1 monoclonal antibody for comparative translational research in dogs with spontaneous tumors

doi: 10.1080/19420862.2023.2287250

Figure Lengend Snippet: Binding of randomly selected scFv-expressing monoclonal phage clones from panning rounds 3 and 4 to cPD-1. ELISA plates were coated with recombinant cPD-1 and human CD19 (left) or canine IL-13 Rα2 (right) as controls. Polyclonal phage from the fourth round of panning against cPD-1 served as positive control phage on both plates. Polyclonal phage from the third round of panning against human CD19 (left graph) and fourth round of panning against canine IL-13Ra2 (right graph) (both sets of phage from unrelated projects) served as positive phage controls. Each monoclonal phage was screened once for binding to cPD-1.

Article Snippet: Briefly, the non-suppressor TOP10F’ Chemically Competent E. coli strain (ThermoFisher Scientific/Invitrogen, C303003) was infected with phage clones that were confirmed to bind to streptavidin immobilized and biotinylated avi-tagged cPD-1 soluble protein (Sino Biological 70,109-D27H-B) by phage ELISA.

Techniques: Binding Assay, Expressing, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control

Journal: mAbs

Article Title: Development and pharmacokinetic assessment of a fully canine anti-PD-1 monoclonal antibody for comparative translational research in dogs with spontaneous tumors

doi: 10.1080/19420862.2023.2287250

Figure Lengend Snippet: The ability of unique soluble scFv clones to bind to cPD-1 by ELISA. 19 unique soluble scF-vs were tested for binding to cPD-1 by ELISA. Increasing amounts of biotinylated cPD-1 were added to streptavidin coated ELISA plate wells and incubated for 1 hour. 0.25 μg/ml soluble HA-tagged scFvs were added to the wells and bound scFvs were detected using an AP-conjugated anti-HA antibody. An irrelevant soluble HA-tagged scFv against MERS (Middle Eastern Respiratory Syndrome) virus from an unrelated project was used as a negative control. A. unique clones identified in the initial survey. B. additional unique clones identified via high throughput screen. Evaluation of each unique soluble clone against increasing amounts of biotinylated cPD-1 was performed once.

Article Snippet: Briefly, the non-suppressor TOP10F’ Chemically Competent E. coli strain (ThermoFisher Scientific/Invitrogen, C303003) was infected with phage clones that were confirmed to bind to streptavidin immobilized and biotinylated avi-tagged cPD-1 soluble protein (Sino Biological 70,109-D27H-B) by phage ELISA.

Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Virus, Negative Control, High Throughput Screening Assay

Journal: mAbs

Article Title: Development and pharmacokinetic assessment of a fully canine anti-PD-1 monoclonal antibody for comparative translational research in dogs with spontaneous tumors

doi: 10.1080/19420862.2023.2287250

Figure Lengend Snippet: PD-1:PD-L1 inhibition assay. A. Schematic of experimental setup. Soluble biotinylated cPD-1 was incubated with increasing concentrations of cPD-L1-Fc either alone or in the presence of a selected soluble anti-cPD-1 scFv or an irrelevant soluble scFv against Middle Eastern Respiratory Syndrome (MERS) virus. Incubation mixtures were added to ELISA plates pre-coated with streptavidin to capture cPD-1:cPD-L1-Fc complexes. Bound complexes were detected using an anti-Fc-AP conjugate and AP colorimetric substrate. B. soluble anti-cPD-1 scFvs show varying degrees of inhibition of cPD-1 binding to cPD-L1 as revealed by a loss of detectable signal.

Article Snippet: Briefly, the non-suppressor TOP10F’ Chemically Competent E. coli strain (ThermoFisher Scientific/Invitrogen, C303003) was infected with phage clones that were confirmed to bind to streptavidin immobilized and biotinylated avi-tagged cPD-1 soluble protein (Sino Biological 70,109-D27H-B) by phage ELISA.

Techniques: Inhibition, Incubation, Virus, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: mAbs

Article Title: Development and pharmacokinetic assessment of a fully canine anti-PD-1 monoclonal antibody for comparative translational research in dogs with spontaneous tumors

doi: 10.1080/19420862.2023.2287250

Figure Lengend Snippet: Soluble canine anti-cPD-1 scFv binding to cell surface expressed cPD-1. The human erythroleukemic cell line K562 was gene edited to eliminate the FcγRII (CD32) (KTδ32). Retroviral transduction of edited cells to express cPD-1 was performed, and cPD-1 positive target cells were selected in puromycin. Soluble, purified, HA-tagged scFvs were incubated with target cells, and bound scFvs were detected using a fluorescent anti-HA antibody. The soluble scFv against MERS and scFv 3–7 (negative binders to cPD-1 by ELISA), were used as negative controls.

Article Snippet: Briefly, the non-suppressor TOP10F’ Chemically Competent E. coli strain (ThermoFisher Scientific/Invitrogen, C303003) was infected with phage clones that were confirmed to bind to streptavidin immobilized and biotinylated avi-tagged cPD-1 soluble protein (Sino Biological 70,109-D27H-B) by phage ELISA.

Techniques: Binding Assay, Transduction, Purification, Incubation, Enzyme-linked Immunosorbent Assay

Journal: mAbs

Article Title: Development and pharmacokinetic assessment of a fully canine anti-PD-1 monoclonal antibody for comparative translational research in dogs with spontaneous tumors

doi: 10.1080/19420862.2023.2287250

Figure Lengend Snippet: A. Primary structures of lead canine anti-cPD-1 antibodies. A. (top) heavy chain alignments of P4B1, P3C6 and its variants along with canine germline gene IGHV3-38*01. (bottom) light chain alignments of P4B1 and P3C6. Framework and CDR regions use IMGT nomenclature. B. Evaluation of full-length IgG D (P3C6mut3.1 and mut3.2) canine anti-cPD-1 binding to cell surface-expressed cPD-1 by flow cytometry. Canine IgG D -HA tagged clones against cPD-1 or the irrelevant MERS antigen (negative control) were used to stain KTδ32 and KTδ32.cPD-1 cells. Bound IgG D molecules were detected using an anti-HA antibody. Plots are gated on live, 7AAD- cells.

Article Snippet: Briefly, the non-suppressor TOP10F’ Chemically Competent E. coli strain (ThermoFisher Scientific/Invitrogen, C303003) was infected with phage clones that were confirmed to bind to streptavidin immobilized and biotinylated avi-tagged cPD-1 soluble protein (Sino Biological 70,109-D27H-B) by phage ELISA.

Techniques: Binding Assay, Flow Cytometry, Clone Assay, Negative Control, Staining