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: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Sequences of selected clones and their binding specificity. ( A ) Selected amino acid sequences of clones from PURE frex ® RD for CTLA-4-Fc. ( B ) Binding specificity of clones in ( A ) was evaluated as follows: mRNA–ribosome–peptide complex of each clone was reacted with each biotinylated protein (CTLA-4-Fc, dotted box; Human-Fc, black box; (-), white box) immobilized on streptavidin magnetic beads. Number of isolated mRNAs was determined by qPCR.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Clone Assay, Binding Assay, Magnetic Beads, Isolation

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Inhibition of interaction between CTLA-4 and B7-1 by C4-302. Inhibitory activity of C4-302 was examined. C4-351, which was found to bind to Fc region of CTLA-4, was used as negative control. After mixing mRNA–ribosome–peptide complex of each clone with CTLA-4, B7-1 immobilized on streptavidin magnetic beads via biotin was added to the mixture. ( A ) Because C4-351 bound to the Fc region of CTLA-4, mRNA was collected at nearly 10 10 . ( B ) In C4-302, mRNA collection was reduced considerably to just under 10 7 . This finding suggests that the C4-302 mRNA–ribosome–peptide complex inhibited the interaction of CTLA-4 with B7-1.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Inhibition, Activity Assay, Negative Control, Magnetic Beads

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Characterization of C4m-3127 synthetic peptide. Triangles indicate luminescence ( A ) and fluorescence ( B ) values for each concentration of C4m-3127 synthetic peptide. ( A ) Half-maximal inhibitory concentration (IC 50 ) value of synthetic cyclic peptide was measured using CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay Kit (BPS Bioscience Inc.) in 11-point two-fold serial dilution from 500 to 0.0050 μM by assay buffer. ( B ) Flow cytometry affinity analysis. CTLA-4 overexpressing CHO cell line was prepared at 1 × 10 5 cells/100 mL and reacted with synthetic peptide C4m-3127 with biotin-PEG4-Lys to N-terminus. Peptide in 10-point two-fold serial dilution from 4.4 to 0.0086 μM by PBS was used for this measurement. Cells with biotinylated peptide bound to their surface were detected with streptavidin–IFluor 647 conjugate. Fluorescence intensity depending on the amount of bound peptide shown on vertical axis.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Fluorescence, Concentration Assay, Screening Assay, Serial Dilution, Flow Cytometry

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Sequence mimic: Strategy of transforming cyclic peptides to small molecules. ( A ) Conceptual diagram of interaction between C4m-3127 and CTLA-4. Dashed lines indicate hydrogen bonds. C, cysteine. Numbers from 1 to 12 correspond to sequence 1 MHPFLPIVSHHF 12 . ( B ) Chemical structure of β-turn. Dashed lines indicate hydrogen bonds. ( C ) Representative PepMetics ® scaffold used in this research, among more than 40 kinds of scaffolds. For synthetic reasons, PepMetics ® scaffold cannot mimic histidine at the 3-position and proline at 6- and 8-positions. ( D ) Conceptual diagram of sequence mimic, with 10 patterns for mimicking sequences from (1, 2, 3) to (10, 11, 12). Because of the limitation of PepMetics ® for proline mimicking, this scaffold did not mimic the four sequences (M1, H2, P3), (H2, P3, F4), (F4, L5, P6), and (L5, P6, I7).

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Sequencing

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Chemical structures of key compounds. IC 50 values show CTLA-4/B7-1 interaction inhibitory activity. Three amino acids (V10, H11, F12) on PGF00432 correspond to C4m-3127 H10V random loop: 1 MHPFLPIVSVHF 12 .

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Activity Assay

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Predicted binding interaction of PGF00432 and PGF00506 with CTLA-4. ( A ) Crystal structure of B7-1/CTLA-4 complex (PDB code: 1I8L). Docking regions on CTLA-4 are derived from binding interface of the complex. ( B ) Predicted binding interaction of PGF00432 with CTLA-4. ( C ) Predicted binding interaction of PGF00506 with CTLA-4. Carbon, nitrogen, oxygen, and hydrogen are shown, respectively, in cyan (CTLA-4) or magenta (B7-1, PGF00432, and PGF00506), blue, red, and white. CTLA-4 and B7-1 are depicted as cyan and magenta ribbons, respectively. Dashed lines represent hydrogen bond interactions.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Binding Assay, Derivative Assay