Journal: Frontiers in Immunology

Article Title: Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo

doi: 10.3389/fimmu.2023.1072810

Figure Lengend Snippet: Design, pharmacokinetic and pharmacodynamic profiles of novel ACC TA99-HL2-KOA1. (A) Model depiction of engineered cytokine mimic HL2-KOA1 binding to IL-2Rα. IL-2 shown in blue, IL-2Rα shown in grey. Positions in contact with F42 shown as grey spheres. F42 and F42V shown as pink and red spheres respectively. Clash scores provided in total Rosetta energy units (REU). (B) Reducing SDS-PAGE analysis comparing the migration patterns of TA99-WT, TA99-Neo2/15, TA99-HL2-KOA1, pVAX backbone control transfection supernatants. (C) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human Tyrp1 compared to a murine isotype IgG2a control. (D) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rα compared to human IL2-Fc control. (E) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rβ compared to human IL2-Fc control. (F) Geometric mean fluorescence intensity of phosphorylated STAT5 in CD25+ CD4+ CD19- T cells from naïve mice (n=6) treated with indicated ACC. (G) Pharmacokinetic profile of recombinant TA99-WT and TA99-Neo-2/15 antibodies; C57BL/6 mice were injected intraperitoneally with 100µg of ACC constructs (n=5 mice per group). Serum at the indicated timepoints was assessed for TYRP1 binding by ELISA. (H) Total serum cytokine level over 14-day period in terms of area under the curve (AUC) in mice treated with ACC. (I) Timecourse of serum TNFα levels in sera of mice administered 100µg ACC. Two sera pools were used for (H, I) . (J) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-Neo2/15 (50µg) + TriVax (10µg each of Trp2, Gp100 and Tyrp1), or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax. 1x10 5 B16F10 cells subcutaneously into the right flank of C57BL/6 mice on Day 0. Green arrows indicate administration of treatment. (K) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax, or anti-PD1 + TA99 (50µg) + Human IL2 (5µg) + TriVax. Challenge and treatment scheme was performed as in (J) Error bars represent standard deviation; non-parametric Mann Whitney T test compared with TA99-WT used in (F) ; non-parametric Kruskal-Wallis ANOVA was used in (I) ; log-rank test was used to compare between differences in all survival curves; *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: In addition, serially diluted recombinant protein variants of antibody-cytokine chimera or recombinant mouse Fc-tagged IL2 (IL2-Fc) (Molecular Innovations, Cat# MIL2-FC-0.05MG) were used in place of serially diluted mouse sera.

Techniques: Binding Assay, SDS Page, Migration, Control, Transfection, Recombinant, Fluorescence, Injection, Construct, Enzyme-linked Immunosorbent Assay, Standard Deviation, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE blockade of PD-1/PD-L1 interaction in coculture cell-based luciferase assay. (A, B) Cytotoxicity assay performed using Cell Counting Kit-8 (CCK) assay. The hPD-1/NFAT Jurkat T cells (A) and hPD-L1/TCR CHO-K1 cells (B) after treatment with SRE for 24 hours. (C, D) The PD-1/PD-L1 blockade bioassay was performed using the Bio-Glo™ luciferase assay system. After addition of hPD-1/NFAT Jurkat T cells and SRE (C) and anti-PD-1 antibodies (αPD-1) (D) , hPD-L1/TCR CHO-K1 cells were seeded for 20 hours. Data are presented as the mean ± SD. * p < 0.05 and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Luciferase, Cytotoxicity Assay, Cell Counting

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE-induced activation of T cells and cytotoxic effect of T cell-mediated cancer cells. (A, B) The cell viability was performed using the CCK-8 assay. Splenocytes were isolated from hPD-L1 MC38 cell-bearing hPD-1 knockin mice. Murine CRC hPD-L1 MC38 cells (A) and hPD-1 mice splenocytes (B) were treated with SRE for 72 hours. (C) Cocultured hPD-L1 MC38 cell viability tested by crystal violet staining; (D) Lactate dehydrogenase (LDH) released by damaged cells, detected via LDH cytotoxicity assay; (E) Relative interleukin-2 (IL-2) level, determined using the mouse IL-2 ELISA set. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, CCK-8 Assay, Isolation, Knock-In, Staining, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE elevated the activation of hPD-1 + CD8 + T cells and the CD8 + T cell-mediated killing effect on hPD-L1 MC38 cancer. (A) Cocultured hPD-L1 MC38 cell viability, tested by crystal violet staining. Cocultured hPD-L1 MC38 cells detected with fluorescence microscopy (× 200) (B) and determined by fluorescent-activated cell sorting analysis (C) . (D) LDH released from damaged cells; (E) Relative perforin 1 (PRF1) level, determined with use of the mouse PRF1 ELISA kit. Data are presented as the mean ± SD. ** p < 0.01 and *** p < 0.001 compared to the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, FACS, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: Sanguisorbae Radix extract reduced tumor growth in the hPD-L1 MC38 cell allograft hPD-1 mouse model. (A) Body weight (grams); (B) Tumor volume after 18 days; (C) Tumor weight after 18 days; (D) Images of tumor tissues (bar indicates 5 mm); (E) hPD-L1 MC38 tumor-bearing mice 18 days after treatment; (F) Representative microscopic images (×400) of CD8 and PRF1-positive area of tumor tissues calculated using immunohistochemical analysis. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Immunohistochemical staining, Standard Deviation