Journal: Frontiers in Immunology

Article Title: The application of Interleukin-2 family cytokines in tumor immunotherapy research

doi: 10.3389/fimmu.2023.1090311

Figure Lengend Snippet: Signaling network of the IL-2 cytokine family. Receptors of IL-2, IL-15, IL-21, IL-4, IL-7 and IL-9 share a common γ chain subunit. They phosphorylate various STAT proteins downstream by activating JAK/STAT signaling pathway.

Article Snippet: For example, the IL-15 super-agonist, N-803 (formerly ALT-803), developed by ImmunityBio assembles an IL-15 mutant protein (IL15N72D), an activating mutation of IL-15Rα sushi domain, and IgG1-Fc together to allow trans- presentation and decreased renal clearance.

Techniques:

Journal: Frontiers in Immunology

Article Title: The application of Interleukin-2 family cytokines in tumor immunotherapy research

doi: 10.3389/fimmu.2023.1090311

Figure Lengend Snippet: Summary of clinical trials involving IL-15 and IL-7.

Article Snippet: For example, the IL-15 super-agonist, N-803 (formerly ALT-803), developed by ImmunityBio assembles an IL-15 mutant protein (IL15N72D), an activating mutation of IL-15Rα sushi domain, and IgG1-Fc together to allow trans- presentation and decreased renal clearance.

Techniques: Infection

Journal: bioRxiv

Article Title: Targeted LNPs deliver mRNA encoding IL-15 superagonists to balance efficacy and toxicity in cancer therapy

doi: 10.1101/2024.01.11.575299

Figure Lengend Snippet: (A) The schematic illustration of structural optimization of the IL-15 superagonist via mRNA format. (B) Detection of activating signal of IL2Rβ-STAT5 in TF-1 IL2Rβ-STAT5 reporter cells. Reporter cells were co-cultured with cell medium containing either N-803 or IL-15 superagonist protein for 24 h before detection. (C-F) Assessment of the activation of peripheral blood mononuclear cells (PBMC) in a co-culture system with N-803 or mRNA-encoded IL-15 superagonists supernatant. PBMC were co-cultured with N-803 (4.5 nM) or mRNA-encoded IL-15 superagonists supernatant (0.1 nM), as well as 10 ug/ml CD3Ab (OKT3) for 3 days, then several parameters were detected, including ( C ) pSTAT5 levels in the cell lysate, ( D ) IFNγ levels in the supernatant, ( E ) cell proliferation rate, and ( F ) changes in CD45+CD3+CD8+ and CD45+CD3+CD56+ cell subtype proportion. The PBMC samples were collected from lung cancer patients ( C, D ) and healthy individuals ( E, F ). Data is shown as mean ± SEM and asterisks indicate significant differences between the N-803 and mRNA-encoded IL-15 superagonist groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Human IL-15 Quantikine ELISA Kit (R&D Systems, MN, USA), 96-well ELISA plates (Corning, NY, USA), recombinant human IL-15Rbeta/gamma Heterodimer Protein (Sino Biological, Beijing, China), N-803 (Sino Biological, Beijing, China), Anti-Human IgG-Fc Secondary Antibody (Sino Biological, Beijing, China), ELISA basic kit (Multi Science, Hangzhou, China), One-Lite Luciferase Assay System (Vazyme, Nanjing, China), ImmunoCultTM-XF T Cell Expansion Medium (StemCell Technologies, Vancouver, Canada), CD3Ab OKT3 (Sino Biological, Beijing, China), Human IFN-gamma Quantikine ELISA Kit (R&D systems, MN, USA), Human IL-6 Quantikine ELISA Kit (R&D systems, MN, USA), Mouse PBMC Isolation Kit (Solarbio, Beijing, China), CellTiter-Glo® Luminescent Cell Viability Assay (Promega, WI, USA), STAT5 alpha/beta (Phospho) [pY694/pY699] Human InstantOneTM ELISA Kit (ThermoFisher, MA, USA), anti-mouse CD3 antibody (Proteintech, Rosemont, USA), anti-mouse CD8 antibody (Abclona, Woburn, USA) and anti-mouse CD161 antibody (Abcam, Cambridge, UK).

Techniques: Cell Culture, Activation Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: Targeted LNPs deliver mRNA encoding IL-15 superagonists to balance efficacy and toxicity in cancer therapy

doi: 10.1101/2024.01.11.575299

Figure Lengend Snippet: (A) This schematic illustrates the design and process of STR-4 mRNA sequence mutation, codon optimization, and in vitro and in vivo screening. (B) The schematic diagram of assessing the ability to activate IL2Rβ-STAT5 signaling in TF-1 reporter cells. (C) The EC 50 heat map of 108 different STR-4 mutants, which was generated by one-by-one “to D” AA mutation from the N-terminus to the C-terminus in the IL-15 portion. (D , E) Assessment of the activation of peripheral blood mononuclear cells (PBMC) in a co-culture system with top STR-4 mutants. The changes in CD45+CD3+CD8+ (D) and CD45+CD3+CD56+ (E) cell subtypes were analyzed, respectively. (F) The schematic diagram of STR-4-D43 and STR-4-D61 mRNAs for anti-tumor assay delivered by LNP Local in MC38 model. Mice were inoculated subcutaneously with 5E5 MC38 cells and randomized into groups when tumors reached 80-120mm 3 . Then mice were treated by various groups, vehicle (empty LNP, i.t.), N-803 (0.2 mpk, i.v.), SRT-4-D43 (2 mpk, i.t.) and SRT-4-D61 (2 mpk, i.t.), at day 0, day 3, day 7, day 10, and day14. Tumor growth (G , K) and body weight changes ( H ) were monitored during whole experiment, and (I , J) blood samples were collected on day 17 for Complete Blood Count. Data is shown as mean ± s.e.m. (n = 3-5 biologically independent samples or animals). Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Human IL-15 Quantikine ELISA Kit (R&D Systems, MN, USA), 96-well ELISA plates (Corning, NY, USA), recombinant human IL-15Rbeta/gamma Heterodimer Protein (Sino Biological, Beijing, China), N-803 (Sino Biological, Beijing, China), Anti-Human IgG-Fc Secondary Antibody (Sino Biological, Beijing, China), ELISA basic kit (Multi Science, Hangzhou, China), One-Lite Luciferase Assay System (Vazyme, Nanjing, China), ImmunoCultTM-XF T Cell Expansion Medium (StemCell Technologies, Vancouver, Canada), CD3Ab OKT3 (Sino Biological, Beijing, China), Human IFN-gamma Quantikine ELISA Kit (R&D systems, MN, USA), Human IL-6 Quantikine ELISA Kit (R&D systems, MN, USA), Mouse PBMC Isolation Kit (Solarbio, Beijing, China), CellTiter-Glo® Luminescent Cell Viability Assay (Promega, WI, USA), STAT5 alpha/beta (Phospho) [pY694/pY699] Human InstantOneTM ELISA Kit (ThermoFisher, MA, USA), anti-mouse CD3 antibody (Proteintech, Rosemont, USA), anti-mouse CD8 antibody (Abclona, Woburn, USA) and anti-mouse CD161 antibody (Abcam, Cambridge, UK).

Techniques: Sequencing, Mutagenesis, In Vitro, In Vivo, Generated, Activation Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: Targeted LNPs deliver mRNA encoding IL-15 superagonists to balance efficacy and toxicity in cancer therapy

doi: 10.1101/2024.01.11.575299

Figure Lengend Snippet: (A) The comparison of STR-4-D61 protein in blood after the mRNA was delivered with LNP liver and LNP lung by i.v. injection. (B) The schematic diagram of STR-4-D61 for lung metastatic tumor therapy. Mice were inoculated intravenously with luciferase-expressing B16F10 and randomized into groups. After 6h, mice were i.v. treated by STR-4-D61 LNP Lung for twice with mRNA dose of 1 mg/kg. (C, D) Tumor growth of each group was monitored by IVIS system and quantification data was analyzed. (E) At day 14, mice were sacrificed and lung images were recorded. (F) The antitumor effect of STR-4-D43 mRNA on lung metastatic tumors was also evaluated through experiments similar to those described above. (G-I) Obvious tumor inhibition of STR-4-D43 group was observed by IVIS imaging, luciferase quantification and lung tissue photographs. (J, L) Treated by STR-4-D43 LNP, the proportion of CD161+ NK cells in blood were significantly increased, and the increase infiltration of CD3+T cells and CD8+ T cells in the lung was observed. ( K ) H&E staining confirmed the anti-tumor efficacy that STR-4-D43 group showed a close-to-healthy lung tissue. Scale bar = 100 μm. Data is shown as mean ± s.e.m. (n = 5 biologically independent animals).

Article Snippet: Human IL-15 Quantikine ELISA Kit (R&D Systems, MN, USA), 96-well ELISA plates (Corning, NY, USA), recombinant human IL-15Rbeta/gamma Heterodimer Protein (Sino Biological, Beijing, China), N-803 (Sino Biological, Beijing, China), Anti-Human IgG-Fc Secondary Antibody (Sino Biological, Beijing, China), ELISA basic kit (Multi Science, Hangzhou, China), One-Lite Luciferase Assay System (Vazyme, Nanjing, China), ImmunoCultTM-XF T Cell Expansion Medium (StemCell Technologies, Vancouver, Canada), CD3Ab OKT3 (Sino Biological, Beijing, China), Human IFN-gamma Quantikine ELISA Kit (R&D systems, MN, USA), Human IL-6 Quantikine ELISA Kit (R&D systems, MN, USA), Mouse PBMC Isolation Kit (Solarbio, Beijing, China), CellTiter-Glo® Luminescent Cell Viability Assay (Promega, WI, USA), STAT5 alpha/beta (Phospho) [pY694/pY699] Human InstantOneTM ELISA Kit (ThermoFisher, MA, USA), anti-mouse CD3 antibody (Proteintech, Rosemont, USA), anti-mouse CD8 antibody (Abclona, Woburn, USA) and anti-mouse CD161 antibody (Abcam, Cambridge, UK).

Techniques: Comparison, Injection, Luciferase, Expressing, Inhibition, Imaging, Staining