Journal: Science Advances

Article Title: Messenger RNA delivery of a dual CD25/CD122 affinity-tuned IL-2 variant prolongs exposure and potentiates antitumor T cell immunity

doi: 10.1126/sciadv.aeh9215

Figure Lengend Snippet: ( A ) IL-2 variants evaluated in this study. ( B ) Affinity measurements of IL-2 variants to IL-2Rα and IL-2Rβ as determined by biolayer interferometry. K d , dissociation constant. ( C ) Signal transducer and activator of transcription 5 (STAT5) phosphorylation in T cell subsets within human peripheral blood mononuclear cells (PBMCs; n = 5 donors) was analyzed following stimulation with serial dilutions of human embryonic kidney (HEK) 293T supernatants containing Alb–IL-2 variants, as determined by flow cytometry. The potency of Alb–IL-2 variants is expressed as reciprocal median effective concentration (EC 50 ). For potencies below the lower limit of quantification (LLOQ), LLOQ/2 values were used for EC 50 . ( D ) STAT5 phosphorylation in IL-2Rα RNA-electroporated or nonelectroporated CD8 + T cells isolated from PBMCs ( n = 2 donors) upon incubation with HEK293T supernatants containing Alb–IL-2 variants as determined by flow cytometry. ( E ) RiboCytokine platform technology. Albumin-cytokine fusion protein sequences are encoded by modified single-stranded RNA and formulated into LNPs. ( F ) Bioluminescence in vivo imaging of BALB/c mice ( n = 3) injected intravenously with 1 μg of firefly luciferase RNA-LNP. Alb RNA-LNP served as control. h, hours. ( G ) Serum levels of translated Alb-IL–2var protein in BALB/c mice ( n = 3) injected intravenously with 10 μg of Alb–IL-2var RNA-LNP (Alb–IL-2var). Alb RNA-LNP served as the control. ( H ) Flow cytometry analysis of CD8 + and CD4 + T cells, T reg cells and CD8 + T–to–T reg cell ratio in blood of C57BL/6 mice ( n = 3) 7 days after dosing with 10 μg of Alb–IL-2 RNA-LNP (Alb–IL-2) or Alb–IL-2var RNA-LNP (Alb–IL-2var). Alb RNA-LNP served as the control. Horizontal dotted lines represent the means of the control group (H). One-way analysis of variance (ANOVA) with Dunnett’s post hoc test (C and H). Not significant (n.s.), P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: Genes encoding the different IL-2 variants with an N-terminal His 6 -tag were synthesized and cloned into pTwist_CMV_BetaGlobin_WPRE_Neo expression vector (Twist Bioscience).

Techniques: Phospho-proteomics, Flow Cytometry, Concentration Assay, Isolation, Incubation, Modification, In Vivo Imaging, Injection, Luciferase, Control

Journal: Science Advances

Article Title: Messenger RNA delivery of a dual CD25/CD122 affinity-tuned IL-2 variant prolongs exposure and potentiates antitumor T cell immunity

doi: 10.1126/sciadv.aeh9215

Figure Lengend Snippet: ( A to D ) CT26 tumor-bearing BALB/c mice ( n = 11) were treated with gp70 RNA-LPX vaccine (vaccine), Alb–IL-2 RNA-LNP (Alb–IL-2), or Alb–IL-2var RNA-LNP (Alb–IL-2var), starting on day 10 post–tumor inoculation (average tumor volume, 16 mm 3 ). Alb RNA-LNP served as the control. (A) Experimental design. (B) gp70-specific CD8 + T and T reg cells, and (C) gp70-specific CD8 + T–to–T reg cell ratio in blood 7 days after the first treatment, as determined by flow cytometry. (D) Survival and individual tumor growth curves. Horizontal dotted lines represent the means of the control group (B and C), and vertical dotted lines represent treatments (D). ( E to G ) B16F10 tumor-bearing C57BL/6 mice ( n = 15) were treated with Trp-1 RNA-LPX vaccine (vaccine), Alb–IL-2 RNA-LNP (Alb–IL-2), and/or Alb–IL-2var RNA-LNP (Alb–IL-2var), starting on day 8 post–tumor inoculation (average tumor volume, 3 mm 3 ). Irrelevant RNA-LPX and hAlb RNA-LNP served as a control. (E) Experimental design. (F) Trp-1–specific CD8 + T cells 7 days after the second treatment, T reg cells 7 days after the first treatment, and CD8 + T–to–T reg cell ratio 7 days after the second treatment, in blood, as determined by flow cytometry. (G) Survival and individual tumor growth curves. Horizontal dotted lines represent the means of the control group (F), and vertical dotted lines represent treatments (G). Statistical tests: Kruskal-Wallis test with Dunn’s multiple comparison test (B, C, and F), log-rank test (D, left; and G, left), and two-way ANOVA with Dunnett’s multiple comparison test (G, right). Not significant (n.s.), P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. CR, complete regression; sc, subcutaneous; Trp-1, tyrosinase-related protein 1.

Article Snippet: Genes encoding the different IL-2 variants with an N-terminal His 6 -tag were synthesized and cloned into pTwist_CMV_BetaGlobin_WPRE_Neo expression vector (Twist Bioscience).

Techniques: Control, Flow Cytometry, Comparison

Journal: Science Advances

Article Title: Messenger RNA delivery of a dual CD25/CD122 affinity-tuned IL-2 variant prolongs exposure and potentiates antitumor T cell immunity

doi: 10.1126/sciadv.aeh9215

Figure Lengend Snippet: ( A ) Serum levels of translated Alb–IL-2var in female cynomolgus monkeys ( n = 3) injected intravenously with 50 μg (mean of 19.45 μg/kg) of Alb–IL-2var RNA-LNP (Alb–IL-2var). ( B to E ) Lymphocytes as assessed by hematocytometry (means + SEM) (B), CD8 + T cells, T reg cells, and NK cells pre- and postdose (7 days after treatment) (C) as well as CD8 + T–to–T reg cell ratios pre- and postdose (D) and over time (means + SEM) (E) as determined by flow cytometry in female cynomolgus monkeys ( n = 3) treated with Alb–IL-2 RNA-LNP (Alb–IL-2) or Alb–IL-2var RNA-LNP (Alb–IL-2var) (60 or 180 μg/kg). Empty LNPs (lipid dose adapted to 120 μg of RNA/kg) served as control. LLOQ, lower limit of quantification.

Article Snippet: Genes encoding the different IL-2 variants with an N-terminal His 6 -tag were synthesized and cloned into pTwist_CMV_BetaGlobin_WPRE_Neo expression vector (Twist Bioscience).

Techniques: Injection, Flow Cytometry, Control

Journal: iScience

Article Title: In vitro recapitulation of intramuscular mRNA vaccination with naive and recall antigens using a human lymphoid follicle chip platform

doi: 10.1016/j.isci.2026.116416

Figure Lengend Snippet: Induction of LFs and anti-RABV-G IgG production by SAM-LNP1 in human LF chips (A) Schematic of the LF Chip created with monocytes and conditioned medium from the intramuscular vaccination-mimicking module created with BioRender.com . (B) Representative 3D confocal microscopic stacks showing pseudo-colored follicles (blue) and cell nuclei (green) present within extracellular matrix (ECM) gels cultured for 4 days within a perfused LF Chip when vaccinated with SAM-LNP1 in the absence or presence of IL-2 and IL-4 (SAM-LNP1 + IL-2&IL-4); scale bars, 100 μm. (C) Quantification of the number (left) and size (right) of LFs in LF chips of one donor based on immunostaining followed by confocal imaging. Each data point represents one field of view (left) or an individual follicle (right); 2 independent chips were analyzed per condition. (D) Anti-RABV-G IgG levels in effluents of no treatment LF chips or chips vaccinated with or without IL-2 and IL-4 and cultured for 14 days were detected using a cell-based assay; 3–4 independent chips were analyzed per condition. (E) Quantification of the number (left) and size (right) of LFs in LF chips from four different donors, with each color representing a different donor. Each data point corresponds to one field of view (left) or an individual follicle (right). (F) Anti-RABV-G IgG levels in effluents of LF chips. Each data point represents one chip, with different symbols indicating chips from three independent donors. (G) Anti-RABV-G IgG levels in effluents of LF chips seeded with naive B + bulk T + monocyte mixture. Each data point represents one chip, with different symbols indicating chips from three independent donors. (H) Heatmap showing average log2 fold changes in cytokine levels in the effluents of three LF chips created using bulk lymphocytes from donors whose prior exposure to rabies was unknown, measured using a Luminex Multiplex Assay at various time points (4, 7, 15, 22, and 28 days post-vaccination) compared to the average levels of each cytokine found at day 1. Representative results from one donor are shown in (B)–(D) and (H), with similar outcomes observed in two donors. Data shown are mean ± SD; (C, left; D; and E) one way ANOVA test followed by Tukey’s multiple comparisons test, (C, right; and E) Mann-Whitney U test, (F and G) Welch’s t test.

Article Snippet: After allowing the matrix to gel for 30 min at 37°C, the top channel was filled with 1 part conditioned medium from the 2D intramuscular module, and 2 parts fresh medium supplemented with IL-2 (Mitenyi Biotec, 130-097-742) and IL-4 (Mitenyi Biotec, 130-093-922) for experiments with RABV-G mRNA vaccines.

Techniques: Cell Culture, Immunostaining, Imaging, Cell Based Assay, Luminex, Multiplex Assay, MANN-WHITNEY