Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin-7-loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma

doi: 10.1007/s00262-021-02856-0

Figure Lengend Snippet: oAD-IL7 successfully replicate in glioma. a Schematic representation of genome of the oncolytic viruses used in this study. b Monolayers of glioblastoma cell lines were infected by oAD-IL7 at the MOI of 10. Cells were imaged with a fluorescence microscope 24, 48, and 72 h after infection. Scale bar, 500 μm. c Cells were collected 72 h after infection for plaque assay. Each number of the virus tilter were calculated on average of three independent wells. d Oncolytic virus-induced GBM apoptosis was confirmed by western blot. Scale bar, 100 μm. e Cells were stained with IL7 antibody 72 h after infection and imaged with a fluorescence microscope. Green fluorescence represents the oAD-IL7 infection; red fluorescence represents the IL7 distribution; blue fluorescence represents the cell nuclei stained with DAPI. Scale bar, 50 nm. f Supernatants were collected for IL7 quantification. g The median tumor volume of the xenograft models treated by PBS, oAD, and oAD-IL7 was recorded. h Survival analysis of the three group of the xenograft models. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed paired t test)

Article Snippet: A cDNA encoding human IL7 purchased from Sinobiological (Beijing, China) was cloned and then inserted the E1 region, and an E1A gene under the control of hTERT, followed by a E1B gene under the control of TATA box was synthesized by General Biosystems (Chuzhou, Anhui province, China) and inserted into the E3 region.

Techniques: Infection, Fluorescence, Microscopy, Plaque Assay, Virus, Western Blot, Staining, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin-7-loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma

doi: 10.1007/s00262-021-02856-0

Figure Lengend Snippet: oAD-IL7 enhanced the survival and efficacy of B7H3-CAR-T. a 1 × 10 B7H3-CAR-T or untransduced T cells underwent serial coculturing with 2 × 105 oncolytic virus-infected U87 cells for a total of 7 days. The GBM cells were refreshed on the 4th day of the serial coculture. b Cumulations of T cells expansion. c For proliferation analysis, T cells were stained with CytoTell Blue before serial coculture and collected for flow cytometry at the end of day 3 and day 7 to monitor the results of division. d For survival analysis, T cells were collected and stained with Annexin V and 7-AAD. e 51Cr-release assays were used to evaluate the lysis of GBM during the second round of coculture at the end of day 7. Data represent the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01 (two-tailed paired t test)

Article Snippet: A cDNA encoding human IL7 purchased from Sinobiological (Beijing, China) was cloned and then inserted the E1 region, and an E1A gene under the control of hTERT, followed by a E1B gene under the control of TATA box was synthesized by General Biosystems (Chuzhou, Anhui province, China) and inserted into the E3 region.

Techniques: Virus, Infection, Staining, Flow Cytometry, Lysis, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin-7-loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma

doi: 10.1007/s00262-021-02856-0

Figure Lengend Snippet: oAD-IL7 prolonged the survival of tumor-infiltrating CAR-T cells in xenograft models. In a parallel experiment, the tumor-bearing mice underwent a delayed treatment since the 21st day after tumor inoculation. a The tumors of the mice treated with single B7H3-CAR-T therapy or combined therapy were imaged by MRI scanning at day 28. b The tumors were removed from the treated mice at day 30, and the immune infiltrate was evaluated by immunohistochemical staining according to the distribution of CD3+ cells. Scale bar, 200 μm. c Titration of oAD-IL7 in glioblastoma tissue by plaque assay. d The ratio of CD3 positive cells in tumor tissues removed from mice treated by combined therapy or single B7H3-CAR-T therapy according to the count of average CD3 + cells in each single field. e Representative flow cytometry analysis of the T cells in the tumor removed from mice underwent different therapies. f The expression of PD1 and LAG-3 of the CD3 positive cells in two groups. g The level of LAG-3 expression on the tumor-infiltrating T cells of two groups. h The level of PD1 expression on the tumor-infiltrating T cells of two groups. i Ki67 expression of the CD3 positive cells in two groups. j Expression of the B7H3 and CXAR of the tumors removed from tumor-bearing mice on day 21. Scale bar, 50 μm. k Expression of the B7H3 and CXAR of the tumors removed from tumor-bearing mice on day 30. Scale bar, 50 μm

Article Snippet: A cDNA encoding human IL7 purchased from Sinobiological (Beijing, China) was cloned and then inserted the E1 region, and an E1A gene under the control of hTERT, followed by a E1B gene under the control of TATA box was synthesized by General Biosystems (Chuzhou, Anhui province, China) and inserted into the E3 region.

Techniques: Immunohistochemical staining, Staining, Titration, Plaque Assay, Flow Cytometry, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin-7-loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma

doi: 10.1007/s00262-021-02856-0

Figure Lengend Snippet: Combination of oAD-IL7 and B7H3-CAR-T improved the anti-tumor effect in vivo. 2 × 105 GBM-LUCF cells were orthotopically injected into the NCG mice, and the bioluminescent imaging was performed twice a week since the 7th day after injection. The oncolytic virus was orthotopically injected on day 7, and the B7H3-CAR-T cells were systematically applied on day 9. a Experimental timeline for in vivo studies. b The representative bioluminescent images of the growth of glioblastoma over time were shown. c The expression of B7H3 and CXAR in the glioblastoma tissue of patient. Scale bar, 50 μm. d, e, and f Quantitated GBM-LUCF bioluminescence from each treatment group is displayed over time. g Average radiance of the three groups of the mice during the first 3 weeks. h Kaplan–Meier survival analysis of GBM-LUCF challenged mice after treatment with oncolytic virus and T cells. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed paired t test). i H&E staining of the specimen removed from tumor-bearing mice that underwent a parallel experiment at day 30

Article Snippet: A cDNA encoding human IL7 purchased from Sinobiological (Beijing, China) was cloned and then inserted the E1 region, and an E1A gene under the control of hTERT, followed by a E1B gene under the control of TATA box was synthesized by General Biosystems (Chuzhou, Anhui province, China) and inserted into the E3 region.

Techniques: In Vivo, Injection, Imaging, Virus, Expressing, Two Tailed Test, Staining

Journal: STAR Protocols

Article Title: Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a

doi: 10.1016/j.xpro.2022.101321

Figure Lengend Snippet:

Article Snippet: IL-7 Protein, Human, Recombinant , SinoBiological , Cat# 11821-HNAE.

Techniques: Recombinant, Electroporation, Modification, SYBR Green Assay, Transfection, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, DNA Extraction, Software, Real-time Polymerase Chain Reaction, Protein Purification, Plasmid Preparation, Synthesized, Flow Cytometry, Spectrophotometry

Journal: bioRxiv

Article Title: Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

doi: 10.1101/2025.06.03.657627

Figure Lengend Snippet: Schematic illustrating the computational pipelines for Neo-7 engineering from wild type IL7 sequence. In general, non-receptor interacting loops were deleted from the WT-IL7 sequence and loops connecting the adjacent helixes were modeled using Rosetta Loop Remodeler and Rosetta fix backbone design function. The sequence of the designed model were extracted and submitted to alphafold (monomer and multimer mode for structure and protein-receptor binding prediction respectively) as an preliminary validation of the Rosetta-remodeled protein. Iterations of the bad models (models that do not fold into the expected structure or models that did not predicted to bind to the receptors) back to the design stage were performed. Models that passed the Alphafold validation proceeded to subsequent in vitro assay using yeast display system and flow cytometry to determine their relative binding affinity to IL-7 receptors in comparison to WT-IL7.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7 dependent murine B-cell cell line were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20 % FBS.

Techniques: Sequencing, Binding Assay, Biomarker Discovery, In Vitro, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

doi: 10.1101/2025.06.03.657627

Figure Lengend Snippet: Blueprint of Neo-7 design. Blueprint of the WT-IL7 were shown on the left of the figure. The connectivity of the functioning helixes were connected in a manner that requires extremely long protein loops by design (i.e. helices were not connected to the closest adjacent helixes but to the opposite helix). Loops that were not interacted with the IL-7 receptors were deleted and the helixes were reconnected in a clockwise manner via new protein linkers connecting to the adjacent helixes. The blueprint of the redesigned protein was shown at the right side of the figure. Protein structures are colored as rainbow ( from N-to-C terminus with the order of Blue-Green-Yellow-Red).

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7 dependent murine B-cell cell line were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20 % FBS.

Techniques:

Journal: bioRxiv

Article Title: Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

doi: 10.1101/2025.06.03.657627

Figure Lengend Snippet: (A) Alphafold validation of the first loop design version of neo7 (Neo-7 LDv1) using the default (left) and single sequence mode (right). (B) Alphafold validation of the second loop design version of neo 7 (left; Neo-7 LDv2) and Neo7 LDv2 with mutations (right) favored by Rosetta fix backbone design. (C) Crystal structure of human IL-7 in complexation to human IL-7 receptor alpha (PDB ID = 3DI2). (D) Superimposition of Neo-7 structures (with or without additional disulfide bridge) predicted by Alphafold. (E) Yeast display and flow cytometry validation of IL-7/Neo-7 bindings towards the IL-7 receptors. The yeast-displayed protein (different redesigned IL-7s) carries a HA-tag while the recombinant IL-7 receptors carries either a HIS tag (IL-7 receptor alpha) or a FC-tag (common-IL-2 family receptor gamma). The signal intensity of the X-axis (confers by the binding of anti-HA mab) correlates with the expression level of the displayed protein while the signal intensity of the Y-axis (confers by the binding of the anti-HIS/anti-FC mab to the recombinant receptors bound to the displayed proteins) correlates with the binding affinity of the displayed proteins towards the IL-7 receptors. (D) Site-directed mutagenesis residues recommended by Rosetta to improve the folding stability of the Neo-7s.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7 dependent murine B-cell cell line were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20 % FBS.

Techniques: Biomarker Discovery, Sequencing, Flow Cytometry, Recombinant, Binding Assay, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

doi: 10.1101/2025.06.03.657627

Figure Lengend Snippet: (A) Inspection of structural and binding interactions of residue mutations Q6P and T45I on Neo-7 towards the murine IL-7R alpha. (B) Yeast display and flow cytometry validation of the binding ability of IL-7/Neo-7 variants toward the IL-7 receptors.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7 dependent murine B-cell cell line were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20 % FBS.

Techniques: Binding Assay, Residue, Flow Cytometry, Biomarker Discovery

Journal: bioRxiv

Article Title: Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

doi: 10.1101/2025.06.03.657627

Figure Lengend Snippet: FPLC profile of E. coli expressed (A) WT-IL7 (B) refolded WT-IL7 (C) Neo-7-Q6P and (D) Neo-7-Q6P-T45I. Percentage of purity is calculated from the SEC-FPLC peak profile via Cytiva Unicorn TM 7 software after affinity chromatography purification. SPR (Biacore) characterization of the binding kinetics of (E) Neo-7-Q6P (F) Neo-7-Q6P-T45I and (G) WT-IL7 towards murine IL-7R alpha. (H) 2E8 proliferation assay to investigate the biological activity of the IL-7/Neo-7s expressed by E. coli .

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7 dependent murine B-cell cell line were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20 % FBS.

Techniques: Software, Affinity Chromatography, Purification, Binding Assay, Proliferation Assay, Activity Assay

Journal: bioRxiv

Article Title: Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

doi: 10.1101/2025.06.03.657627

Figure Lengend Snippet: FPLC profile of CHO-S expressed (A) WT-IL7 (B) Neo-7-Q6P and (C) Neo-7-Q6P-T45I. Percentage of purity is calculated from the SEC-FPLC peak profile via Cytiva Unicorn TM 7 software after affinity chromatography purification. Murine spleenocyte proliferation assay to investigate the biological activity of the Fc-fused IL-7/Neo-7s at (D) day 3 and (E) day 7 post-treatment. In vivo immune stimulatory ability of the Fc-fused cytokines on murine PBMCs at day 0 to day 12 post-treatment. The data are presented as count of (F) total viable CD45+ cells (G) viable CD45+ CD3+ CD4+ T-cells (H) viable CD45+ CD3+ CD8+ T-cells (I) viable CD45+ CD3-NK1.1+ NK-cells. All data were presented as individual data plots with error bars (SEM). Statistical differences among groups were determined using one-way ANOVA with Turkey’s multiple comparison test. Significance levels are defined as follow *p < 0.05; **p = 0.01–0.05; ***p = 0.0001–0.001; and ****p < 0.0001.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7 dependent murine B-cell cell line were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20 % FBS.

Techniques: Software, Affinity Chromatography, Purification, Proliferation Assay, Activity Assay, In Vivo, Comparison

Journal: bioRxiv

Article Title: Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

doi: 10.1101/2025.06.03.657627

Figure Lengend Snippet: (A) gene ontology analysis of the gene expression data from RNA sequencing (n = 3; three independent biological donor for each group). (B) Gene Set Enrichment Analysis (GSEA) of splenic CD8+ T-cells treated by Fc-Neo-7 versus Fc-WT-IL7. (C) principal component analysis and (D) Gene expression heatmap derived from Z-scores calculated from the RNA sequencing data.

Article Snippet: 2E8 cells (ATCC TIB-239), an IL-7 dependent murine B-cell cell line were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Cat: 12440053) supplemented with 0.05 mM 2-mercaptoethanol 2 ng/mL mouse IL7 (Sino Biological) and 20 % FBS.

Techniques: Gene Expression, RNA Sequencing, Derivative Assay

Journal: Developmental Cell

Article Title: Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus

doi: 10.1016/j.devcel.2023.08.017

Figure Lengend Snippet:

Article Snippet: HumanKine® recombinant human IL-7 protein , Proteintech , cat# HZ-1281.

Techniques: Recombinant, Modification, Selection, RNA Extraction, Polymer, Cell Culture, Derivative Assay, Software