Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: In general, non-receptor interacting loops were deleted from the WT-IL7 sequence and loops connecting the adjacent helices were modeled using Rosetta Loop Remodeler and Rosetta fix backbone design function. The sequence of the designed model was extracted and submitted to AlphaFold (monomer and multimer mode for structure and protein-receptor binding prediction respectively) as a 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 predict 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: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: Blueprint of the WT-IL7 was shown on the left of the figure. The connectivity of the functioning helixes was 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: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) AlphaFold validation of the first loop design version of Neo-7 (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 Neo-7 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 carry either a HIS tag (IL-7 receptor alpha) or a FC-tag (common-IL-2 family receptor gamma; for detection of IL-2Rγ binding, yeast cells were first incubated with recombinant IL-7Rα, washed, and subsequently incubated with IL-2Rγ.) The signal intensity of the X-axis (conferred by the binding of anti-HA mab) correlates with the expression level of the displayed protein while the signal intensity of the Y-axis (conferred 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.

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, Incubation, Expressing

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

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: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

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 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 . Error bars represent standard deviation (n=3).

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, Standard Deviation

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: In silico immunogenicity prediction of ( A ) WT-IL7 ( B ) Neo-7 ( C ) Neo-7 single mutant ( D ) Neo-7 double mutant. ( E ) Sequence comparison of Neo-7 and WT-IL7, helices of the cytokine are colored as blue, green, yellow and red; loops are colored as gray. ( F ) Alphafold predicted structural model of FC-Neo-7.

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: In Silico, Immunopeptidomics, Mutagenesis, Sequencing, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

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 7 software after affinity chromatography purification. (D–E) Murine splenocyte proliferation assays performed at day 3 and day 7 following treatment with Fc-control (gray), Fc-WT-IL7 (red), Fc-Neo-7-Q6P (blue), or Fc-Neo-7-Q6P-T45I (green). 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 a 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. Treatment groups are colored as Fc-control (gray), Fc-WT-IL7 (red), Fc-Neo-7-Q6P (blue), and Fc-Neo-7-Q6P-T45I (green). All data were presented as individual data plots with error bars (SEM) (n=3). Statistical differences among groups were determined using one-way ANOVA with Turkey’s multiple comparison test. Significance levels are defined as follows *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, Control, In Vivo, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) Gene ontology analysis of the gene expression data from RNA sequencing (n=3; three independent biological donors 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. The gene expression heatmap is derived from Z-scores calculated from the RNA sequencing data, with expression levels color-coded from high (red) to low (blue).

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, Expressing