DOI: 10.5061/dryad.9zw3r22v5
title: Data from: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

description: Interleukin-7 (IL-7) plays a central role in maintaining T cell
 development and immune homeostasis, and enhancing the cytokine’s
 immune-stimulatory functionality has broad therapeutic implications
 against various oncological malignancies. Herein, we show a
 computationally designed IL7 superkine, Neo-7, which exhibits enhanced
 folding efficiency and superior binding affinity to its cognate receptors.
 To streamline the protein candidate prediction and validation process, the
 loop region of IL7 was strategically targeted for redesign while most of
 the receptor-interacting regions were preserved. Leveraging advanced
 computational tools such as AlphaFold2, we show loop remodeling to rectify
 structural irregularities that allows for iterative stabilization of
 protein backbone and leads to identification of beneficial mutations
 conducive to receptor engagement. Neo-7 superkine shows improved
 thermostability and production yield, and it exhibits heightened
 immune-stimulatory and anticancer effect. These findings underscore the
 utility of a targeted computational approach for de novo cytokine
 development.
description: # Data from: Targeted computational design of an interleukin-7 superkine
 with enhanced folding efficiency and immunotherapeutic efficacy Dataset
 DOI: [10.5061/dryad.9zw3r22v5](https://doi.org/10.5061/dryad.9zw3r22v5) ##
 Description of the data and file structure File:
 eLife-VOR-RA-2025-107671-sourcedata3.zip The source data is divided into
 different folders as follows: ### Computational protein design
 (delineating the important models used as design blueprint and outputs
 from the Alphafold design; readers are encouraged to use publicly
 available visualization software such as Pymol (for .pse format) and
 Biovia Discovery Studio (for.dsv format) to visualize the dataset. 
 -Figure2-source data.pse = 3DI2 (3D structure for WT-IL7; PDB ID 3DI2)
 superimposed with Neo-7 Helix only (Helix section from 3DI2 used to design
 Neo-7) and Neo-7 skeleton (Neo-7 structure with connecting loops designed
 to connect the helices).  -Figure3A-source data.pse = Superimposition of
 Neo7-LD1-AF-singleseq (3D structure for Neo-7-LD1 modeled using alphafold
 single sequence mode), Neo-7 Helix only (Helix section from 3DI2 used to
 design Neo-7    ); Neo7-LD1-AF-default (3D structure for Neo-7-LD1 modeled
 using alphafold default mode) -Figure3B-source data.pse = Superimposition
 of Neo7-LD2 (3D structure for Neo-7-LD2 design modeled using alphafold
 single sequence mode), Neo-7 cw Helix only (Helix section from 3DI2 used
 to design Neo-7); Neo7-LD2_R5K_T44I (3D structure for Neo-7-LD2 design
 with R5K and T44I mutations modeled using alphafold single sequence mode).
 -Figure 3C source data = Alphafold model of human IL-7 in complexation to
 human IL-7 receptor alpha -Figure 3D source data = Superimposition of
 Neo-7 structures (with or without additional disulfide bridge) predicted
 by Alphafold.    -Figure4A-source data 1.pdb = PDB file listing 3D
 structure of Neo-7 Q6P and T45I mutants in complex with IL7 receptor
 alpha. Alphafold multimer  -Figure4A-source data 2.dsv = PDB file listing
 molecular interaction of Q6P mutation with IL7 receptor alpha modeled by
 Alphafold multimer. -Figure4A-source data 3.dsv = PDB file listing
 molecular interaction of T45I  mutation with IL7 receptor alpha modeled by
 Alphafold multimer. ### Yeast display flow cytometry data: Yeast display
 data of IL-7 and Neo-7 variants against IL7R-alpha and IL2R-Gamma
 (pre-binding to IL7R-alpha is required for IL2R-G binding). All data is
 provided as a standard flow cytometry file format (.fcs). Readers are
 encourage to use FlowJo to open the workspace file for deeper inspection
 of flow cytometry raw data and the gating strategies.  **Description:** 
 **Yeast display flow-cytometry data:** -m-alpha (murine IL-7 receptor
 alpha); m-gamma (murine IL-2 family common receptor gamma)  -X-axis =
 signal intensity coresponding to the expression/displayed level of the
 displayed protein  -Y-axis = signal intensity corresponding to the binding
 of the displayed protein to the indicated receptor.  **-Subfolder1:
 Figure3E-flow cytometry raw data\Figure3E-sourcedata** **-m-alpha:** yeast
 display data of displayed IL7/Neo7 using mouse IL7 receptor alpha as 
 antigen **--Sample naming :** (064 = Neo7-LD1/ 065=Neo7-LD1-R5K-T44I-I45T/
 075 50a=Neo7-LD2-R5K-T44I-I45T/ 076 50a=Neo7-LD2/ 095A=Neo7-LD1-Disulfide
 stapled-R5K-T44I-I45T/ 096A=Neo7-LD2-Disulfide stapled-R5K-T44I-I45T/
 100A=WT-IL7 with N-terminal Cys truncation/ 147 50 A = Wild type IL7/
 yeast IL-7 libraryS0_Group_eby100 s= wild type control stained with
 antigens and secondary antibody/ yeast IL-7 libraryS0_Group_eby100 2only=
 wild type control stained with only secondary antibody/ yeast IL-7
 libraryS0_Group_eby100 us= wild type unstain control) **-m-gamma** : yeast
 display data of displayed IL7/Neo7 using mouse IL7 receptor alpha and
 mouse IL2 receptor gamma as  antigen **--Sample naming :** (064 =
 Neo7-LD1/ 065=Neo7-LD1-R5K-T44I-I45T/ 075 50a=Neo7-LD2-R5K-T44I-I45T/ 076
 50a=Neo7-LD2/ 095A=Neo7-LD1-Disulfide stapled-R5K-T44I-I45T/
 096A=Neo7-LD2-Disulfide stapled-R5K-T44I-I45T/ 100A=WT-IL7 with N-terminal
 Cys truncation/ 147 50 A = Wild type IL7/ yeast IL-7
 libraryS0_Group_eby100 s= wild type control stained with antigens and
 secondary antibody/ yeast IL-7 libraryS0_Group_eby100 2only= wild type
 control stained with only secondary antibody/ yeast IL-7
 libraryS0_Group_eby100 us= wild type unstain control) **-Figure4-source
 data mAlpha:** yeast display data of displayed IL7/Neo7 using mouse IL7
 receptor alpha as  antigen **--Sample naming :** [
 108A50=Neo7-LD2-Disulfide
 stapled-R5K-T44I-I45T-(Q6P)/ 109A50=Neo7-LD2-Disulfide stapled-R5K-T44I/
 11A50=Neo7-LD2-Disulfide stapled-R5K-T44I-(Q6P) ] **-Figure4-source data
 mGamma:** yeast display data of displayed IL7/Neo7 using mouse IL2
 receptor gamma (g-only) or mouse IL7 receptor alpha and mouse IL2 receptor
 gamma (g malpha + mgamma) as  antigen. **--Sample naming :** [108g
 only=Neo7-LD2-Disulfide stapled-R5K-T44I-I45T-(Q6P) stained with mouse IL2
 receptor gamma only/ 109 g only=Neo7-LD2-Disulfide stapled-R5K-T44I
 stained with mouse IL2 receptor gamma only/ 110 g only=Neo7-LD2-Disulfide
 stapled-R5K-T44I-(Q6P) stained with mouse IL2 receptor gamma only/ 108 50a
 g=Neo7-LD2-Disulfide stapled-R5K-T44I-I45T-(Q6P) stained with mouse IL7
 receptor alpha and mouse IL2 receptor gamma/ 109 50a g=Neo7-LD2-Disulfide
 stapled-R5K-T44I stained with mouse IL7 receptor alpha and mouse IL2
 receptor gamma/ 110 50a g=Neo7-LD2-Disulfide stapled-R5K-T44I-(Q6P)
 stained with mouse IL7 receptor alpha and mouse IL2 receptor gamma] ###
 **In vitro data:** This section covers the SPR data from Biacore and
 cell-based data done in the study. All data is documented in excel sheet
 and readers are encouraged to use Microsoft Excel to inspect the data.
 **-Biacore_SPR_wdetails.xlsx**  **--Sample naming:** NEO7-Q6P:
 Neo7-LD2-Disulfide stapled-R5K-T44I-I45T-(Q6P)/
 NEO7-Q6PT45I: Neo7-LD2-Disulfide stapled-R5K-T44I-(Q6P)/ WT-IL7: Wild type
 IL7 | Parameter | Unit | Meaning | | :---------- | :----- |
 :------------------------------------------------------- | | **ka** |
 1/M·s | Association rate constant (binding speed) | | **kd** | 1/s |
 Dissociation rate constant (complex stability) | | **KD** | M |
 Equilibrium dissociation constant (affinity = kd/ka) | | **Rmax** | RU |
 Maximum theoretical response if all sites bound | | **Conc** | M |
 Injected analyte concentration series | | **tc** | s | Contact time during
 association phase | | **Flow** | µL/min | Sample flow rate through sensor
 chip | | **kt** | RU/M·s | Mass transport coefficient (diffusion rate to
 surface) | | **RI** | RU | Refractive index shift from buffer mismatch | |
 **Chi²** | RU² | Fit quality indicator (deviation between data and model)
 | | **U-value** | — | Randomness of residuals (model validity check) |
 **In vitro cell-based data** * \--**Tab Figure 5H 2E8 cell**: 2E8 cells,
 data collected using CCK8 assay (Absorbance at 460 nm).  * **--Sample
 naming:** NEO7-Q6P: Neo7-LD2-Disulfide stapled-R5K-T44I-I45T-(Q6P)/
 NEO7-Q6PT45I: Neo7-LD2-Disulfide stapled-R5K-T44I-(Q6P)/ IL7-WT: Wild type
 IL7 * **--Tab Figure 6D-E spleenocyte** : Data expressed as count of
 cells/100 uL of cell culture.  * **--Sample naming:** Isotype (FC only)
 control: Fc only recombinant protein/ FC-WT-Il7: Fc-wild type IL7 fusion/
 Fc-NEO7-Q6P: Fc-Neo7-LD2-Disulfide stapled-R5K-T44I-I45T-(Q6P) fusion/
 Fc-NEO7-Q6PT45I: Fc-Neo7-LD2-Disulfide stapled-R5K-T44I-(Q6P) fusion. ###
 **In vivo data:** The in vivo data covers all data obtained using mice
 subjects, for GrpahPad Prism 8 XML files readers are recomended to
 visualize the data using GraphPad Prism (version 8 or above). For RNA
 sequencing data, the source data for heatmap generation is included in
 Excel file format. The full report of RNA seq is included as  Chrome HTML
 document format. Readers can assess those files using google chrome.  **In
 vivo data:**  Figure 6F-I N7 variants on mice PBMC: data expressed as
 count of cells per uL of mice peripheral blood Figure7A tumor growth
 curve: tumor volume were expressed in the unit of mm3, body weights were
 expressed in grams.  Figure7B-C TILs analysis: data expressed as
 percentage of the indicated cell population per total cells analyzed. 
 **RNA-seq report:**  -**DGE full table.xlsx:** all gene analyzed from the
 RNA seq data for differential gene expression **-Figure8B source data
 NEO7_RNASEQ_GENEONTOLOGY_LOG2FOLD.pzfx:** Top 10 annotated gene ontology
 that diiferentially expressed comparing the Neo-7 group to Isotype control
 group; Neo-7 group to wild type IL7 group; and wild type IL7 group to
 isotype group. The Neo-7 correspond to the following
 variant: Neo7-LD2-Disulfide stapled-R5K-T44I-(Q6P).  **-Figure8C RNA seq
 Heatmap source data.xlsx :** Sourced data for heat map generation using
 Z-score.  **--Sample name:** Isotype (IST) -1/2/3 ; Neo7-Double
 mutation(N7)-1/2/3; Wild type interleukin-7(I7)-1/2/3 **--Tab:**
 DGE-WT-IST (differential gene expression raw data comparing WT-IL7 and
 isptype control); DGE-N7-WT (differential gene expression raw data
 comparing Neo-7 and WT-IL7); | Column | Meaning | | :--------------- |
 :----------------------------------------------------------------------- |
 | `row.names` | Gene identifier (e.g., gene symbol or Ensembl ID) | |
 `baseMean` | Mean normalized expression of the gene across all samples | |
 `log2FoldChange` | Estimated log₂ fold change between conditions (e.g.,
 treated vs control) | | `lfcSE` | Standard error of the log₂ fold change |
 | `pvalue` | Wald test *p*-value for the null hypothesis (log₂FC = 0) | |
 `padj` | Adjusted *p*-value (Benjamini–Hochberg FDR correction) | Gene
 ontology enrichment analysis from DAVID:  | Column | Meaning | |
 :------------ |
 :----------------------------------------------------------------------------------------------------------------------------------------------------- | | **Sublist** | The subset of genes analyzed (e.g., upregulated, downregulated, or user-defined list). | | **Category** | The database or ontology category, such as **GOTERM\_BP\_DIRECT**, **KEGG\_PATHWAY**, **INTERPRO**, etc. | | **Term** | The specific biological term or pathway name (e.g., “cell cycle,” “oxidative phosphorylation”). | | **RT** | (Optional field — in DAVID this sometimes means “Record Type” or an internal code used for referencing the database term.) | | **Count** | Number of genes from your sublist associated with this term. | | **%** | The percentage of your input genes associated with this term (`Count / total genes × 100`). | | **P-Value** | Raw enrichment *p*-value (usually from a hypergeometric or Fisher’s exact test). | | **Benjamini** | *Benjamini–Hochberg*–corrected *p*-value (false discovery rate, FDR). This column is the one to use for assessing significance (typically **&lt; 0.05**). | **--Tab:** DGE-N7-IST (differential gene expression raw data comparing Neo-7 and isotype control); DGE-FC-WT-N7 (differential gene expression comparison of isotype contol, wild type IL7 and Neo-7 using log2fold change);  Diff-gene (transformation of data from DGE-FC-WT-N7 tab for Z-score calculation, the calculation formula is showed below); Cell cycle (genes related to cell cycle); EFF (genes related to immune effector effects); EXINB (genes related to immune exhaustion inhibition); metabolism (genes related to T-cell metabolism); heatmap (heat map generated based on RNA expression profile); heatmap all (organized heat map generated based on RNA expression profile). Zscore were calculated as using **TPM-normalized RNA-seq data**: Z=(X−μ)/σ * X​ = TPM of **gene ** * μ​ = **mean TPM** across all samples * σ= **standard deviation** of gene TPM values across all samples * Z&gt;0: higher-than-average expression of that gene in that sample * Z&lt;0: lower-than-average expression * Z=0: expression equals the dataset mean for that gene | Column | Meaning | | :------------------------------------- | :---------------------------------------------------------------------------------- | | **Geneid** | Unique gene identifier from annotation file (e.g., GTF). | | **Symbol** | Standard gene symbol (e.g., *IL7R*). | | **Description** | Brief gene function/annotation. | | **EntrezID** | NCBI Entrez Gene ID. | | **Counts\_**\* | Raw read counts per sample (integer values before normalization). | | **TPM\_**\* | “Transcripts Per Million” — normalized expression for comparability across samples. | | **log2FC\_Neo7\_DM\_Isotype\_control** | Log₂ fold change between Neo-7 DM and Isotype Control groups. | | **log2FC\_Neo7\_DM\_WT\_IL7** | Log₂ fold change between Neo-7 DM and WT IL-7 groups. | | **log2FC\_WT\_IL7\_Isotype\_control** | Log₂ fold change between WT IL-7 and Isotype Control groups. | | **padj\_Neo7\_DM\_Isotype\_control** | Adjusted p-value (FDR-corrected) for Neo-7 DM vs Isotype Control. | | **padj\_Neo7\_DM\_WT\_IL7** | Adjusted p-value for Neo-7 DM vs WT IL-7. | | **padj\_WT\_IL7\_Isotype\_control** | Adjusted p-value for WT IL-7 vs Isotype Control. | | **pvalue\_Neo7\_DM\_Isotype\_control** | Raw p-value (unadjusted) for Neo-7 DM vs Isotype Control. | | **pvalue\_Neo7\_DM\_WT\_IL7** | Raw p-value for Neo-7 DM vs WT IL-7. | | **pvalue\_WT\_IL7\_Isotype\_control** | Raw p-value for WT IL-7 vs Isotype Control. | **multiqc_report.html:** The full report of RNA seq in HTML document format. Readers can assess those files using Google Chrome.  ## Code/software -pymol session file (.pse): Pymol (free for academic)  -Biovia discovery studio file (.dsv): Biovia Discovery studio (free version visualizer)  -Microsoft Excel file (.xlsx): Microsoft Excel  -GraphPad Prism 8 XML file: GraphPad Prism (version 8 and above) -Flow cytometry (.fcs) and flowjo workspace (.wsp) file: Flowjo  -Chrome HTML file: any browser ## Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA