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.

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).

Validation of Neo-7 designs from Rosetta loop remodeling and fix backbone design.

(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.

Site-directed mutagenesis residues recommended by Rosetta to improve the folding stability of the Neo-7s.

Validation of amino acid mutations that confers to the binding affinity of Neo-7 towards IL-7 receptor alpha and gamma.

(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.

Characterization of E. coli expressed IL-7 and Neo-7s and the in vitro biological activities.

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 UnicornTM 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.

Yield, purity and thermostability data of the E. coli expressed IL-7/Neo-7s, yield was presented as mg of recombinant protein per 200 mL of E. coli culture.

Characterization of CHO-S cells expressed FC-fused cytokines and their in vitro and in vivo biological activities.

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 UnicornTM 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.

Yield and purity data of the CHO-S expressed Fc-fused IL-7/Neo-7s, yields were presented as mg of recombinant protein per 100 mL of CHO cell culture.

In vivo anti-tumor activity of Fc-fused WT-IL7/Neo-7s towards MC38 syngeneic models.

(A) Tumor growth curve of MC38 tumor after treatment with the Fc-fused cytokines. Proportion of total leukocytes and different immune cell types within the MC38 tumor of mice at day 7 post-treatment. (B) Proportion of intratumoral CD4+ and CD8+ T-cells (C) Proportion of other immune cell types present in the TME at day-7 post treatment. All data were presented as individual data plots with error bars (SEM). Statistical differences among groups were determined using two-way ANOVA and one-way ANOVA with Turkey’s multiple comparison test for tumor growth curve and TILs analysis respectively. Significance levels are defined as follow *p < 0.05; **p = 0.01–0.05; ***p = 0.0001–0.001; and ****p < 0.0001.

RNA-sequencing of splenic CD8+ T-cells isolated from Fc-IL7/Neo-7s treated mice.

(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.