Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy
- Institute of Biomedical Sciences, Academia Sinica, Taiwan
- Department of Chemistry, National Taiwan University, Taiwan
Figures
Figure 1
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 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.
Figure 2
Blueprint of Neo-7 design.
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).
Figure 3 with 1 supplement
Validation of Neo-7 designs from Rosetta loop remodeling and fix backbone design.
(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.
Figure 3—figure supplement 1
Yeast cell flow cytometry gating strategy.
Figure 4
Validation of amino acid mutations that confer 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.
Figure 5 with 1 supplement
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 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).
Figure 5—figure supplement 1
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.
Figure 6
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 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.
Figure 7 with 5 supplements
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 follows *p < 0.05; **p = 0.01–0.05; ***p = 0.0001–0.001; and ****p < 0.0001.
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Figure 7—source data 1
Absolute count of immune cells determined from TILs/mg of the tumor excised.
- https://cdn.elifesciences.org/articles/107671/elife-107671-fig7-data1-v1.docx
Figure 7—figure supplement 1
Immune cell profiling of treated mice.
(A) Immune cell composition in the peripheral blood of treated mice. (B) T-cell subset composition in peripheral blood. (C) T-cell subset composition in tumor-infiltrating lymphocytes (TILs).
Figure 7—figure supplement 2
Tumor growth, body weight, and treatment schedule.
(A) Individual tumor growth curves for mice treated with Fc (isotype) control and Fc-Neo-7 variants. (B) Body weight changes of mice in each treatment group throughout the treatment period. (C) Treatment timeline of the study.
Figure 7—figure supplement 3
Tumor growth, body weight, and treatment schedule of mice harboring MC38 tumor treated with the combination of Fc-Neo-7 and oxaliplatin.
(A) Tumor growth curve of mice treated with isotype control, Fc-Neo-7, oxaliplaitn and Fc-Neo-7 + oxaliplatin. (B) Individual tumor growth curves for mice treated with Fc (isotype) control and Fc-Neo-7 variants. (B) Body weight changes of mice in each treatment group throughout the treatment period. (C) Treatment timeline of the study.
Figure 7—figure supplement 4
Gating strategy for flow cytometry studies of immune cell profiling conducted in this study.
Figure 7—figure supplement 5
Absolute count of the tumor-infiltrated immune cells.
Figure 8
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 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).
Tables
Table 1
Site-directed mutagenesis residues recommended by Rosetta to improve the folding stability of the Neo-7s.
| Residue number | Wild type | Mutant | Remarks | Rosetta’s choice |
|---|---|---|---|---|
| 5 | K | R | Cytokine-receptor binding interface | R |
| 6 | Q | P | Cytokine-receptor binding interface | Q |
| 44 | T | I | Cytokine core-surface interface | I |
| 45 | I | T | Borderline of cytokine binding interface | T |
Table 2
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.
| Culture volume | Expression mode | Yield after Ni-NTA purification | Yield after FPLC purification | Purity from FPLC (%) | Thermostability (melting temperature) | |
|---|---|---|---|---|---|---|
| Neo-7 (Q6P) | 200 mL | Soluble | 3.76 | 1.56 | 78.26 | 71.12 |
| Neo-7 (Q6P+T45 I) | 200 mL | Soluble | 1.67 | 0.36 | 58.24 | 70.39 |
| WT-IL7 | 200 mL | Refolding | 7.41 | 0.23 | 23.24 | 56.35 |
Table 3
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.
| Culture volume | Expression host | Yield after protein A affinity purification/100 mL culture | Yield after FPLC purification/100 mL culture | Purity from FPLC curve (%) | |
|---|---|---|---|---|---|
| Neo-7-Fc | 130 mL | CHO | 15.73 | 10.44 | 80.88 |
| Neo-7 T43I-Fc | 130 mL | CHO | 16.66 | 6.42 | 55.15 |
| WT-IL7-Fc | 130 mL | CHO | 17.22 | 3.37 | 31.84 |
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Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy
eLife 14:RP107671.
https://doi.org/10.7554/eLife.107671.3
