Fig 1.
Sequences and schematic structures of the compounds.
(A) Amino acid sequences of MDK1248, MDK1188, and MDK1472 with intrachain disulfide bonds shown as dashed lines. Schematic representations of (B) peptide MDK1472 and (C) IgG2Fc-1472 fusion MDK-703.
Fig 2.
Binding of MDK1472, MDK-703, and component peptides to human and cynomolgus IL-7R subunits.
(A, B) Determination of IC50 values in a competition ELISA for compound binding to human and cyno IL-7Rα and γc. The assay format is IL-7Rα-(His)6-tagged ECD or Fc-γc ECD immobilized on 96-well plates. Tracers are C-terminal biotinylated forms of the reference IL-7Rα and γc peptide ligands, each pre-complexed with Neutravidin-HRP (NA-HRP). (C, D) Label-free measurement of the equilibrium dissociation constant (KD) for peptide binding to IL-7Rα and γc extracellular domains with peptides was performed by biolayer interferometry as detailed in methods. Red lines depict 1:1 binding fit from Gator™ software. Blue lines represent individual BLI sensogram data taken every 0.1 seconds. Inset table displays comparison of ELISA data and BLI kinetic data.
Fig 3.
IL-7Rα dependence of agonist activity induced by MDK1472 and MDK-703 in TF-1 and TF1-7Rα cells.
Compound-induced STAT5 phosphorylation in (A) TF-1 (γc+) and (B) TF1-7Rα (IL-7Rαγc+) cells. (C) pSTAT5 accumulation in TF1-7Rα cells treated with IL-7 or MDK-703 in the presence of an IL-7 neutralizing antibody. Cells were pre-incubated for 15 min with 1 pM to 30 nM anti-IL-7, and test compounds were then added at concentrations equivalent to EC75 and incubated for an additional 30 min. Reactions were stopped and cell lysates prepared for pSTAT5 analysis.
Fig 4.
Effects of MDK1472, MDK-703, and their subdomain peptides on activation of receptors sharing subunits with IL-7R.
(A) pSTAT5 dose response of IL-7 and the test compounds in TF1-7Rα cells (10 μM MDK1188 or MDK1248 alone indicated by arrow). (B) Response to IL-7 in the presence of 10 μM of each of the subunit-binding peptides MDK1248 and MDK1188 (the peptide fragments of MDK1472, which bind to IL-7Rα and γc, respectively). (C) Dose-response of IL-2, IL-4, IL-9, IL-15, and IL-21-induced STAT activation in TF-1 cells expressing the respective α or β receptor subunits. Inhibition by (D) MDK1472, (E) MDK-703, of the activation of each member of the γc receptor family by its respective cytokine agonist, and (F) IC50 of MDK1188 on IL-2. (G) Dose responses of IL-7 and MDK-703 induction of pSTAT5 in TF-1-7Rα/TSLPR cells, and MDK-703 in the presence or absence of MDK2058, a high affinity (IC50 = 300pM) competitive inhibitor of MDK-703 binding to the γc subunit. (H) Dose response of TSLP induction of pSTAT5 in TF-1-7Rα/TSLPR cells in the presence or absence of 20 μM MDK2058 and 600 nM MDK-703.
Fig 5.
MDK1472 and MDK-703 behave as full agonists in activating the JAK-STAT5 pathway.
Naive CD4+ and CD8+ T-cells, were separately isolated by negative selection from PBMCs, and rested overnight prior to compound exposure. (A) Cells were treated with saturating concentrations of IL-7 (10 nM), MDK1472 (1 μM), and MDK-703 (1 μM) for up to 2 hours and scored for pSTAT5 accumulation by ELISA. (B) The dose response for each compound was measured in human naive CD4+ and CD8+ cells, utilizing an exposure time of 15 min (Tmax).
Fig 6.
Induction of pSTAT5 in PBMCs treated with MDK-703 or IL-7.
Frozen PBMCs from 5 healthy human donors (A, B) and cynomolgus monkey (C) were rested (A, C) or activated with anti-CD3/CD28 (B). Cells were stained for viability, followed by cell surface antibody staining on ice. Cells were washed and incubated with MDK-703 or IL-7 for 30 min at 37°C to activate the IL-7 receptor. After washing, cells were fixed, permeabilized, stained with anti-pSTAT5 antibody, and analyzed immediately by flow cytometry. The mean fluorescence intensity (MFI) of pSTAT5 detection was shown as mean ± SEM. (D) EC50 values of MDK-703 vs. IL-7 in immune subsets in human and cynomolgus PBMCs are shown in S1 Table. Flow cytometry gating data shown in S2 Fig.
Fig 7.
Proliferation of PBMC subpopulations following treatment with MDK-703 or IL-7.
Frozen PBMCs from 5 healthy donors were rested overnight and treated with 100 nM MDK-703 or 1nM IL-7, or no added compound, and cultured for 30 days in the presence of compounds. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry for (A) Ki-67 expression and (B) absolute numbers of CD8+, CD4+, Treg, and NK cells. Data are shown as mean ± SEM. Flow cytometry gating data shown in S2 Fig.
Fig 8.
Test compounds induce decrease in cell surface IL-7Rα. Rested human CD8+ T-cells were treated with saturating concentrations of the test agonists (10 nM IL-7; 1 μM MDK1472 or MDK-703) or the negative control compounds (untreated, 1 μM MDK1169, or MDK-202) for 20 minutes on ice, then incubated at (A) 37°C or (B) 0°C for varying times to monitor uptake of IL-7Rα from the cell surface. Following timed incubations, the samples were stained, fixed, and analyzed by flow cytometry; and data was collected as median fluorescence intensity (MFI), as detailed in Methods. These data were normalized with the blank value (no added compound) set at 100% surface IL-7Rα, and the signal baseline set at 0. The primary flow data (MFI) is shown in S3 Fig.
Fig 9.
The expansion of CD8+ T memory subpopulations following treatment with MDK-703 or IL-7.
Frozen PBMCs from 5 healthy donors were rested overnight and left untreated or treated with 100 nM MDK-703 or 1 nM IL-7 in culture. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry of CD8+ T naïve and memory populations. (A) Schematic of the putative differentiation pathway of the memory T cell compartment. (B) differential gating of naïve and early memory (Tscm) subpopulations. (C) Cell counts of treated and untreated memory subpopulations over time. (D) Stacked bar representation of changes in total memory T cell subpopulations over time. (E) Comparison of CD8+ Tscm expansion on day 16 and day 30 of culture with MDK-703 or IL-7. Data are shown as mean ± SEM. Detailed gating information is provided in S4 Fig.
Fig 10.
Expansion of immune cell subpopulations in humanized mice treated with MDK-703.
NSG mice (n = 10 per treatment) engrafted with human CD34+ cells from two donors (5 mice/donor) were dosed once intravenously with 1 mg/kg Fc (gray bars) or MDK-703 (blue bars), and peripheral blood (B-D) and spleen (E-G) were collected and analyzed at the indicated times by flow cytometry. (A) Diagram of the experimental plan. (B) Frequencies of Ki-67+ in CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 7. (C) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 12. (D) CD8+ T memory subpopulations in peripheral blood on day 12. (E) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in the spleen on day 12. (F) CD8+ T memory subpopulations in the spleen on day 12. (G) TCF1 expression in CD8+ T memory subpopulations in the spleen on day 12. Population gates were drawn based on FMO controls. Statistical analysis was done using Student’s T-Test. *p<0.05, **p<0.005, and ***p<0.0005. Detailed gating information is provided in S6 Fig.
Fig 11.
Pharmacokinetic (PK) and pharmacodynamic (PD) properties of MDK-703 in cynomolgus macaques.
(A) PK of MDK-703: Animals (n = 3) were administered a single dose of 1 mg/kg MDK-703 via IV, SC, or IM. The serum concentration of MDK-703 at the indicated time points was determined by sandwich ELISA. (B and C) PD effect of MDK-703: Three animals were dosed once subcutaneously with 0.3 mg/kg, and blood samples were collected at the indicated time points for absolute lymphocyte counts (B) and immune profiling of CD8, CD4, Treg, and NK cells by flow cytometry (C). Data show mean ±SEM.