METHOD FOR MONITORING CATHEPSIN S INHIBITION

Abstract

The present invention relates to a method for monitoring Cathepsin S inhibitor activity in tissue samples of animals comprising: a) providing a tissue sample of an animal to whom a Cathepsin S inhibitor has been administered or providing a tissue sample of an animal that was contacted in vitro with a Cathepsin S inhibitor, wherein the tissue samples comprise white blood cells, b) measuring li p10 peptide level in white blood cells of the tissue sample of step a) by flow cytometry and c) correlating li p10 peptide level in white blood cells to Cathepsin S inhibitor dose, wherein a Cathepsin S inhibitor leads to increased level of li p10 peptide in the white blood cells.

Claims

1. A method for monitoring Cathepsin S inhibitor activity in a tissue sample of animals comprising: a) providing a tissue sample of an animal to whom a Cathepsin S inhibitor has been administered or providing a tissue sample of an animal that was contacted in vitro with a Cathepsin S inhibitor, wherein the tissue sample comprise white blood cells; b) measuring lip10 peptide level in white blood cells of the tissue sample of step a) by flow cytometry; and c) correlating li p10 peptide level in white blood cells to Cathepsin S inhibitor dose, wherein a Cathepsin S inhibitor leads to increased level of li p10 peptide in white blood cells.

2. A method for the identification of a Cathepsin S inhibitor comprising: a) contacting a non-human animal with a test compound; b) providing a tissue sample of the animal of step a) comprising white blood cells; and c) measuring li p10 peptide level in white blood cells of the tissue sample of step b) by flow cytometry; wherein an increased level of li p10 peptide in white blood cells in the sample of step a) compared to a level of li p10 peptide in white blood cells in a tissue sample of an untreated animal is indicative for a Cathepsin S inhibitor.

3. The method of claim 1 wherein the tissue sample is blood.

4. The method of claim 2 wherein the tissue sample is blood.

5. The method of claim 1, wherein the white blood cells are Antigen Presenting Cells (APCs).

6. The method of claim 2, wherein the white blood cells are Antigen Presenting Cells (APCs).

7. The method of claim 5, wherein the APCs are selected from B cells and monocytes.

8. The method of claim 6, wherein the APCs are selected from B cells and monocytes.

9. The method of claim 1 wherein the animal is a human being.

10. The method of claim 2, wherein the non-human animal is a rodent.

11. The method of claim 1, wherein the white blood cells are permeabilized to measure li p10 peptide by flow cytometry.

12. The method of claim 2, wherein the white blood cells are permeabilized to measure li p10 peptide by flow cytometry.

13. The method of claim 1, wherein an antibody directed to li p10 is used in flow cytometry to measure li p10 in white blood cells.

14. The method of claim 2, wherein an antibody directed to li p10 is used in flow cytometry to measure li p10 in white blood cells.

15. An anti li p10 antibody comprising a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5, CDR3 sequence comprising the amino acid sequence of SEQ ID NO:6., and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:8, a CDR3 sequence comprising the amino acid sequence of SEQ ID NO:9.

16. The anti li p10 antibody of claim 9, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:10 and the VL domain comprises the amino acid sequence of SEQ ID NO:11.

17. The method of claim 13, wherein said antibody is the antibody of claim 15.

18. The method of claim 13, wherein said antibody is the antibody of claim 16.

19. The method of claim 14, wherein said antibody is the antibody of claim 15.

20. The method of claim 14, wherein said antibody is the antibody of claim 16.

Description

SHORT DESCRIPTION OF THE FIGURES

[0034] FIG. 1: Detection of Ii p10 expression on B cells and T cells using a neoepitope antibody. PBMCs isolated from blood donation of a healthy donor (R213) were incubated for 20 h with increasing concentrations of Cathepsin S inhibitor 1 or vehicle. Ii p10 detection on B cells (CD20+) and T cells (CD3+) was determined by flow cytometry using a different neoepitope antibody 7B8 (black).

[0035] FIG. 2: Detection of Ii p10 expression on B cells using a neoepitope antibody. PBMCs isolated from blood donation of a healthy donor (R172) were incubated for 20 h with increasing concentrations of Cathepsin S inhibitor 1 or vehicle. Ii p10 detection on B cells (CD20+) and T cells (CD3+) was determined by flow cytometry using two different neoepitope antibodies, namely 7B8 (black) and 13C4 (grey). Stain index=Median Fluorescence Intensity (MFI).sub.B cells/MFI.sub.Tcells

[0036] FIG. 3 A: Ii p10 accumulates in B cells upon treatment with Cathepsin S inhibitors. PBMCs isolated from blood donations of eleven healthy donors (R39; R185; R198; R204; R209; R140; R154; R175; R213; R4;R214) were incubated for 20 h with increasing concentrations of Cathepsin S inhibitor 1 or vehicle. Ii p10 detection on B cells (CD20+) and T cells (CD3+) was determined by flow cytometry using 7B8 neoepitope antibody. Stain index=MFI.sub.B cells/MFI.sub.Tcells.

[0037] FIG. 3 B: Ii p10 accumulates in B cells upon treatment with Cathepsin S inhibitors. PBMCs isolated from blood donations of eleven healthy donors (R39; R185; R198; R204; R209; R140; R154; R175; R213; R4;R214) were incubated for 20 h with increasing concentrations of Cathepsin S inhibitor 2 or vehicle. Ii p10 detection on B cells (CD20+) and T cells (CD3+) was determined by flow cytometry using 7B8 neoepitope antibody. Stain index=MH.sub.B cells/MFI.sub.Tcells.

[0038] FIG. 4: Cathepsin S inhibitor IC50 concentrations. PBMCs isolated from blood donations of eleven healthy donors (R39; R185; R198; R204; R209; R140; R154; R175; R213; R4;R214) were incubated for 20 h with increasing concentrations of Cathepsin S inhibitor 1, Cathepsin S inhibitor 2 or vehicle. Ii p10 detection on B cells (CD20+) and T cells (CD3+) was determined by flow cytometry using 7B8 neoepitope antibody. Response curves and IC50 values have been calculated with GraphPad Prism software.

[0039] FIGS. 5 A-F: Ii p10 neoepitope assay longitudinal variability. PBMCs isolated from blood donations of eleven healthy donors (R198; R209; R154; R213; R214) at two time points and were incubated for 20 h with increasing concentrations of Cathepsin S inhibitor 1, or vehicle. Ii p10 detection on B cells (CD20+) and T cells (CD3+) was determined by flow cytometry using 7B8 neoepitope antibody.

[0040] FIG. 6 A: Ii p10 accumulation in B cells is detected by the neoepitope assay. Ii p10 detection on B cells (CD20+) was determined by flow cytometry using 7B8 neoepitope antibody clone. PBMCs isolated from 3 cynos (Macaca fascicularis) blood donations were incubated for 20 h with increasing concentrations of Cathepsin S inhibitor 1 or vehicle. The p10 stain index of Macaca fascicularis samples was determined by dividing the Median Fluorescence Intensity (MFI) of B cells by the MFI of Forward Scatter Low CD20 negative (FSCloCD20) cells.

[0041] FIG. 6 B: Ii p10 accumulation in B cells is detected by the neoepitope assay after a single oral dose of Cathepsin S inhibitor. Ii p10 detection on B cells (CD20+) was determined by flow cytometry using 7B8 neoepitope antibody clone. Blood was collected from animals 2 h and 7 h after a single dose of 50 mg/kg. Ii p10 neoepitope assay was performed in whole blood. The p10 stain index of Macaca fascicularis samples was determined by dividing the Median Fluorescence Intensity (MFI) of B cells by the MFI of Forward Scatter Low CD20 negative (FSCloCD20) cells.

[0042] FIG. 7A-7D: Quantification of p10 on samples obtained from subjects participating in a single center, randomized, double-blind, placebo-controlled, single ascending dose study to evaluate the safety, tolerability, pharmacokinetic and pharmacodynamic effects of single doses of Cathepsin inhibitor 1 in healthy male and female volunteers. To account for and limit the impact of the expected inter-subject variability as observed in preclinical experiments, an interleaved cohort (leapfrog) design was employed. Subjects were recruited into two cohorts of eight subjects each (Cohorts A and B). For each individual within a cohort, the study was a randomized, placebo controlled, four treatment, four periods, four-way crossover. The single dose applied shortly after time point 0 hours is indicated on each FIG. 7A to 7D (cohort A) and FIG. 8A to 8D (cohort B).

[0043] FIG. 8A-8D: Cohort B data, see FIG. 7 description.

EXPERIMENTAL PART

[0044] In Vitro Assay with Cathepsin S Inhibitor [0045] Use a sterile Polystyrene 24 well plate [0046] Add 500 l complete RPMI+Cathepsin S inhibitor with increasing concentrations (0 to 10 M) [0047] Add 500 l of PBMC suspension (210.sup.6) to each well [0048] Mix the content of the wells and incubate the plate for 20 h at +37 C., 5% CO2 in a humidified incubator

FACS Staining:

[0049] prepare 1 lyse/fix solution [0050] add 4 mL of pre-warmed 1 lyse/fix solution per 210.sup.6 PBMCs or 200 l blood [0051] incubate for 10 min at 37 C, wash the cells 1 with PBS (250g, 5 minutes, RT), remove supernatant [0052] add 2 mL of 1 permeabilising solution per tube [0053] incubate for 20 min at RT [0054] wash the cells 2 with 2 ml Cell Stain Buffer (250g, 5 minutes, RT), remove supernatant [0055] add 10 g/mL unlabeled anti-p10 antibody in Cell Staining Buffer for 1 h at RT [0056] wash the cells 1 with 2 ml Cell Stain Buffer (250g, 5 minutes, RT), remove supernatant [0057] add 1:125 PE-conjugated goat anti-rabbit IgG antibody in Cell Staining for 30 min at RT in the dark [0058] wash the cells 2 with 2 ml Cell Stain Buffer (250g, 5 minutes, RT), remove supernatant [0059] add the antibodies (comp. control) or antibody mixture (diluted in 100 l/sample Cell Stain Buffer) to each tube [0060] incubate for 20 min at +4 C. in the dark [0061] wash the cells 1 with 2 ml Cell Stain Buffer (250g, 5 minutes, RT), remove supernatant [0062] resuspend pellet in 200 l Cell Stain Buffer and keep the cells in the dark until the analysis on the FACS

[0063] Antibodies:

TABLE-US-00001 goat anti-rabbit IgG PE SouthernBiotech Cat# 4050-09 1:125/sample CD3 Brilliant Violet 421 BioLegend Cat# 300434 5 l/sample CD14 APC Beckman Coulter Cat# IM2580 5 l/sample CD20 AlexaFluor 488 BD Biosciences Cat# 558056 20 l/sample CD45 PerCP/Cy5.5 BioLegend Cat# 304028 2 l/sample

[0064] Materials and Buffers:

[0065] 24-well plates, BD, order ID: 351147Storage: room temperature, sterile, polystyrene non tissue culture treated

[0066] 10 BD Phosphoflow Lyse/Fix buffer lysis buffer (BD, order ID: 558049). Storage: +4 C. Dilute the 5 BD Phosphoflow Lyse/Fix buffer with H.sub.2O bidest. to 1 and prewarm it to the required temperature prior to use 10 Phosflow Perm Buffer IV permeabilisation buffer (BD, order ID: 560746). Storage at RT. Dilute 10 BD Phosphoflow Perm Buffer IV with PBS to 1.

[0067] 1 Cell Satining Buffer (BioLegend, order ID: 420201. Storage: +4 C the 1 Cell Satining Buffer is ready to use. 1 PBS without CaCl.sub.2 and without MgCl.sub.2 (Gibco, order ID: 14190-094). Storage: room temperature. The PBS is ready to use. 5 ml Polystyrene round-bottom tube (FACS tube, BD, order ID: 352052). Storage: room temperature.

Amino Acid Sequences

[0068]

TABLE-US-00002 Sequence name Seq. Id. No. Human Cathepsin S 1 Human invariant chain li 2 Human li p10 3 VH CDR 1 of anti li p10 antibody 7B8 4 VH CDR 2 of anti li p10 antibody 7B8 5 VH CDR 3 of anti li p10 antibody 7B8 6 VL CDR 1 of anti li p10 antibody 7B8 7 VL CDR 2 of anti li p10 antibody 7B8 8 VL CDR 3 of anti li p10 antibody 7B8 9 VH chain of anti li p10 antibody 7B8 10 VL chain of anti li p10 antibody 7B8 11