METHOD FOR DETERMINING THE AMOUNT OF A THERAPEUTIC ANTIBODY IN THE BRAIN

Abstract

Herein is reported a method for determining the concentration of a therapeutic antibody in a tissue of an experimental animal to whom the therapeutic antibody had been administered, wherein the interference from residual blood in a tissue sample of the experimental animal, which is used for determining the concentration of the therapeutic antibody in said tissue, is reduced, wherein the concentration of the therapeutic antibody in the tissue of the experimental animal is calculated with the following formula:

[00001]$C_{\mathrm{tmAb},\mathrm{tissue}}=\frac{C_{tmAb,\mathrm{tissue},\det .}}{C_{tis\mathrm{sue},\mathrm{sample}}}-\frac{\frac{C_{refmAb,\mathrm{tissue},\det .}}{C_{tis\mathrm{sue},\mathrm{sample}}}}{C_{refmAb,\mathrm{plasma}.\det .}}*C_{tmAb,\mathrm{plasma},\det }.$

wherein C.sub.tmAb,tissue,det.=obtained by determining the concentration of the therapeutic antibody in the tissue sample of the experimental animal, C.sub.tmAb,plasma,det.=obtained by determining the concentration of the therapeutic antibody in a blood sample of the experimental animal directly prior to taking the tissue sample, C.sub.refmAb,tissue,det.=obtained by determining the concentration of the inert reference antibody in the tissue sample of the experimental animal, C.sub.refmAb,plasma,det.=obtained by determining the concentration of an inert reference antibody in the blood sample of the experimental animal directly prior to taking the tissue sample, C.sub.tissue,sampie=obtained by determining the tissue concentration in the tissue sample, whereby the inert reference antibody does not penetrate into said tissue, whereby the inert reference antibody is administered 2 to 10 minutes prior to obtaining the tissue and blood sample.

Claims

1. A method or assay for determining the concentration of a therapeutic antibody in a tissue of an experimental animal, whereby the tissue has a barrier to the blood circulation of said animal and whereby the therapeutic antibody had been administered to said experimental animal, wherein the interference from residual blood in a tissue sample of the experimental animal, which is used for determining the concentration of the therapeutic antibody in said tissue, is reduced, the method comprising the following steps i) determining the concentration of the therapeutic antibody in a blood sample of the experimental animal, ii) determining the concentration of the therapeutic antibody in the tissue sample of the experimental animal, iii) determining the concentration of an inert reference antibody in the blood sample of the experimental animal, iv) determining the concentration of the inert reference antibody in the tissue sample of the experimental animal, v) determining the tissue concentration in the tissue sample, and determining the concentration of the therapeutic antibody in the tissue of the experimental animal with the following formula: $$\begin{gathered} C_{\mathrm{tmAb},\mathrm{tissue}}=\frac{C_{tmAb,\mathrm{tissue},\det .}}{C_{tis\mathrm{sue},\mathrm{sample}}}-\frac{\frac{C_{refmAb,\mathrm{tissue},\det .}}{C_{tis\mathrm{sue},\mathrm{sample}}}}{C_{\mathrm{refmAb},\mathrm{plasma}.\det .}}*C_{tmAb,\mathrm{plasma},\det }.\mathrm{with}{C}_{\mathrm{tmAb},\mathrm{plasma},\det .}=\mathrm{concentration}\mathrm{of}\mathrm{the}\mathrm{therapeutic}\mathrm{antibody}\mathrm{of}i)C_{\mathrm{tmAb},\mathrm{tissue},\det .}=\mathrm{concentration}\mathrm{of}\mathrm{the}\mathrm{therapeutic}\mathrm{antibody}\mathrm{of}\mathrm{ii})C_{\mathrm{refmAb},\mathrm{tissue},\det .}=\mathrm{concentration}\mathrm{of}\mathrm{the}\mathrm{therapeutic}\mathrm{antibody}\mathrm{of}\mathrm{iii}) \\ {C}_{\mathrm{refmAb},\mathrm{plasma},\det .}=\mathrm{concentration}\mathrm{of}\mathrm{the}\mathrm{therapeutic}\mathrm{antibody}\mathrm{of}\mathrm{iv}) \\ {C}_{\mathrm{tissue},\mathrm{sample}}=\mathrm{tissue}\mathrm{concentration}\mathrm{of}v) \end{gathered}$$ whereby the inert reference antibody does not cross said barrier between the tissue and the blood circulation, whereby the inert reference antibody had been administered i) either together with the therapeutic antibody in case the sample is to be taken within 5 minutes after the administration of the therapeutic antibody, or ii) 2 to 10 minutes prior to taking the tissue sample, whereby the blood sample is taken directly prior to the tissue sample.

2. The Method according to claim 1, wherein the tissue is either brain tissue and the therapeutic antibody can cross the blood-brain-barrier or ocular tissue and the therapeutic antibody can cross the blood-ocular-barrier.

3. The method according to any one of claims 1 to 2, wherein the therapeutic antibody is a bispecific antibody.

4. The method according to any one of claims 1 to 3, wherein the therapeutic antibody is specifically binding to human transferrin receptor and a brain target.

5. The method according to claim 4, wherein the brain target is human CD20 or human Abeta or human alpha-synuclein or human tau or human glucocerebrosidase or human lingo-1 or human huntingtin.

6. The method according to any one of claims 1 to 5, wherein the experimental animal is selected from mouse, rat, rabbit, dog, sheep, ape, and monkey.

7. The method according to any one of claims 1 to 6, wherein the experimental animal is a non-human experimental animal with a body weight of more than 100 g and less than 15 kg.

8. The method according to any one of claims 1 to 7, wherein the experimental animal is a cynomolgus monkey.

9. The method according to any one of claims 1 to 8, wherein the inert reference antibody is a human germline antibody.

10. The method according to any one of claims 1 to 9, wherein the inert reference antibody is DP47GS.

11. The method according to any one of claims 1 to 10, wherein the inert reference antibody does not cross said barrier in detectable amounts within 15 minutes after its application.

12. The method according to claim 11, wherein the inert reference antibody does not cross said barrier in detectable amounts within 10 minutes after its application.

13. The method according to any one of claims 1 to 12, wherein the inert antibody is administered about 5 minutes prior to taking the tissue sample.

14. The method according to any one of claims 1 to 10, wherein the tissue is perfused with an aqueous solution directly after taking the blood sample and prior to taking the tissue sample.

15. The method according to any one of claims 1 to 11, wherein the determining of the concentrations is by a bridging ELISA.

Description

DESCRIPTION OF THE FIGURES

[0454] FIG. 1 Exemplary calculation for determining residual-plasma-corrected brain lysate concentration of a tmAb.

[0455] FIG. 2 Structure of the exemplary brain-shuttle construct used in the examples of the method according to the invention.

[0456] FIG. 3 Detection assay for therapeutic antibody and reference antibody used in the Examples.

[0457] FIG. 4 Overlay of the calibration curves of the detection assay according to Example 1 for the inert reference antibody in the presence of 1% cynomolgus and mouse, respectively, brain lysate.

[0458] FIG. 5 Ratio of corrected concentration to non-corrected concentration of antibody 2 in mouse brain lysate.

[0459] FIG. 6 ELISA for the determination of antibody 2.

[0460] FIG. 7 Calibration curve of complement factor H Elecsys assay.

[0461] FIG. 8 ELISA for the quantification of α2-macroglobulin in cCSF. The capture antibody is a murine anti-human α2-macroglobulin antibody; the detection antibody is a biotinylated goat anti-human α2-macroglobulin antibody.

[0462] FIG. 9 Calibration curve of the ELISA of Figure Z.

[0463] FIG. 10 Calibration curve of complement component 5a ELISA assay.

[0464] FIG. 11 Time course of Magnevist in PK study in plasma and brain tissue.

[0465] FIG. 12 ELISA for the quantification of cynomolgus IgG in cCSF. The capture mAb is an anti-cyno IgG antibody; the detection mAb is an anti-cyno IgG antibody binding to a not interference epitope with respect to the first antibody.

[0466] FIG. 13 Calibration curve of the ELISA of Figure X.

General Methods

Preparation of Cynomolgus Brain Tissue Homogenates

[0467] Frozen cynomolgus/mouse brain tissue samples of 300 mg were thawed at room temperature for 2 h. 800 μL of lysis buffer, one tablet of cOmplete protease inhibitor cocktail (Roche Diagnostics GmbH) dissolved in 50 mL Tissue Extraction Reagent I (Invitrogen), were added to the thawed brain tissue. Next, the sample was homogenized in a MagNA Lyser instrument (Roche Diagnostics) for 20 seconds at 6500 rpm. The tissue homogenate was then centrifuged for 10 minutes at 12,000 rpm using a Centrifuge 5430 (Eppendorf). Finally, the supernatant was transferred to a 1.5 mL vial for further analysis or stored at −80° C.

Example 1

ELISA for the Quantification of DP47GS-PGLALA in Brain Lysates

[0468] To quantify the inert reference monoclonal antibody DP47GS-PGLALA (SEQ ID NO: 69 and 70) in cynomolgus brain lysate samples a serial sandwich enzyme linked immunosorbent assay (ELISA) was used. In the ELISA procedure, all samples and controls are subjected to an initial 1:100 pre-dilution in Assay Diluent to the desired 1% final assay concentration.

[0469] Capture Antibody (anti-DP47GS antibody, biotinylated), diluted Calibrators (DP47GS-PGLALA) as well as diluted Quality controls and samples, detection reagent (anti-PGLALA antibody clone M-1.7.24, digoxygenylated) and anti-digoxygenin-antibody-POD-conjugate are added successively to a streptavidin coated microtiter plate (SA-MTP). The reagents were incubated for 1 hour on a MTP shaker at 500 rpm and after each step the MTP was washed three times with 300 washing buffer (1×PBS, 0.05% Tween) and residual fluids were removed. After that, the formed immobilized immune complexes were visualized by addition of ABTS solution, a horseradish POD substrate, which was converted to a colored reaction product. Finally, the color intensity was photometrically determined (absorption at 405 nm-490 nm reference wavelength). The signal is proportional to the analyte concentration in the brain lysate sample. The quantification of DP47GS-PGLALA was performed by back calculation of the absorbance values using the corresponding calibration curve with a non-linear 4-parameter Wiemer-Rodbard curve fitting function with weighting.

TABLE-US-00010 brain lysate assay concentration concentration DP47GS- DP47GS- PGLALA PGLALA Quality control Calculation [ng/mL] [ng/mL] ULQC highest calibrator 250 2.5 Upper limit of quantification HQC highest calibrator × 185 1.85 High range 0.75 MQC geometric mean of 80 0.80 Medium range HQC and LQC LQC LLQC × 3 25 0.25 Low range LLQC lowest calibrator 8 0.08 Lower limit of quantification

[0470] Coating with capture reagent is achieved by pipetting 100 μL of a solution comprising 500 ng/mL biotinylated anti-DP47GS antibody into each SA-MTP well. Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05% Tween). Residual washing buffer is carefully removed.

[0471] Then 100 μl of the respective calibrators, quality controls and samples to the designated wells of the coated MTP. Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer is carefully removed.

[0472] Then 100 μL of digoxygenylated anti-PGLALA antibody clone M-1.7.24 at a concentration of 125 ng/mL is added to each MTP well. Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer is carefully removed.

[0473] The 100 μL of an anti-digoxygenin antibody-POD-conjugate at a concentration of 50 mU/mL is added to each MTP well. Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer is carefully removed.

[0474] Then 100 μL ABTS solution is added to each MTP well. The optical density is measured until the average signal of the duplicates of Calibrator sample 1 reaches 1.8-2.2 AU at a measuring wavelength of 405 nm (reference wavelength 490 nm).

Example 2

ELISA for the Quantification of Cynomolgus IgG in CSF

[0475] To quantify cynomolgus IgG in cynomolgus cerebrospinal fluid a serial sandwich enzyme linked immunosorbent assay (ELISA) was used. In the ELISA procedure, all samples and controls are subjected to an initial pre-dilution in Assay Diluent to the desired 1% final assay concentration.

[0476] Capture Antibody (anti-cynomolgus IgG antibody 1; epitope 1; biotinylated), diluted Calibrators as well as diluted Quality controls and samples, detection reagent (anti-cynomolgus IgG antibody 2; epitope 2, not interfering with epitope 1; digoxygenylated) and anti-digoxygenin-antibody-POD-conjugate are added successively to a streptavidin coated microtiter plate (SA-MTP). The reagents were incubated for 1 hour on a MTP shaker at 500 rpm and after each step the MTP was washed three times with 300 μL washing buffer (1×PBS, 0.05% Tween) and residual fluids were removed. After that, the formed immobilized immune complexes were visualized by addition of ABTS solution, a horseradish POD substrate, which was converted to a colored reaction product. Finally, the color intensity was photometrically determined (absorption at 405 nm-490 nm reference wavelength). The signal is proportional to the analyte concentration in the brain lysate sample. The quantification of cynomolgus IgG was performed by back calculation of the absorbance values using the corresponding calibration curve with a non-linear 4-parameter Wiemer-Rodbard curve fitting function with weighting.

TABLE-US-00011 cCSF assay concentration concentration cynomolgus cynomolgus Quality control Calculation IgG [ng/mL] IgG [ng/mL] ULQC highest calibrator 12000 120 Upper limit of quantification HQC highest calibrator × 9000 90 High range 0.75 MQC geometric mean 45000 45 Medium range of HQC and LQC LQC LLQC × 3 2200 22 Low range LLQC lowest calibrator 720 7.2 Lower limit of quantification

[0477] Coating with capture reagent is achieved by pipetting 100 μL of a solution comprising 250 ng/mL biotinylated anti-cynomolgus IgG antibody 1 into each SA-MTP well. Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer is carefully removed.

[0478] Then 100 μl of the respective calibrators, quality controls and samples to the designated wells of the coated MTP Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer is carefully removed.

[0479] Then 100 μL of digoxygenylated anti-cynomolgus antibody 2 at a concentration of 250 ng/mL is added to each MTP well. Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer is carefully removed.

[0480] The 100 μL of an anti-digoxygenin antibody-POD-conjugate at a concentration of 25 mU/mL is added to each MTP well. Thereafter the MTP is covered with adhesive cover foil and incubated for 1 hour on a MTP shaker (500 rpm). The supernatant is removed and each well of the MTP is washed three times with 300 μL washing buffer (PBS, 0.05 Tween). Residual washing buffer is carefully removed.

[0481] Then 100 μL ABTS solution is added to each MTP well. The optical density is measured until the average signal of the duplicates of Calibrator sample 1 reaches 1.8-2.2 AU at a measuring wavelength of 405 nm (reference wavelength 490 nm).

Example 3

Production of Brain Tissue Lysates

[0482] First, the lysis buffer was freshly prepared according to the manufacturer's instructions (Invitrogen; tissue extraction reagent I; Cat.-No. FNN0071). Per 50 ml of lysis buffer 1 tables of Complete is added (Roche Diagnostics GmbH, Mannheim, Germany; Cat.-No. 11697498001).

[0483] Second, to the brain tissue sample, approx. 100-300 mg, between 600 μL and 800 were lysis buffer is added. Optionally MagNA Lyser Green Beads are added.

[0484] Third, the samples were placed for 20 sec. at 6500 rpm in the MagNA Lyser (Roche Diagnostics GmbH, Mannheim, Germany).

[0485] Fourth, after incubation in the MagNA Lyser the samples are centrifuged for 10 min. at 12,000 rpm (Eppendorf Centrifuge 5430).

[0486] Fifth, the supernatant (500-700 μL) was recovered and stored at −80° C. until further analysis.