INTERFERENCE-SUPPRESSED PHARMACOKINETIC IMMUNOASSAY

Abstract

Herein is reported a method for the determination of a bispecific antibody in a serum-containing sample comprising the steps of first incubating a solid phase to which a capture antibody specifically binding to the bispecific antibody has been immobilized with the sample to form an immobilized capture antibody-bispecific antibody-complex, thereafter incubating the solid phase with a replacement antibody that competes with the capture antibody for binding to the bispecific antibody and thereby forming a solution comprising a replacement antibody-bispecific antibody-complex, and determining in the solution the replacement antibody-bispecific antibody-complex.

Claims

1. A method for the determination of a bispecific antibody in a serum-containing sample comprising the following steps: a) incubating a solid phase to which a capture antibody that is specifically binding to the bispecific antibody has been immobilized with the serum-containing sample to form an immobilized capture antibody-bispecific antibody-complex, b) optionally washing the solid phase whereby the capture antibody-bispecific antibody-complex remains bound to the solid phase, c) incubating the solid phase with a replacement antibody that competes with the capture antibody for binding to the bispecific antibody and thereby forming a solution comprising a replacement antibody-bispecific antibody-complex, d) determining in the solution obtained in step c) the replacement antibody-bispecific antibody-complex and thereby determining the bispecific antibody in a serum-containing sample.

2. The method according to claim 1, wherein the replacement antibody is labelled and the determining of the replacement antibody-bispecific antibody-complex is by detecting said label.

3. The method according to any one of claims 1 to 2, wherein the replacement antibody is the capture antibody.

4. The method according to any one of claims 1 to 3, wherein the solid phase in step c) is incubated with the replacement antibody at a concentration of 6.67 nM or more.

5. The method according to any one of claims 1 to 4, wherein the serum-containing sample comprises the bispecific antibody and a monospecific antibody comprising one binding site of the bispecific antibody, and the method is a method with reduced interference of the monospecific antibody in the determination of the bispecific antibody.

6. The method according to any one of claims 1 to 5, wherein the bispecific antibody is an anti-CD3/CD20 bispecific antibody.

7. The method according to claim 6, wherein the bispecific anti-CD3/CD20 antibody is RG6026.

8. The method according to any one of claims 5 to 7, wherein the monospecific antibody is an anti-CD20 antibody.

9. The method according to claim 8, wherein the anti-CD20 antibody is obinutuzumab.

Description

DESCRIPTION OF THE FIGURES

[0162] FIG. 1 Scheme of an immunoassay for the determination of a bispecific antibody: immobilization of the bispecific antibody to the solid phase (SA-MTP; streptavidin-coated micro titer plate) by a capture reagent and determination of the bound bispecific antibody by detection reagent.

[0163] FIG. 2 Scheme of biased immunoassay: direct interaction of the monospecific antibody with the solid phase and thereby biasing the specific capture step.

[0164] FIG. 3 Concentration-response curve for a bridging immunoassay employing an anti-idiotypic anti-CD3 binding site antibody as capture antibody and an anti-idiotypic anti-CD20 binding site antibody as detection antibody for the determination of a bispecific CD20-TCB in the presence of the respective monospecific anti-CD20 antibody.

[0165] FIG. 4 Concentration-response curve for a bridging immunoassay employing an anti-idiotypic anti-CD3 binding site antibody as capture antibody and an anti-idiotypic anti-CD20 binding site antibody as detection antibody for the determination of a bispecific CD20-TCB in the absence of the respective monospecific anti-CD20 antibody.

[0166] FIG. 5 Concentration-response curve for a bridging immunoassay employing an anti-idiotypic anti-CD3 binding site antibody as capture antibody and an anti-P329G antibody as detection antibody for the determination of a bispecific CD20-TCB in the presence and absence of the respective monospecific anti-CD20 antibody.

[0167] FIG. 6 Concentration-response curve for a bridging immunoassay according to the current invention comprising an additional displacement step in the presence as well as in the absence of the respective monospecific anti-CD20 antibody.

EXAMPLE 1

[0168] Bridging Immunoassay with Double Anti-Idiotypic Antibody

General Procedure:

[0169] Murine monoclonal antibodies were produced by Roche Diagnostics GmbH (Germany). As anti-idiotypic anti-CD3 binding site antibody biotinylated M-4.25.93-IgG was used and as anti-idiotypic anti-CD20 binding site antibody digoxigenylated M-6.15.528-IgG-Dig was used.

[0170] CD20-TCB calibrators or QCs (quality controls) as well as samples with CD20-TCB and anti-CD20 antibody were prepared in 100% pooled human serum obtained from Trina Bioreactives, Switzerland or 100% Cynomolgus pooled serum (Seralab, UK).

[0171] Antibody preparations and serum sample dilutions were made in Universal Buffer (Roche Diagnostics GmbH, Germany). Each washing included 3 steps with 300 μL PBS, 0.05% Tween 20 (Sigma Aldrich, Germany), incubation steps were carried out at room temperature with shaking at 500 rpm and the working volume per well was 100 μL.

[0172] For capturing Streptavidin plates (high binding capacity, Microcoat GmbH, Bemried, Germany) were coated with 0.2 μg/mL M-4.25.93-IgG-Bi for one hour and unbound antibody was removed by washing followed by addition of diluted (1:10) serum samples and incubation for one hour. After washing analyte detection was performed by successive addition of M-6.15.528-IgG-Dig (0.2 μg/mL, 50 min) and <Dig>-Fab fragments covalently bound to horseradish peroxidase (Roche Diagnostics GmbH, Germany; 40 mU, 50 min). A washing step was applied in between and after this step. ABTS (2′2′-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid; Roche Diagnostics GmbH, Germany) was added as substrate for color reaction, which was monitored by photometrical read-out at 405 nm (reference wavelength 490 nm). Samples were analyzed in duplicates and mean absorbance values were calculated and further processed. Concentrations were determined by back calculation from the calibration curve with a non-linear 4-parameter curve-fitting function (Wiemer-Rodbard). Accuracy was calculated in relation to nominal concentrations in the spiked serum samples.

[0173] a) Sample Comprising CD20-TCB and 200 μg/mL Anti-CD20 Antibody:

TABLE-US-00003 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 100.00 142.47 142 HQC 75.00 125.36 167 MQC 15.00 69.10 461 LQC 3.00 61.12 2037 LLQC 1.00 59.00 5900 blank 0 56.92 —

[0174] b) Sample Comprising CD20-TCB Only:

TABLE-US-00004 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 100.00 104.95 105 HQC 75.00 80.83 108 MQC 15.00 15.58 104 LQC 3.00 3.42 114 LLQC 1.00 1.07 107 blank 0 b.l.q. —

[0175] c) Sample Comprising Anti-CD20 Antibody Only (Values Back Calculated on a CD20-TCB Calibration Curve in 10% Cynomolgus Monkey Serum):

TABLE-US-00005 measured concentration [ng/mL] nominal solid phase coated with anti- solid phase without anti- concentration idiotypic anti-CD3 binding idiotypic anti-CD3 binding [μg/mL] site antibody site antibody 500.00 47.97 52.30 50.00 23.84 26.59 5.00 7.26 7.40 0 b.l.q. b.l.q.

EXAMPLE 2

[0176] Bridging Immunoassay with Anti-Idiotypic Antibody Capture and Anti-P329G Antibody Detection

General Procedure:

General: 4 Step Sandwich ELISA

[0177] Incubation at room temperature with shaking at 500 rpm [0178] Working value: 100 μL [0179] Washing (denoted as w hereafter): 3×300 μL PBS, 0.05% Tween 20 [0180] Replicates n=2, calculation of mean absorbance value [0181] Calibration curve fit: non-linear 4-parameter curve-fitting function (Wiemer-Rodbard) [0182] Assay range: 1 ng/mL-100 ng/mL

Reagents:

[0183] Human pooled serum; Trina Bioreactives, Switzerland [0184] Murine monoclonal antibodies; Roche Diagnostics GmbH, Germany [0185] Anti-idiotypic anti-CD3 binding site antibody, biotinylated: M-4.25.93-IgG-Bi [0186] Anti-P329G antibody, digoxigenylated: M-1.7.24-IgG-Dig [0187] Streptavidin plate (high binding capacity); Microcoat GmbH, Bernried, Germany [0188] Universal Buffer (denoted as UB hereinafter); Roche Diagnostics GmbH, Germany [0189] Washing buffer; 1×PBS, 0.05 Tween (Sigma Aldrich, Germany) [0190] <Dig>-Fab fragments covalently bound to horseradish peroxidase (<Dig>-Fab-POD); Roche Diagnostics GmbH, Germany [0191] 2′2′-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS); Roche Diagnostics GmbH, Germany

Assay Conduct:

[0192] Coating: 0.2 μg/mL M-4.25.93-IgG-Bi, 1 hour, w [0193] Sample: 1 to 10 in UB, 1 hour, w [0194] Detection 1: 0.2 μg/mL M-1.7.24-IgG-Dig, 50 min, w [0195] Detection 2: 50 mU<Dig>-Fab-POD, 50 min, w [0196] Substrate reaction: ABTS, repeated absorbance measurement at 405 nm (reference wavelength 490 nm)
a) Sample Comprising CD20-TCB and 200 μg/mL Anti-CD20 Antibody:

TABLE-US-00006 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 100.00 95.41 95 HQC 75.00 72.48 97 MQC 15.00 13.17 88 LQC 3.00 3.09 103 LLQC 1.00 1.07 107 blank 0 b.l.q. —

b) Sample Comprising CD20-TCB Only:

[0197]

TABLE-US-00007 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 100.00 96.40 96 HQC 75.00 73.77 98 MQC 15.00 13.33 89 LQC 3.00 3.14 105 LLQC 1.00 1.14 114 blank 0 b.l.q. —

EXAMPLE 3

[0198] Bridging Immunoassay with Double Anti-Idiotypic Antibody at Reduced Immobilization Densities

General Procedure:

[0199] Same procedure as described in Example 1, except for use of Streptavidin plate with medium binding capacity, Microcoat GmbH, Bernried, Germany

a) Sample Comprising CD20-TCB and 200 μg/mL Anti-CD20 Antibody:

TABLE-US-00008 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 100.00 102.01 102 HQC 75.00 78.84 105 MQC 15.00 16.59 111 LQC 3.00 4.48 149 LLQC 1.00 2.41 241 blank 0 1.27 —

b) Sample Comprising CD20-TCB Only:

[0200]

TABLE-US-00009 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 100.00 101.17 101 HQC 75.00 78.43 105 MQC 15.00 15.67 104 LQC 3.00 3.33 111 LLQC 1.00 1.05 105 blank 0 b.l.q. —

EXAMPLE 4

[0201] Bridging Immunoassay with Double Anti-Idiotypic Antibody at Reduced Immobilization Densities with 1% Casein in PBS as Assay Buffer

General Procedure:

General: 4 Step Sandwich ELISA

[0202] Incubation at room temperature with shaking at 500 rpm [0203] Working value: 100 μL [0204] Washing (denoted as w hereinafter): 3×300 μL PBS, 0.05% Tween 20 [0205] Replicates n=2, calculation of mean absorbance value [0206] Calibration curve fit: non-linear 4-parameter curve-fitting function (Wiemer-Rodbard) [0207] Assay range: 1 ng/mL-64 ng/mL

Reagents:

[0208] Human pooled serum; Trina Bioreactives, Switzerland [0209] Murine monoclonal antibodies; Roche Diagnostics GmbH, Germany [0210] Anti-idiotypic anti-CD3 binding site antibody, biotinylated: M-4.25.93-IgG-Bi [0211] Anti-idiotypic anti-CD20 binding site antibody, digoxigenylated: M-6.15.528-IgG-Dig [0212] Streptavidin plate (medium binding capacity); Microcoat GmbH, Bernried, Germany [0213] Casein (Hammersten); VWR Prolabo Chemicals, Germany [0214] Assay buffer (denoted as AB hereinafter): 1% Casein in 1×PBS (Roche Diagnostics GmbH, Germany) [0215] Washing buffer; 1×PBS, 0.05% Tween (Sigma Aldrich, Germany) [0216] <Dig>-Fab fragments covalently bound to horseradish peroxidase (<Dig>-Fab-POD); Roche Diagnostics GmbH, Germany [0217] 2′2′-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS); Roche Diagnostics GmbH, Germany

Assay Conduct:

[0218] Coating: 0.25 μg/mL M-4.25.93-IgG-Bi, 1 hour, w [0219] Sample: 1 to 10 in AB, 1 hour, w [0220] Detection 1: 0.25 μg/mL M-6.15.528-IgG-Dig, 60 min, w [0221] Detection 2: 40 mU<Dig>-Fab-POD, 60 min, w [0222] Substrate reaction: ABTS, repeated absorbance measurement at 405 nm (reference wavelength 490 nm)
a) Sample Comprising CD20-TCB and 200 μg/mL Anti-CD20 Antibody:

TABLE-US-00010 measured measured measured measured nominal conc. conc. 1 conc. 2 conc. 3 conc. 4 [ng/mL] [ng/mL] [ng/mL] [ng/mL] [ng/mL] precision [%] ULQC 64.00 66.40 80.55 52.93 43.76 26 HQC 48.00 52.90 59.85 39.51 34.73 25 MQC 12.00 12.29 13.13 8.17 7.58 27 LQC 3.00 3.72 3.85 2.31 2.34 28 LLQC 1.00 1.39 1.60 0.99 1.07 23 nominal conc. accuracy 1 accuracy 2 accuracy 3 accuracy 4 [ng/mL] [%] [%] [%] [%] precision [%] ULQC 64.00 104 126 83 68 26 HQC 48.00 110 125 82 72 25 MQC 12.00 102 109 68 63 27 LQC 3.00 124 128 77 78 28 LLQC 1.00 139 160 99 107 23

b) Sample Comprising CD20-TCB Only:

[0223]

TABLE-US-00011 measured measured measured measured nominal conc. conc. 1 conc. 2 conc. 3 conc. 4 [ng/mL] [ng/mL] [ng/mL] [ng/mL] [ng/mL] precision [%] ULQC 64.00 57.53 61.02 56.93 49.57 9 HQC 48.00 45.43 51.75 47.17 44.61 7 MQC 12.00 10.46 11.98 8.07 10.05 16 LQC 3.00 3.03 3.11 2.10 2.65 17 LLQC 1.00 1.00 1.11 0.69 0.98 19 nominal conc. accuracy 1 accuracy 2 accuracy 3 accuracy 4 [ng/mL] [%] [%] [%] [%] precision [%] ULQC 64.00 90 95 89 77 9 HQC 48.00 95 108 98 93 7 MQC 12.00 87 100 67 84 16 LQC 3.00 101 104 70 88 17 LLQC 1.00 100 111 69 98 19

EXAMPLE 5

[0224] Bridging Immunoassay with Double Anti-Idiotypic Antibody at Reduced Immobilization Densities Under Acidic (pH 5.5) Washing Conditions

General Procedure:

General: 4 Step Sandwich ELISA

[0225] Incubation at room temperature with shaking at 500 rpm [0226] Working value: 100 μL [0227] Washing (denoted as w hereinafter): 3×300 μL PBS, 0.05% Tween 20, pH 5.5 [0228] Replicates n=2, calculation of mean absorbance value [0229] Calibration curve fit: non-linear 4-parameter curve-fitting function (Wiemer-Rodbard) [0230] Assay range: 1 ng/mL-64 ng/mL

Reagents:

[0231] Human pooled serum; Trina Bioreactives, Switzerland [0232] Murine monoclonal antibodies; Roche Diagnostics GmbH, Germany [0233] Anti-idiotypic anti-CD3 binding site antibody, biotinylated: M-4.25.93-IgG-Bi [0234] Anti-idiotypic anti-CD20 binding site antibody, digoxigenylated: M-6.15.528-IgG-Dig [0235] Streptavidin plate (medium binding capacity); Microcoat GmbH, Bernried, Germany [0236] Assay buffer: Universal Buffer (denoted UB hereinafter); Roche Diagnostics GmbH, Germany [0237] Washing buffer; 1×PBS, 0.05% Tween, pH 5.5 (Sigma Aldrich, Germany, pH adjusted with 2 M HCl, Sigma Aldrich, Germany) [0238] <Dig>-Fab fragments covalently bound to horseradish peroxidase (<Dig>-Fab-POD); Roche Diagnostics GmbH, Germany [0239] 3,3′,5,5′-Tetramethyl benzidine (TMB); Roche Diagnostics GmbH, Germany [0240] 1 M H2504, Carl Roth, Germany

Assay Conduct:

[0241] Coating: 0.25 μg/mL M-4.25.93-IgG-Bi, 1 hour, w [0242] Sample: 1 to 10 in UB, 1 hour, w [0243] Detection 1: 0.25 μg/mL M-6.15.528-IgG-Dig, 60 min, w [0244] Detection 2: 10 mU<Dig>-Fab-POD, 60 min, w [0245] Substrate reaction: TMB, stopped with 50 μL 1 M H2504 [0246] Photometrical read-out at 450 nm (reference wavelength 690 nm)
a) Sample Comprising CD20-TCB and 200 μg/mL Anti-CD20 Antibody:

TABLE-US-00012 measured measured measured measured nominal conc. conc. 1 conc. 2 conc. 3 conc. 4 [ng/mL] [ng/mL] [ng/mL] [ng/mL] [ng/mL] precision [%] ULQC 64.00 55.54 35.06 58.56 56.44 21 HQC 48.00 42.89 30.55 44.88 47.12 18 MQC 12.00 10.10 6.74 9.80 11.95 22 LQC 3.00 2.74 1.85 2.57 3.24 22 LLQC 1.00 1.05 0.81 1.09 1.70 33 nominal conc. accuracy 1 accuracy 2 accuracy 3 accuracy 4 [ng/mL] [%] [%] [%] [%] precision [%] ULQC 64.00 87 55 91 88 21 HQC 48.00 89 64 94 98 18 MQC 12.00 84 56 82 100 22 LQC 3.00 91 62 86 108 22 LLQC 1.00 105 81 109 170 33

b) Sample Comprising CD20-TCB Only:

[0247]

TABLE-US-00013 measured measured measured measured nominal conc. conc. 1 conc. 2 conc. 3 conc. 4 [ng/mL] [ng/mL] [ng/mL] [ng/mL] [ng/mL] precision [%] ULQC 64.00 56.24 44.84 60.61 51.59 13 HQC 48.00 43.67 34.35 46.80 45.26 13 MQC 12.00 11.87 8.47 12.11 11.27 15 LQC 3.00 2.96 2.12 2.97 2.43 16 LLQC 1.00 0.98 0.85 1.34 1.03 20 nominal conc. accuracy 1 accuracy 2 accuracy 3 accuracy 4 [ng/mL] [%] [%] [%] [%] precision [%] ULQC 64.00 88 70 95 81 13 HQC 48.00 91 72 98 94 13 MQC 12.00 99 71 101 94 15 LQC 3.00 99 71 99 81 16 LLQC 1.00 98 85 134 103 20

EXAMPLE 6

Bridging Immunoassay According to the Current Invention

General Procedure:

[0248] Murine monoclonal antibodies were produced by Roche Diagnostics GmbH (Germany). As anti-idiotypic anti-CD3 binding site antibodies biotinylated M-4.35.77-IgG as well as digoxigenylated M-4.25.93-IgG were used and as anti-idiotypic anti-CD20 binding site antibody biotinylated M-6.15.528 was used.

[0249] CD20-TCB calibrators or QCs (quality controls) as well as samples with CD20-TCB and anti-CD20 antibody were prepared in 100% pooled human serum obtained from Trina Bioreactives, Switzerland. Antibody preparations and serum sample dilutions were made in Universal Buffer (Roche Diagnostics GmbH, Germany). Each washing included 3 steps with 300 μL PBS, 0.05% Tween 20 (Sigma Aldrich, Germany). Incubation steps were carried out at room temperature with shaking at 500 rpm. The procedure was divided into two parts conducted on two streptavidin coated microtiter plates (SA-MTP 1 and 2, high binding capacity, Microcoat GmbH, Bernried, Germany).

[0250] Part 1: For capturing SA-MTP 1 was coated with 135 μL/well of 0.21 μg/mL M-4.35.77-IgG-Bi for one hour and unbound antibody was removed by washing followed by successive addition of 67.5 μL/well Universal buffer and 67.5 μL/well serum sample (resulting in 50% Matrix) and incubation for 35 minutes. After washing 135 μL of 1 μg/mL M-4.25.93-Dig (detection antibody) was added and the plate was incubated for 2.5 hours without a following washing step for desorption (replacement step).

[0251] Part 2: 90 minutes after addition of detection antibody to SA-MTP1, SA-MTP2 was coated with 100 μL/well 0.25 μg/mL M-6.28.530-Bi for one hour followed by washing. Thereafter 100 μL of the formed complexes (drug-detection antibody) were transferred from SA-MTP1 to SA-MTP2 and incubated for 35 minutes. As second detection reagent 100 μL/well 50 mU<Dig>-Fab fragments covalently bound to horseradish peroxidase (Roche Diagnostics GmbH, Germany) was added and the plate was incubated for 45 Minutes. A washing step was applied before and after this step. 100 μL/well ABTS (2′2′-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid; Roche Diagnostics GmbH, Germany) was used as substrate for color reaction, which was monitored by photometrical read-out at 405 nm (reference wavelength 490 nm). Samples were analyzed in duplicates and mean absorbance values were calculated and further processed. Concentrations were determined by back calculation from the calibration curve with a non-linear 4-parameter curve-fitting function (Wiemer-Rodbard). Accuracy was calculated in relation to nominal concentrations in the spiked serum samples.

a) Sample Comprising CD20-TCB and 200 μg/mL Anti-CD20 Antibody:

TABLE-US-00014 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 40.00 41.31 103 HQC 30.00 30.58 102 MQC 7.50 7.13 95 LQC 1.88 1.72 92 LLQC 0.63 0.51 82 blank 0 b.l.q. —

b) Sample Comprising CD20-TCB Only:

[0252]

TABLE-US-00015 nominal measured concentration concentration accuracy [ng/mL] [ng/mL] [%] ULQC 40.00 41.90 105 HQC 30.00 31.07 104 MQC 7.50 7.05 94 LQC 1.88 1.77 95 LLQC 0.63 0.54 86 blank 0 b.l.q. —