SPR-based dual-binding assay for the functional analysis of multispecific molecules

Abstract

Herein is reported a method for determining the binding of an antibody, which comprises a first binding site specifically binding to a first antigen and a second binding site specifically binding to a second antigen, to said first and said second antigen, wherein the method comprises the steps of capturing the antibody on a solid phase using a capture reagent specifically binding to a constant domain of the antibody, incubating the captured antibody with the first or the second antigen to form a captured antibody-antigen complex and determining a first binding signal, either i) incubating the captured antibody-antigen complex with the antigen not used for the formation of the captured antibody-antigen complex to form a captured antibody-antigen-antigen complex and determining a second binding signal, or ii) regenerating the surface, capturing the antibody on a solid phase using a capture reagent specifically binding to a constant domain of the antibody, incubating the captured antibody with the antigen not used for the formation of the captured antibody-antigen complex in step b) to form a captured antibody-antigen-antigen complex and determining a third binding signal, and determining the overall or individual binding of the antibody to the first and the second antigen from the first binding signal and the second or third binding signal.

Claims

1. A method for determining the binding of an antibody, which comprises a first binding site specifically binding to a first antigen and a second binding site specifically binding to a second antigen, to said first and said second antigen, wherein the method comprises the following steps: a) capturing the antibody on a solid phase using a capture reagent specifically binding to a constant domain of the antibody, b) incubating the captured antibody with the first or the second antigen to form a captured antibody-antigen complex and determining a first binding signal, c) either incubating the captured antibody-antigen complex with the antigen not used for the formation of the captured antibody-antigen complex to form a captured antibody-antigen-antigen complex and determining a second binding signal, or regenerating the surface, capturing the antibody on a solid phase using a capture reagent specifically binding to a constant domain of the antibody, incubating the captured antibody with the antigen not used for the formation of the captured antibody-antigen complex in step b) to form a captured antibody-antigen complex and determining a third binding signal, and d) determining the overall or individual binding of the antibody to the first and the second antigen from the first binding signal and the second or third binding signal.

2. The method according to claim 1, wherein step d) is d) determining the overall or individual binding of the antibody to the first and the second antigen if i) the formation of the captured antibody-antigen complex results in a first binding signal and ii) the formation of the captured antibody-antigen-antigen complex or the formation of the captured antibody-antigen complex after regeneration results in a second or third binding signal that is increased with respect to the first binding signal.

3. A method for determining overall and individual binding of an antibody, which comprises a first binding site specifically binding to a first antigen and a second binding site specifically binding to a second antigen, to said first and said second antigen, wherein the method comprises the following steps: capturing the antibody on a solid phase using a capture reagent specifically binding to a constant region of the antibody, incubating the captured antibody with the first or second antigen to form an immobilized antibody-antigen complex, incubating the captured antibody-antigen complex with the antigen not used for the formation of the captured antibody-antigen complex to form a captured antibody-antigen-antigen complex, and determining the individual and based on this, also the overall binding of the antibody to the first and the second antigen if the formation of the captured antibody-antigen complex results in a first binding signal and the formation of the captured antibody-antigen-antigen complex results in a second binding signal that is increased with respect to the first binding signal.

4. The method according to claim 1, wherein the solid phase is a surface plasmon resonance chip, the first binding signal is a surface plasmon resonance response and the second binding signal is a surface plasmon resonance response.

5. The method according to claim 1, wherein the antibody is a bispecific antibody.

6. The method according to claim 5, wherein the bispecific antibody is a CrossMab or a DutaFab.

7. The method according to claim 1, wherein the capture reagent is an anti-Fc-region antibody or an anti-Fab antibody.

8. The method according to claim 7, wherein the anti-Fab antibody is an anti-kappa-light-chain antibody or an anti-lambda-light-chain antibody.

9. The method according to claim 1, wherein the antibody is a DutaFab, the solid phase is a surface plasmon resonance chip, the capture reagent is an anti-kappa-light chain antibody or an anti-lambda light chain antibody, the first binding signal is a surface plasmon resonance response and the second binding signal is a surface plasmon resonance response.

10. The method according to claim 1, wherein the capturing is by injecting the antibody for 45 to 720 seconds, at a flow rate of 2.5 to 10 μL/min, and at a concentration of 0.6 μg/mL to 10 μg/mL.

11. The method according to claim 10, wherein the capturing is by injecting the antibody for about 60 seconds, at a flow rate of about 5 μL/min, and at a concentration of 0.6 μg/mL to 10 μg/mL.

12. The method according to claim 1, wherein the incubating is by an injection of the respective antigen at a concentration of 0.5 to 5 μg/mL for 20 to 90 seconds.

13. The method according to claim 12, wherein the incubating is by an injection of the respective antigen at a concentration of about 2 μg/mL for 30 or 60 seconds.

14. The method according to claim 1, wherein the incubating is in a buffer comprising PBS-T and 300 mM NaCl.

Description

DESCRIPTION OF THE FIGURES

[0132] FIG. 1 Capturing and interaction of the two antigens to a bispecific antibody, exemplified by a CrossMab. (A) First, the CrossMab is captured by Fab-capture-Antibody (capture signal Rc). Second, antigen-2 (Ra) and/or antigen-1 (Rv) can be recruited from solution to the captured CrossMab. (B) Sensorgram displaying the individual interaction. Rc: Binding response corresponding to capture signal. Ra: Binding response corresponding to antigen-2 binding. Rv: Binding response corresponding to antigen-1 binding.

[0133] FIG. 2 Independent binding of antigen-2 and antigen-1 determined with a DutaFab captured at 1.5 μg/mL (lower graphs) and at 5 μg/mL (upper graph) by injection of antigen-2 and antigen-1 simultaneously or sequentially; the graphs are triplicates whereof the upper one was obtained by the simultaneous injection of antigen-2 and antigen-1, the middle one was obtained by injection of antigen-1 followed by the injection of antigen-2, the lower one was obtained by injection of antigen-2 followed by injection of antigen-1.

[0134] FIG. 3 Linear dose-response-curve out of a full dose-response curve with different bispecific antibody (CrossMab) concentrations. For a final readout parallel line model was used plotting the antigen-binding signal of (A) antigen-2 against the bispecific antibody concentration on a logarithmic scale and (B) antigen-1 against the bispecific antibody concentration on a logarithmic scale.

EXAMPLES

[0135] Equipment and Reagents

[0136] All SPR experiments were performed on a BIAcore©T200 instrument (GE Healthcare) at 25° C. VEGFA-121, Ang2, CrossMabs and all used control antibodies were manufactured by Roche Diagnostics GmbH.

Example 1

[0137] Assay Procedure—Dual Binding Assay

[0138] Capturing anti-human Fab antibody (Human Fab Capture Kit, GE Healthcare) was immobilized on the surface of a CMS biosensor chip using the provided amine coupling chemistry from GE Healthcare. Flow cells were activated with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 5 μL/min. Anti-human Fab antibody was injected in sodium acetate, pH 5.0 at 15 μg/ml for 420 sec, which resulted in a surface density of approximately 6000 RU. At least 5000 RU drug should be immobilized to ensure target binding is not limited by the immobilization. A reference control flow cell was treated in the same way as described before. Finally both surfaces were blocked with an injection of 1 M ethanolamine/HCl pH 8.5. As immobilization buffer HBS-N (10 mM HEPES, 150 mM NaCl, pH 7.4, GE Healthcare) was used. The bispecific antibodies were diluted in PBS-T (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4, 0.05% Tween-20; Roche Diagnostics GmbH, Mannheim, Germany) and injected on the second flow cell at various concentrations for 90 sec at a flow rate of 10 μL/min Ang2 was injected for 60 sec with a concentration of 1.1 μg/ml followed by an injection of 1.4 μg/ml VEGFA-121 for 60 sec over both flow cells. The dissociation time (washing with running buffer) was 30 sec at a flow rate of 10 μL/min. All interactions were performed at 25° C. The regeneration solution of Glycine (pH 2.1) was injected for 60 sec at a flow rate of 30 μL/min to remove any non-covalently bound protein after each binding cycle. Signals were detected at a rate of one signal per second. Samples were measured in triplicates and the activity was measured relative to a standard sample.

Example 2

[0139] Assay Procedure—Target-Based Bridging Assay

[0140] The target-based bridging assay is essentially based on the assay described in Gassner et al. [11] with the following modifications: The dose-response curve was adjusted to e.g. 1.92-5 μg/ml. The assay could be qualified in the range of 50% to 150%: Linearity was 0.9987, accuracy was 97% and the precision was 2.9%. The assay is specific for the drug, loss of function indicating and suitable for later stage validation.

Example 3

[0141] Curve Fitting and Statistical Analysis

[0142] For the assays a parallel line model according to USP 1034 [14] for relative binding activity calculation was used. The parallel line model can be described by the following equations:

Y.sub.S=α.sub.S+βlog(x)+e  (equation 3)

Y.sub.T=α.sub.T+βlog(x)+e  (equation 4)

where Y.sub.S is the response to a standard sample with concentration x and Y.sub.T is the response to a test sample with the same concentration x. The potency of a test sample relative to a

[00001]$\mathrm{standard}\mathrm{sample}\mathrm{is}=\exp \left(\frac{{\alpha }_T-{\alpha }_s}{\beta }\right).$

The basic similarity assumption for this model is the equality of slopes β.