Binding assays based on ph gradient psr

Abstract

Herein is reported a method for determining a dissociation rate constant kd of an antibody and its antigen at a dissociation pH, wherein the method comprises the steps of immobilizing the antibody at a first pH on a solid phase of the antigen that has been conjugated with the antibody; applying a pH gradient from the first pH value to the dissociation pH value, and then maintaining the pH value at the dissociation pH value; and recording a binding signal while maintaining the pH value, and calculating the dissociation rate constant kd of the antibody and its antigen at the dissociation pH value therefrom.

Description

[0001] This application is a divisional application of PCT application PCT / EP2019 / 086740, filed on December 20, 2019, with the invention name “Binding assay based on pH gradient SPR”. The date on which the PCT application entered the Chinese national phase is June 23, 2021, and the application number is 201980085597.5. Technical Field

[0002] The present invention belongs to the field of functional assays.Herein a novel SPR-based binding assay is reported for measuring pH-dependent interactions of antibodies. Background Art

[0003] SPR (surface plasmon resonance) is a biosensor-based technology to measure real-time protein-protein interactions. SPR technology has become a standard tool in biopharmaceutical research and development [1-5] and is often used to determine the binding constants of macromolecular interactions. The ability to determine the association and dissociation kinetics of molecular interactions provides detailed insights into the mechanisms of complex formation [6]. This information is becoming an important component of the selection and optimization process for monoclonal antibodies and other biopharmaceutical products [7-10]. In addition, SPR technology allows the determination of, for example, the binding activity (binding capacity) of an antibody binding target.

[0004] The antigen neutralizing ability of a single antibody molecule depends on its affinity. By improving affinity, antigens can be neutralized with a smaller amount of antibodies. A variety of methods can be used to improve antibody affinity. In addition, if affinity can be made infinite by covalently binding the antibody to the antigen, a single antibody molecule can neutralize an antigen molecule (a bivalent antibody can neutralize two antigen molecules). However, due to the stoichiometric neutralization of one antibody to one antigen (a bivalent antibody to two antigens), it is impossible to completely neutralize the antigen with an antibody amount less than the amount of antigen. In order to extend the neutralization of the neutralizing antibody for a certain period of time, the dosage of the antibody must be higher than the amount of antigen produced in the body during the same period of time. Only by relying on the improvement of antibody pharmacokinetics or affinity maturation technology, the reduction of the required antibody dosage is limited.

[0005] Therefore, in order to maintain the antigen neutralization effect of an antibody for a target period of time with an amount of antibody that is smaller than the amount of antigen, a single antibody must neutralize multiple antigens.

[0006] Shank-Retzlaff, ML and Sligar, SG disclosed analyte gradient-surface plasmon resonance as a one-step method for determining kinetic rates and binding affinities of macromolecules (Anal. Chem. 72 (2000) 4212-4220).

[0007] US2005 / 0019933 discloses a method for characterizing an interaction between two substances in a liquid environment, wherein a liquid containing at least one substance passes through a measurement system as a flow, and wherein the interaction occurs within the measurement system. The method comprises generating a concentration gradient of at least one of the substances or at least one other substance that has an effect on the interaction or a component of the interaction.

[0008] Schasfoort, RBM et al., Handbook of Surface Plasmon Resonance (December 31, 2008 (2008-12-31), pp. 354-394, Cambridge), disclose future trends in SPR technology.

[0009] US2010 / 256338 discloses multispecific antibodies, especially bispecific antibodies, comprising full-length antibodies and single-chain Fab fragments, methods for preparing the same, pharmaceutical compositions containing the same, and uses thereof.

[0010] WO 2012 / 023053 discloses novel bispecific monoclonal antibodies having different specificities for each binding site of an immunoglobulin molecule and methods for preparing novel bispecific monoclonal antibodies having different specificities for each binding site of an immunoglobulin molecule.

[0011] Schlothauer, T. et al. disclosed an analytical FcRn affinity chromatography method for functional characterization of monoclonal antibodies (mAbs 5 (2013) 576-586).

[0012] WO 2015 / 140126 discloses a method for determining the presence of antibody-Fab-FcRn interactions in the antibody-Fc-FcRn complex that influence the in vivo half-life using an FcRn affinity chromatography column with positive linear pH gradient elution in the presence of different salt concentrations.

[0013] WO 2015 / 172800 discloses novel multispecific molecules and novel therapeutic methods based on such multispecific molecules.

[0014] Meschendoerfer, W. et al. disclosed an SPR-based assay that enables full functional analysis of bispecific molecules (J. Pharm. Biomed. Anal. 132 (2016) 141-147).

[0015] EP 2 275 443 discloses methods for improving the pharmacokinetics of antigen-binding molecules and methods for increasing the number of antigen bindings of antigen-binding molecules, as well as antigen-binding molecules with improved pharmacokinetics, antigen-binding molecules with increased number of antigen bindings, and methods for preparing such molecules. Summary of the invention

[0016] Here we report a novel SPR-based assay for measuring the pH-dependent binding activity of antibodies.

[0017] One aspect as reported herein is an assay or a method for determining whether the binding of a (monoclonal) antibody to its antigen is pH dependent (i.e. the antibody has pH dependent antigen binding), wherein the method comprises the following steps:

[0018] a) capturing / immobilizing the antibody on a solid phase of an antigen to which the antibody has been conjugated at a first pH value, i.e., capturing / immobilizing the antibody through the interaction of the antibody with its antigen,

[0019] b) applying a pH gradient from a first pH value to a second pH value to the solid phase and monitoring the dissociation of the antibody from the solid phase,

[0020] If dissociation of the antibody from the solid phase can be detected in step b), the binding of the antibody to its antigen is pH-dependent.

[0021] An exemplary SPR sensorgram showing pH-dependent dissociation is shown in Figure 2 Shown in.

[0022] One aspect as reported herein is an assay or method for selecting a (monoclonal) antibody with pH-dependent binding to its antigen (i.e. the antibody has pH-dependent antigen binding), wherein the method comprises the following steps:

[0023] a) capturing / immobilizing the antibody on a solid phase of an antigen to which the antibody has been conjugated at a first pH value, i.e., capturing / immobilizing the antibody through the interaction of the antibody with its antigen,

[0024] b) applying a pH gradient from a first pH value to a second pH value to the solid phase and monitoring the dissociation of the antibody from the solid phase,

[0025] Wherein, if dissociation of the antibody from the solid phase can be detected in step b), the antibody is selected.

[0026] One aspect as reported herein is an assay or method for determining the pH value at which an antibody having pH-dependent binding to its antigen dissociates from its antigen (pH-dependent dissociation), wherein the method comprises the following steps:

[0027] a) capturing / immobilizing the antibody on a solid phase of an antigen to which the antibody has been conjugated at a first pH value, i.e., capturing / immobilizing the antibody through the interaction of the antibody with its antigen,

[0028] b) applying a pH gradient from a first pH value to a second pH value to the solid phase and monitoring the dissociation of the antibody from the solid phase,

[0029] The pH value at which the antibody dissociates from its antigen is the pH value of the pH gradient at which dissociation of the antibody from the solid phase can be detected in step b).

[0030] One aspect as reported herein is a method for determining the dissociation rate constant k of an antibody from its antigen. d An assay or method wherein the antibody has a pH-dependent binding to its antigen at a dissociation pH (the dissociation pH is a pH at which dissociation of the antibody from its antigen can be detected) (pH-dependent dissociation), wherein the method comprises the following steps:

[0031] a) capturing / immobilizing the antibody on a solid phase of an antigen to which the antibody has been conjugated at a first pH value, i.e., capturing / immobilizing the antibody through the interaction of the antibody with its antigen,

[0032] b) applying a pH gradient from a first pH value to a dissociation pH value to the solid phase and then maintaining the pH value at the dissociation pH value,

[0033] c) Record the (binding) signal and calculate the dissociation rate constant k from it d , and thus determine the dissociation rate constant k at the dissociation pH d .

[0034] An exemplary SPR sensorgram showing dissociation at the dissociation pH is shown in Figure 3 Shown in.

[0035] One aspect as reported herein is an assay or method for determining the influence of antibody oligomerization on the pH-dependent binding of a (monoclonal) antibody to its antigen, wherein the method comprises the following steps:

[0036] a) capturing / immobilizing the monomeric antibody on a solid phase to which an antigen has been conjugated at a first pH value, i.e., capturing / immobilizing the monomeric antibody through the interaction between the antibody and its antigen,

[0037] b) applying a pH gradient from a first pH value to a second pH value to the solid phase, monitoring the dissociation of the antibody from the solid phase and thereby calculating the dissociation rate,

[0038] c) repeating steps a) to b) using dimer, trimer, tetramer and / or oligomer antibodies,

[0039] Wherein if the dissociation rates calculated in step b) differ by more than 10% for a monomeric antibody and a dimeric, trimer, tetrameric and / or oligomeric antibody, then the pH-dependent binding of the antibody to its antigen is affected by oligomerization.

[0040] One aspect as reported herein is an assay or method for determining the influence of antigen oligomerization on the pH-dependent binding of a (monoclonal) antibody to its antigen, wherein the method comprises the following steps:

[0041] a) capturing / immobilizing the antibody on a solid phase to which a monomeric antigen to which the antibody has been conjugated at a first pH value, i.e., capturing / immobilizing the antibody through the interaction between the antibody and its antigen,

[0042] b) applying a pH gradient from a first pH value to a second pH value to the solid phase, monitoring the dissociation of the antibody from the solid phase and thereby calculating the dissociation rate,

[0043] c) repeating steps a) to b) with the dimer, trimer, tetramer and / or oligomer antigen immobilized on a solid phase,

[0044] Wherein if the dissociation rates calculated in step b) differ by more than 10% for monomeric antigen and dimeric, trimer, tetrameric and / or oligomeric antigen, then the pH-dependent binding of the antibody to its antigen is affected by oligomerization.

[0045] One aspect as reported herein is a method for separating or isolating a low pH binding (monoclonal) antibody from a mixture of antibodies binding to the same antigen, wherein the method comprises the following steps:

[0046] a) applying the antibody mixture to a solid phase to which the antigen to which the antibody mixture has been conjugated at a first pH value, i.e. capturing / immobilizing the antibodies by interaction with their antigens,

[0047] b) applying a pH gradient from a first pH value to a second pH value to the solid phase and optionally monitoring the dissociation of the antibody from the solid phase,

[0048] c) collecting antibodies that dissociate at low pH,

[0049] The low pH binding (monoclonal) antibody is thereby separated or isolated from a mixture of antibodies that bind the same antigen.

[0050] In one embodiment, the low pH is pH 6.5 or lower. In one embodiment, the low pH is pH 6.0 or lower. In one embodiment, the low pH is pH 5.5 or lower.

[0051] Embodiments of the aspects outlined above are described below. It should be understood that all possible combinations of the embodiments with each other are also disclosed.

[0052] In one embodiment, the conditions in step b) are kept constant except for the pH.

[0053] In one embodiment, the method is a surface plasmon resonance method.

[0054] In one embodiment, dissociation is determined by surface plasmon resonance.

[0055] In one embodiment, the solid phase is a surface plasmon resonance chip.

[0056] In one embodiment, the antigen is immobilized at high density.

[0057] In one embodiment, the solid phase is a surface plasmon resonance chip, dissociation is determined by surface plasmon resonance, and the antigen is immobilized to the surface plasmon resonance chip at least at 500 RU. In a preferred embodiment, the antigen is immobilized at 500-4000 RU.

[0058] In one embodiment, the binding of the antibody to the solid phase is strong.

[0059] In one embodiment, the first pH value is pH 7.4 or higher and the second pH value is pH 5.5 or lower, or vice versa.

[0060] In one embodiment, if the antigen is not monomeric, the antibody is monomeric, and vice versa.

[0061] In one embodiment, the antibody is bivalent with respect to its antigen.

[0062] In one embodiment, dissociation occurs when the binding signal changes by 5% or more. In one embodiment, dissociation occurs when the binding signal changes by 10% or more.

[0063] In one embodiment, the antigen is a soluble antigen.

[0064] In one embodiment, the antibody is a monospecific antibody.

[0065] In one embodiment, the antibody is a bispecific antibody.

[0066] In one embodiment, the antibody is a bispecific antibody having a first binding site that specifically binds to a first epitope on the antigen and a second binding site that specifically binds to a second epitope on the antigen.

[0067] In one embodiment, the capture is performed by injecting the antibody for 45 to 720 seconds at a flow rate of 2.5 to 30 μL / min, in a preferred embodiment at a flow rate of 2.5 to 10 μL / min and at a concentration of 0.6 μg / mL to 30 μg / mL. In a preferred embodiment, the capture is performed 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.

[0068] In one embodiment, the pH gradient lasts from 100 to 10,000 seconds. In one embodiment, the pH gradient lasts from 1000 to 5000 seconds. In one embodiment, the pH gradient lasts from 2000 to 2300 seconds.

[0069] In one embodiment, the dissociation pH is determined in a first step using the method according to the invention.

[0070] In one embodiment, the dissociation pH is maintained for 100 to 10,000 seconds. In one embodiment, the dissociation pH is maintained for 150 to 1,000 seconds.

[0071] One aspect as reported herein is an assay or a method for determining whether the binding of a (monoclonal) antibody to its antigen is pH dependent (pH dependent antigen binding), wherein the method comprises the following steps:

[0072] a) at a first pH value, capturing / immobilizing the antibody on a solid phase using a capture reagent that specifically binds to the constant domain of the antibody,

[0073] b) incubating the captured antibody with its antigen / applying its antigen to the captured antibody to form a captured antibody-antigen complex until a stable / constant binding signal is obtained,

[0074] c) applying a pH gradient from a first pH value to a second pH value to the solid phase and monitoring the dissociation of the antigen from the antibody,

[0075] If dissociation of the antigen from the antibody can be detected in step c), the binding of the antibody to its antigen is pH-dependent.

[0076] One aspect as reported herein is an assay or method for selecting a (monoclonal) antibody (with pH-dependent antigen binding) with pH-dependent binding to its antigen, wherein the method comprises the following steps:

[0077] a) at a first pH value, capturing / immobilizing the antibody on a solid phase using a capture reagent that specifically binds to the constant domain of the antibody,

[0078] b) incubating the captured antibody with its antigen / applying its antigen to the captured antibody to form a captured antibody-antigen complex until a stable / constant binding signal is obtained,

[0079] c) applying a pH gradient from a first pH value to a second pH value to the solid phase and monitoring the dissociation of the antigen from the antibody,

[0080] If dissociation of the antigen and the antibody can be detected in step c), the antibody is selected.

[0081] One aspect as reported herein is an assay or method for determining the pH value at which an antibody having pH-dependent binding to its antigen dissociates from its antigen (pH-dependent dissociation), wherein the method comprises the following steps:

[0082] a) at a first pH value, capturing / immobilizing the antibody on a solid phase using a capture reagent that specifically binds to the constant domain of the antibody,

[0083] b) incubating the captured antibody with its antigen / applying its antigen to the captured antibody to form a captured antibody-antigen complex until a stable / constant binding signal is obtained,

[0084] c) applying a pH gradient from a first pH value to a second pH value to the solid phase and monitoring the dissociation of the antigen from the antibody,

[0085] The pH value at which the antigen and the antibody dissociate is the pH value at which the pH gradient at which the antigen and the antibody dissociate can be detected in step c).

[0086] One aspect as reported herein is a method for determining the dissociation rate constant k of an antibody from its antigen. d An assay or method wherein the antibody has a pH-dependent binding to its antigen at a dissociation pH (the dissociation pH is a pH at which dissociation of the antibody from its antigen can be detected) (pH-dependent dissociation), wherein the method comprises the following steps:

[0087] a) at a first pH value, capturing / immobilizing the antibody on a solid phase using a capture reagent that specifically binds to the constant domain of the antibody,

[0088] b) incubating the captured antibody with its antigen / applying its antigen to the captured antibody to form a captured antibody-antigen complex until a stable / constant binding signal is obtained,

[0089] c) applying a pH gradient from a first pH value to a dissociation pH value to the solid phase and then maintaining the pH value at the dissociation pH value,

[0090] d) Record the binding signal and calculate the dissociation rate constant k from it d , and thus determine the dissociation rate constant k at the dissociation pH d .

[0091] One aspect as reported herein is an assay or method for determining the influence of antibody oligomerization on the pH-dependent binding of a (monoclonal) antibody to its antigen, wherein the method comprises the following steps:

[0092] a) capturing / immobilizing the monomeric antibody on a solid phase at a first pH value using a capture reagent that specifically binds to the constant domain of the antibody,

[0093] b) incubating the captured antibody with its antigen / applying its antigen to the captured antibody to form a captured antibody-antigen complex until a stable / constant binding signal is obtained,

[0094] c) applying a pH gradient from a first pH value to a second pH value to the solid phase, monitoring the dissociation of the antigen from the antibody, and thereby calculating the dissociation rate,

[0095] d) repeating steps a) to c) with dimer, trimer, tetramer and / or oligomer antibodies,

[0096] Wherein if the dissociation rates calculated in step c) differ by more than 10% for a monomeric antibody and a dimeric, trimer, tetrameric and / or oligomeric antibody, then the pH-dependent binding of the antibody to its antigen is affected by oligomerization.

[0097] One aspect as reported herein is an assay or method for determining the influence of antigen oligomerization on the pH-dependent binding of a (monoclonal) antibody to its antigen, wherein the method comprises the following steps:

[0098] a) at a first pH value, capturing / immobilizing the antibody on a solid phase using a capture reagent that specifically binds to the constant domain of the antibody,

[0099] b) incubating the captured antibody with its monomeric antigen / applying its monomeric antigen to the captured antibody to form a captured antibody-antigen complex until a stable / constant binding signal is obtained,

[0100] c) applying a pH gradient from a first pH value to a second pH value to the solid phase, monitoring the dissociation of the antigen from the antibody, and thereby calculating the dissociation rate,

[0101] d) repeating steps a) to c) with dimer, trimer, tetramer and / or oligomer antigens,

[0102] Wherein if the dissociation rates calculated in step c) differ by more than 10% for monomeric antigen and dimeric, trimer, tetrameric and / or oligomeric antigen, then the pH-dependent binding of the antibody to its antigen is affected by oligomerization.

[0103] Embodiments of the aspects outlined above are described below. It should be understood that all possible combinations of the embodiments with each other are also disclosed.

[0104] In one embodiment, all conditions except pH are kept constant during step c).

[0105] In one embodiment, dissociation is determined by surface plasmon resonance.

[0106] In one embodiment, the solid phase is a surface plasmon resonance chip.

[0107] In one embodiment, the antibodies are immobilized at high density.

[0108] In one embodiment, the solid phase is a surface plasmon resonance chip, dissociation is determined by surface plasmon resonance, and the antibody is immobilized to the surface plasmon resonance chip at least at 50 RU. In a preferred embodiment, the antibody is immobilized at 50-400 RU.

[0109] In one embodiment, the first pH value is pH 7.4 or higher and the second pH value is pH 5.5 or lower, or vice versa.

[0110] In one embodiment, if the antigen is not monomeric, the antibody is monomeric, and vice versa.

[0111] In one embodiment, the antibody is bivalent with respect to its antigen.

[0112] In one embodiment, dissociation occurs when the binding signal changes by 5% or more. In one embodiment, dissociation occurs when the binding signal changes by 10% or more.

[0113] In one embodiment, the antigen is a soluble antigen.

[0114] In one embodiment, the antibody is a monospecific antibody.

[0115] In one embodiment, the antibody is a bispecific antibody.

[0116] In one embodiment, the antibody is a bispecific antibody having a first binding site that specifically binds to a first epitope on the antigen and a second binding site that specifically binds to a second epitope on the antigen.

[0117] In one embodiment, the capture is performed by injecting the antibody for 45 to 720 seconds at a flow rate of 2.5 to 30 μL / min, in a preferred embodiment at a flow rate of 2.5 to 10 μL / min and at a concentration of 0.6 μg / mL to 30 μg / mL. In a preferred embodiment, the capture is performed 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.

[0118] In one embodiment, the pH gradient lasts from 100 to 10,000 seconds. In one embodiment, the pH gradient lasts from 1000 to 5000 seconds. In one embodiment, the pH gradient lasts from 2000 to 2300 seconds.

[0119] In one embodiment, the dissociation pH is determined in a first step using the method according to the invention.

[0120] In one embodiment, the dissociation pH is maintained for 100 to 10,000 seconds. In one embodiment, the dissociation pH is maintained for 150 to 1,000 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 Classical SPR-BIAcore kinetic protocol, where only one constant buffer condition is applied for one measurement.

[0122] Figure 2 Exemplary sensorgrams of the dissociation of a pH-dependent antibody from a solid surface using a pH gradient from pH 7.4 to pH 4.9 according to the methods of the present invention.

[0123] Figure 3 Example sensorgram for determining the dissociation rate constant at the dissociation pH. DETAILED DESCRIPTION

[0124] definition

[0125] The term "about" means a range of + / -20% of the value that follows. In one embodiment, the term "about" means a range of + / -10% of the value that follows. In one embodiment, the term "about" means a range of + / -5% of the value that follows.

[0126] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0127] Antibodies generally comprise two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each of the heavy chain polypeptides and the light chain polypeptides contains a variable domain (variable region) (usually the amino terminal portion of the polypeptide chain), which comprises a binding region capable of interacting with an antigen. Each of the heavy chain polypeptides and the light chain polypeptides comprises a constant region (usually the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of antibodies i) to cells (such as phagocytes) carrying Fcγ receptors (FcγR) or ii) to cells carrying neonatal Fc receptors (FcRn) (also referred to as Brambell receptors). It also mediates the binding to some factors, including factors of the classical complement system, such as components (C1q). The constant domain of the heavy chain of an antibody comprises a CH1 domain, a CH2 domain, and a CH3 domain, while the light chain comprises only one constant domain CL, which can be a κ isotype or a λ isotype.

[0128] The variable domain of an immunoglobulin light or heavy chain comprises different segments, namely four framework regions (FR) and three hypervariable regions (HVR).

[0129] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0130] The term "binding to" refers to the binding of a binding site to its target, e.g., the binding of an antibody binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain (VH / VL pair) to a corresponding antigen. This binding can be measured using, e.g. The ELISA kit was used to determine the ELISA result.

[0131] For example, in In a possible embodiment of the assay, the antigen is bound to a surface and the binding of the antibody binding site is measured by surface plasmon resonance (SPR). The affinity of the binding is expressed in terms of ka (association constant: rate constant for association to form a complex), kd (dissociation constant: rate constant for dissociation of the complex), and K D (k d / k a Alternatively, the binding signal of the SPR sensorgram can be directly compared with the response signal of a reference substance with respect to the resonance signal height and dissociation behavior.

[0132] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production process of monoclonal antibody preparations, such variants are usually presented in small amounts), each antibody comprising the population is identical and / or binds to the same epitope. Contrary to the polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0133] As used herein, the term "hypervariable region" or "HVR" refers to each of the following: regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). Typically, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0134] (a) hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0135] (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0136] (c) antigenic contact points present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0137] (d) a combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0138] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al.

[0139] The term "valency" as used in this application indicates the presence of a specified number of binding sites in a (antibody) molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" indicate the presence of two binding sites, four binding sites and six binding sites in a (antibody) molecule, respectively. A bispecific antibody as reported herein is a preferred embodiment of "bivalent".

[0140] In certain embodiments, the antibody is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In certain embodiments, one of the binding specificities is for a first antigen, and the other is for a different second antigen. In certain embodiments, a multispecific antibody can bind to two different epitopes of the same antigen. Multispecific antibodies can also be used to localize cytotoxic agents to cells expressing the antigen. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0141] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" engineering (see, e.g., US 5,731,168). Multispecific antibodies can also be prepared by the following methods: engineering electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553); using the "diabody" technology to prepare bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); using single-chain Fv (scFv) dimers (Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

[0142] The antibody or fragment may also be a multispecific antibody as described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 or WO 2010 / 145793.

[0143] The antibody or fragment thereof may also be a multispecific antibody as disclosed in WO 2012 / 163520.

[0144] Methods reported in this paper

[0145] The increasing complexity of novel biotherapeutics such as antibodies poses new challenges for functional characterization. This is especially true if antibodies that display pH-dependent binding to their corresponding antigens must be characterized.

[0146] For example, exemplary antibodies with pH-dependent antigen binding and their production are reported in EP 2 275 443. In principle, pH-dependent antigen binding can be introduced into any antibody. By introducing pH-dependent antigen binding into an antibody, a single antigen-binding antibody can be converted into a repeated antigen-binding antibody that can bind to multiple antigen molecules. When an antibody binds to multiple antigen molecules, this antibody exerts a more superior in vivo effect than common antigen-binding molecules.

[0147] The method of weakening the antigen-binding activity of an antibody at pH 5.8 compared to the antigen-binding activity at pH 7.4 (the method of imparting pH-dependent binding ability) is not particularly limited and may be any method. Such methods include, for example, a method of weakening the antigen-binding activity at pH 5.8 compared to the antigen-binding activity at pH 7.4 by replacing other amino acids in the antibody with histidine or inserting histidine into the antibody. It is known that pH-dependent antigen-binding activity can be imparted to an antibody by replacing amino acids in an antibody with histidine (FEBS Letter, 309 (1), 8588 (1992)). Such a histidine mutation (substitution) or insertion site is not particularly limited, and any site is acceptable as long as the antigen-binding activity at pH 5.8 is lower than the antigen-binding activity at pH 7.4 (K) compared to before the mutation or insertion. D (pH5.8) / K D (pH 7.4) value becomes larger). In the case of antibodies, such sites include, for example, sites within the variable region of the antibody. A person skilled in the art can appropriately determine the appropriate number of histidine mutation or insertion sites.

[0148] Histidine can be substituted or inserted at a single site or at two or more sites. It is also feasible to introduce non-histidine mutations (amino acid mutations other than histidine) at the same time. In addition, histidine mutations can be introduced simultaneously with histidine insertions. Histidine can be randomly substituted or inserted using methods such as histidine scanning, which uses histidine instead of alanine in alanine scanning known to those skilled in the art. Alternatively, a library of antigen-binding molecules with random histidine mutations or insertions can be selected that is K compared to before mutation. D (pH5.8) / K D (pH 7.4) increased antigen-binding molecules.

[0149] When histidine replaces an amino acid of an antibody or is inserted between amino acids of an antibody, it is preferred but not required that the antigen-binding activity of the antibody at pH 7.4 after the histidine replacement or insertion is comparable to the antigen-binding activity of the antibody at pH 7.4 before the histidine replacement or insertion. "The antigen-binding activity of the antibody at pH 7.4 after the histidine replacement or insertion is comparable to the antigen-binding activity of the antibody at pH 7.4 before the histidine replacement or insertion" means that even after the histidine replacement or insertion, the antibody still retains 10% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more of the antigen-binding activity before the histidine replacement or insertion. When the antigen-binding activity of an antibody is weakened by histidine replacement or insertion, the antigen-binding activity can be adjusted by introducing substitution, deletion, addition and / or insertion of one or more amino acids in the antibody so that the antigen-binding activity becomes comparable to the antigen-binding activity before the histidine replacement or insertion.

[0150] Alternative methods for weakening the antigen binding activity of antibodies at pH 5.8 compared to the antigen binding activity at pH 7.4 include methods of replacing amino acids in antibodies with non-natural amino acids or inserting non-natural amino acids into antibody amino acids. It is known that non-natural amino acids can be used to artificially control pKa (Angew. Chem. Int. Ed. 2005, 44, 34; Chem. Soc. Rev. 2004 Sep 10; 33 (7): 422-30; Amino Acids. 1999; 16 (3-4): 345-79). Therefore, non-natural amino acids can be used to replace the histidine described above. Such non-natural amino acid substitutions and / or insertions can be introduced simultaneously with the histidine substitutions and / or insertions described above. Any non-natural amino acid can be used in the present invention. Non-natural amino acids known to those skilled in the art can be used.

[0151] It is therefore an object of the present invention to provide a method for determining the pH-dependent antigen binding of an antibody multiple times binding to its antigen.

[0152] Standard SPR-based assay setups have been described in the art. These setups allow the binding activity of an antibody to its antigen to be assessed under a set of conditions.

[0153] In the present invention, the absolute difference in antigen-binding activity between acidic and neutral pH is not important as long as the antigen-binding activity at acidic pH is different from that at neutral pH.

[0154] However, in one embodiment, K D (pH5.8) / K D (pH7.4) value (i.e., the dissociation constant (K) for the antigen at pH 5.8 and at pH 7.4) D The ratio of ) is preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. D (pH5.8) / K D The upper limit of the (pH 7.4) value is not particularly limited, and may be any value, such as 400, 1,000 or 10,000, as long as the molecule can be produced by the technique of a person skilled in the art.

[0155] However, in one embodiment, K D (pH7.4) / K D (pH5.8) value (i.e., the dissociation constant (K) for the antigen at pH 7.4 and at pH 5.8) D The ratio of ) is preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. D (pH7.4) / K D The upper limit of the (pH 5.8) value is not particularly limited, and may be any value, such as 400, 1,000 or 10,000, as long as the molecule can be produced by the techniques of those skilled in the art.

[0156] When the antigen is a soluble antigen, the antigen binding activity can be expressed by the dissociation constant (K D Alternatively, when the antigen is a membrane antigen, the antigen binding activity can be represented by the apparent dissociation constant. The dissociation constant (K D ) and the apparent dissociation constant (apparent K D ) can be determined by methods known to those skilled in the art, for example, using BIAcore (GE healthcare), Scatchard plot or FACS.

[0157] In one embodiment, when the dissociation rate constant (k d ) instead of the dissociation constant (K D ) is used as an indicator of binding activity differences, k d (pH5.8) / kd (pH 7.4) value (i.e., the dissociation rate constant (k) for the antigen at pH 5.8 and at pH 7.4) d The ratio of ) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and still more preferably 30 or more. d (pH5.8) / k d The upper limit of the (pH 7.4) value is not particularly limited and may be any value, such as 50, 100 or 200, as long as the molecule can be produced by techniques known to those skilled in the art.

[0158] In one embodiment, when the dissociation rate constant (kd) is used as an indicator of the difference in binding activity instead of the dissociation constant (KD), the kd (pH 7.4) / kd (pH 5.8) value (i.e., the ratio of the dissociation rate constant (kd) for the antigen at pH 7.4 to that at pH 5.8) is preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and still more preferably 30 or more. The upper limit of the kd (pH 7.4) / kd (pH 5.8) value is not particularly limited, and may be any value, such as 50, 100 or 200, as long as the molecule can be produced by a technique known to those skilled in the art.

[0159] When the antigen is a soluble antigen, the antigen binding activity can be expressed as the dissociation rate constant (k d ). Alternatively, when the antigen is a membrane antigen, the antigen binding activity can be represented by the apparent dissociation rate constant (apparent kd). The dissociation rate constant (k d ) and the apparent dissociation rate constant (apparent k d ) can be determined by the method according to the invention directly at the dissociation pH.

[0160] Herein, weakening the antigen-binding activity at acidic pH compared to the antigen-binding activity at neutral pH means weakening the antigen-binding ability of the antibody at pH 4.0 to pH 6.5 compared to the antigen-binding ability at pH 6.7 to pH 10.0, preferably weakening the antigen-binding activity of the antibody at pH 5.5 to pH 6.5 compared to the antigen-binding activity at pH 7.0 to pH 8.0, more preferably weakening the antigen-binding activity of the antibody at pH 5.8 compared to the antigen-binding activity at pH 7.4. Therefore, in the present invention, the acidic pH is generally pH 4.0 to pH 6.5, preferably pH 5.5 to pH 6.5, and more preferably pH 5.8. Alternatively, in the present invention, the neutral pH is generally pH 6.7 to pH 10.0, preferably pH 7.0 to pH 8.0, and more preferably pH 7.4.

[0161] Therefore, in one embodiment, the first pH value is between pH 6.7 and pH 10.0 and includes the end value, and the second pH value is between pH 4.0 and pH 6.5 and includes the end value. In one embodiment, the first pH value is between pH 7.0 and pH 8.0 and includes the end value, and the second pH value is between pH 5.5 and pH 6.5 and includes the end value. In one embodiment, the first pH value is between pH 7.2 and pH 7.6 and includes the end value, and the second pH value is between pH 5.5 and pH 5.8 and includes the end value. In a preferred embodiment, the first pH value is about pH 7.4, and the second pH value is between pH 5.5 and pH 5.8 and includes the end value.

[0162] The present invention is based, at least in part, on the discovery that surface plasmon resonance experiments can be performed using a pH gradient. Thus, by determining the precise pH at which this dissociation of an antibody from its antigen occurs, i.e., the dissociation pH, on the one hand, and by determining the dissociation rate constant at said dissociation pH, on the other hand, the pH-dependent antigen binding of an antibody can be characterized.

[0163] The method according to the present invention includes recording or monitoring the binding signal of the antibody and the antigen as an essential step while applying a pH gradient. At the pH gradient point where the binding signal begins to decrease, the interaction between the antibody and its antigen is weakened due to the protonation or deprotonation of the amino acid residues in the binding site. The pH gradient point defines the dissociation pH value of the pH-dependent interaction between the antibody and its antigen.

[0164] For example, to assess the suitability of an antibody with pH-dependent antigen binding as a repeat antigen-binding antibody capable of binding to multiple antigen molecules, the pH-dependent antigen binding can be determined using the method according to the invention. If binding to the antigen is reduced (or even abolished) at lysosomal pH (i.e., below pH 6.0), preferably at about pH 5.0, while binding is maintained (or even maximal) at physiological pH (i.e., above pH 7.0), preferably at about pH 7.4, the antibody is suitable for the intended purpose.

[0165] Therefore, the method according to the present invention comprises the following core steps

[0166] - exposing the antibody-antigen-complex to a pH gradient from physiological pH to lysosomal pH and determining i) if any,

[0167] or

[0168] ii) The precise pH value for antibody-antigen-complex dissociation.

[0169] Although the above has been outlined for a pH gradient from neutral to acidic pH values, the method according to the invention can be used with any pH gradient, ie from neutral to acidic or from neutral to alkaline or from alkaline to acidic or from acidic to alkaline.

[0170] Thus, one aspect as reported herein is an assay or a method for determining whether the binding of a (monoclonal) antibody to its antigen is pH dependent (pH dependent antigen binding), wherein the method comprises the following steps:

[0171] a) capturing / immobilizing the antibody on a solid phase to which the antigen has been conjugated at a first pH value,

[0172] b) applying a pH gradient from a first pH value to a second pH value to the solid phase and monitoring the dissociation of the antibody from the solid phase,

[0173] If dissociation of the antibody from the solid phase can be detected in step b), the binding of the antibody to its antigen is pH-dependent.

[0174] One aspect as reported herein is a method for determining the dissociation rate constant k of an antibody from its antigen. d An assay or method wherein the antibody has a pH-dependent binding to its antigen at a dissociation pH (the dissociation pH is a pH at which dissociation of the antibody from its antigen can be detected) (pH-dependent dissociation), wherein the method comprises the following steps:

[0175] a) at a first pH value, capturing / immobilizing the antibody on a solid phase using a capture reagent that specifically binds to the constant domain of the antibody,

[0176] b) incubating the captured antibody with its antigen to form a captured antibody-antigen complex until a stable / constant binding signal is obtained,

[0177] c) applying a pH gradient from a first pH value to a dissociation pH value to the solid phase and then maintaining the pH value at the dissociation pH value,

[0178] d) Record the binding signal and calculate the dissociation rate constant k from it d , and thus determine the dissociation rate constant k at the dissociation pH d .

[0179] This approach is advantageous because by lowering the pH from above the dissociation pH to the dissociation pH, the processes during endocytosis and lysosomal acidification can be simulated. In addition, the starting point for determining the dissociation rate constant can be precisely set, allowing a more accurate determination of the dissociation rate constant at the dissociation pH.

[0180] References

[0181] [1]M.A.Cooper,Nature Reviews Drug Discovery 1,(2002)515-528.

[0182] [2]D.G.Myszka,Journal of Molecular Recognition 12,(1999)390-408.

[0183] [3]R.L.Rich,D.G.Myszka,Journal of Molecular Recognition 13,(2000)388-407.

[0184] [4]D.G.Myszka,R.L.Rich,Pharmaceutical science&technology today 3,(2000)310-317.

[0185] [5]R.Karlsson,A.Faelt,Journal of immunological methods 200,(1997)121-133.

[0186] [6]T.A.Morton,D.G.Myszka,Methods in enzymology 295,(1998)268-294.

[0187] [7]K.Nagata,H.Handa,Real-time analysis of biomolecular interactions,Springer,2000.

[0188] [8]R.L.Rich,D.G.Myszka,Current opinion in biotechnology 11,(2000)54-61.

[0189] [9]A.C.Malmborg,C.A.Borrebaeck,Journal of immunological methods 183,(1995)7-13.

[0190]

[10] W.Huber,F.Mueller,Current pharmaceutical design 12,(2006)3999-4021.

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[11] C. Gassner, F. Lipsmeier, P. Metzger, H. Beck, A. Schnueriger, JTRegula, J. Moelleken, Journal of pharmaceutical and biomedical analysis 102, (2015) 144-149.

[0192] The following examples and drawings are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0193] Example

[0194] Equipment and reagents

[0195] All SPR experiments were performed on a Xantec SR7500DC with a sensor chip Xantec SCR CMD500L.

[0196] Examples 1

[0197] Chip preparation

[0198] To immobilize the antigen, two flow cells (Fc) were prepared, Fc1 as a blank control and Fc2 as a specific target binding interaction surface. To immobilize the antigen on flow cell two, its surface had been activated with EDC / NHS to prepare the amine coupling of the dimeric antigen construct. The antigen dimer had been prepared in 10 mM acetate buffer at pH 4.5 at a concentration of 30 μg / ml. This solution was injected onto Fc2 at a flow rate of 20 μl / min for 10 minutes.

[0199] Running a pH gradient

[0200] To set up the gradient, two PBS-P+ buffers were prepared. Buffer A was set to pH 7.4. Buffer B was set to pH 4.9.

[0201] Antibody samples at a concentration of 100 nM were injected in buffer A for 90 seconds at a flow rate of 100 μl / min. After equilibration for 210 seconds, the gradient was started from 100% buffer A to 100% buffer B in 2400 seconds. At the end of the run, the surface was regenerated with 10 mM glycine buffer, pH 1.5.

[0202] The pH at which the pH-dependent binder starts to dissociate was determined by a 25:75 ratio of buffer A to buffer B. The mixture was measured to give a pH of 5.85 to 5.9.

[0203] Example 2

[0204] Chip preparation

[0205] To immobilize the anti-human Fc capture antibody ( <hfc>GE BR-1008-39), two flow cells were prepared, Fc1 as a blank control, and Fc2 as a specific target binding interaction surface. In order to immobilize the capture antibody on the flow cell two, its surface had been activated with EDC / NHS to prepare amine coupling. <hfc>It has been prepared in 10 mM acetate buffer at pH 4.5 at a concentration of 30 μg / ml. This solution was injected over Fc2 at a flow rate of 20 μl / min for 10 minutes.

[0206] Run pH gradient after antibody

[0207] To establish the gradient, two PBS-P+ buffers were prepared. The pH of buffer A was set to 7.4. Buffer B was set to pH 4.9. The antibody in question was injected into buffer A at a concentration of 100 nM for 90 seconds at a flow rate of 100 μl / min. In the second step, different antigen preparations (dimer, wild type and monomer) were injected into buffer A at a concentration of 100 nM for 90 seconds. After 210 seconds of equilibrium, the gradient started from 100% buffer A to 100% buffer B in 2400 seconds. At the end of the run, the surface was regenerated with a 3M MgCl2 solution.< / hfc> < / hfc>

Claims

1. A method for determining the dissociation rate constant k between an antibody and its antigen at the dissociation pH value d A surface plasmon resonance method, wherein the method comprises the following steps: a) immobilizing the antibody on a solid phase of the antigen to which the antibody has been conjugated at a first pH value, b) applying a pH gradient from said first pH value to said dissociation pH value and then maintaining the pH value at said dissociation pH value, c) recording the binding signal during the period of maintaining the pH value, and calculating the dissociation rate constant k between the antibody and its antigen at the dissociation pH value d .

2. A method for determining the dissociation rate constant k between an antibody and its antigen at the dissociation pH value d A surface plasmon resonance method, wherein the method comprises the following steps: a) immobilizing the antibody on a solid phase at a first pH value using a capture reagent that specifically binds to a constant domain of the antibody, b) incubating the captured antibody with its antigen to form a captured antibody-antigen complex, c) applying a pH gradient from the first pH value to the dissociation pH value to the solid phase and then maintaining the pH value at the dissociation pH value, d) recording the binding signal during the period of maintaining the pH value, and calculating the dissociation rate constant k of the antibody and its antigen at the dissociation pH value therefrom d .

3. The method according to any one of claims 1 to 2, wherein the dissociation pH is determined by a method comprising the following steps: i) immobilizing the antibody on a solid phase of the antigen to which the antibody has been conjugated at a first pH value, ii) applying a pH gradient from the first pH value to a second pH value to the solid phase and monitoring the dissociation of the antibody from the solid phase, The pH value at which the antibody dissociates from its antigen is the pH value of the pH gradient at which the antibody can be detected to dissociate from the solid phase in step ii).

4. The method according to any one of claims 1 to 2, wherein the dissociation pH is determined by a method comprising the following steps: i) immobilizing the antibody on a solid phase at a first pH value using a capture reagent that specifically binds to a constant domain of the antibody, ii) incubating the captured antibody with its antigen to form a captured antibody-antigen complex, iii) applying a pH gradient from the first pH value to a second pH value to the solid phase and monitoring the dissociation of the antigen from the antibody, The pH value at which the antigen dissociates from the antibody is the pH value of the pH gradient at which the dissociation of the antigen from the antibody can be detected in step iii).

5. The method according to any one of claims 1 to 4, wherein the dissociation is determined by surface plasmon resonance.

6. The method of claim 1 or any one of claims 3 to 5, wherein the antigen is immobilized at a high density.

7. The method according to any one of claims 1 to 6, wherein the first pH value is between pH 6.7 and pH 10.0, inclusive, and the second pH value is between pH 4.0 and pH 6.5, inclusive.

8. The method of any one of claims 1 to 7, wherein the first pH value is about pH 7.4 and the second pH value is between pH 5.5 and pH 5.8, inclusive.

9. The method of any one of claims 1 to 8, wherein dissociation occurs when the binding signal changes by 5% or more.

10. The method according to any one of claims 1 to 9, wherein the antigen is a soluble antigen.

11. The method according to any one of claims 1 to 10, wherein the antibody is a monospecific antibody.

12. The method according to any one of claims 1 to 11, wherein capturing is performed by injecting the antibody at a flow rate of 2.5 to 30 μL / min and at a concentration of 0.6 μg / mL to 30 μg / mL for 45 to 720 seconds.

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