Methods of determining potency of a human neonatal FC receptor antagonist

Abstract

Methods of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of human IgG to FcRn using a competition binding assay are provided. Kits for carrying out said methods, and methods of manufacturing a FcRn antagonist composition comprising said methods of determining potency are also provided.

Description

[0001] METHODS OF DETERMINING POTENCY OF A HUMAN NEONATAL FC RECEPTOR ANTAGONIST

[0002] FIELD OF THE INVENTION

[0003] 5 The present invention relates to methods of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of human IgG to FcRn using a competition binding assay. In further aspects, the present invention provides kits for carrying out said methods, and methods of manufacturing a FcRn antagonist composition comprising said methods of determining potency.

[0004] BACKGROUND OF THE INVENTION

[0005] The human neonatal Fc receptor (FcRn) mediates IgG homeostasis. Specifically, FcRn acts as the salvage receptor for IgG; the receptor binds to the Fc portion of IgG in the early endosome and traffics IgG away from lysosomal degradation and back to the extracellular space. This

[0006] 15 mechanism extends the serum half-life of IgG. It also permits IgG transport across cellular barriers (such as the epithelium in the gut or lung). Thus, inhibiting the interaction of endogenous IgG with FcRn has therapeutic potential for the treatment of antibody-mediated autoimmune disease due to the accelerated clearance and degradation of pathogenic autoreactive IgGs.

[0007] 20 Several FcRn antagonists have been developed to outcompete human IgG for binding to FcRn, including anti-FcRn antibodies and peptides. More recently, modified Fc regions have been developed that have increased affinity for FcRn compared to the Fc region of wild-type IgG. For example, efgartigimod is a modified Fc fragment that shows clinical benefit for several IgG- mediated autoimmune diseases, such as generalized myasthenia gravis (gMG) and chronic

[0008] 25 inflammatory demyelinating polyneuropathy (CIDP).

[0009] The manufacturing and purification of biological drug substances for clinical use is often a complex process, requiring careful control so as to yield a product that is stable, safe for administration to humans and that retains the required level of identity and biological activity. It is necessary for biological drugs, such as FcRn antagonists, to be examined before release for clinical use. All manufacturing lots or batches must typically undergo testing for features of the product such as physiochemical properties, biological activity and purity. Only those that fulfil defined release criteria can be made available for clinical use.

[0010] 35 Biological drugs that bind to a particular target are often tested for binding activity using techniques such as surface plasmon resonance or Western Blot. However, these assays are limited in the information that they can provide and are often not representative of the in vivo biological mode of action of the drug.

[0011] SUMMARY OF THE INVENTION

[0012] It is an object of the present invention to provide methods for testing FcRn antagonist compositions so as to better assess the biological activity of the compositions in outcompeting human IgG for binding to FcRn. The present application discloses, for the first time, analytical methods for measuring the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of human IgG or an IgG Fc region to FcRn. By measuring this competition or inhibition, the methods provide significantly more information than simply measuring the binding affinity of the FcRn antagonist composition for FcRn. In particular, the methods model the in vivo biological effect of FcRn antagonists in blocking recycling of endosomal IgG, which results in an increased rate of IgG degradation and, concomitantly, a reduced serum level of IgG. Thus, the methods are of particular benefit in determining the potency of FcRn antagonist compositions for treating IgG-mediated autoimmune diseases.

[0013] It follows that, in a first aspect, the invention provides a method of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of human IgG to FcRn using a competition binding assay, the assay comprising the steps of: a) incubating the FcRn antagonist composition with FcRn in the presence of human IgG such that FcRn antagonist and human IgG compete for binding to FcRn; b) separating any FcRn - human IgG complexes from any unbound human IgG; and c) detecting the quantity of FcRn - human IgG complexes.

[0014] In some embodiments of this first aspect, the human IgG with which the FcRn antagonist competes is human lgG3. The present inventors have found that lgG3 has the largest assay window (signal-to-noise ratio) of any human IgG and is therefore better at detecting small differences in potency between test FcRn antagonist compositions (e.g., obtained from different manufacturing lots). Nevertheless, any other human IgG may be used, such as human lgG1 , lgG2 or lgG4.

[0015] In some embodiments, steps a) - c) of the assay are carried out at a pH of about 5 to about 8. Advantageously, conducting the assay at this pH models in vivo inhibition of endogenous IgG- FcRn interactions.

[0016] In some embodiments, steps a) - c) are carried out at a pH of about 5.5 to about 6.5. In some embodiments, steps a) - c) are carried out at a pH of about 6.0. Conducting the assay at a pH of 5.5 to 6.5, or about 6.0, models the competition or inhibition of endogenous IgG-FcRn interactions in the early endosome, and therefore the ability of FcRn antagonists to block IgG trafficking back to the cell surface.

[0017] In some embodiments, the FcRn antagonist composition is serially diluted such that two or more samples having different concentrations of the FcRn antagonist composition are incubated with FcRn in the presence of human IgG. As such, the results of the assay may be used to generate a dose response curve of the FcRn antagonist composition inhibiting the binding of human IgG to FcRn. The results of the assay may also or alternatively be used to calculate a half maximal inhibitory concentration (IC50) of the FcRn antagonist composition. Both dose response curves and IC50 values can easily and quickly be compared by a person skilled in the art, for example to identify variability in potency between test FcRn antagonist compositions.

[0018] In some embodiments, results of the assay are compared with a reference standard (“efgartigimod gold standard” or “error free standard” or a standard with known results). The reference standard is typically a FcRn antagonist composition suitable for clinical use. For example, the reference standard may be suitable for use in the treatment of an antibody- mediated autoimmune diseases, such as myasthenia gravis (MG), optionally generalized myasthenia gravis (gMG), or chronic inflammatory demyelinating polyneuropathy (CIDP), or immune thrombocytopenia (ITP) or pemphigus vulgaris (PV).

[0019] The assay may be repeated with the reference standard in place of the test FcRn antagonist composition. Repeating the assay with the reference standard FcRn antagonist composition is beneficial to minimise the impact of any differences in assay set-up or conditions, for example between two manufacturing sites.

[0020] In some embodiments, a relative potency of the FcRn antagonist composition is calculated as: (1050 of the reference standard / IC50 of the FcRn antagonist composition) x 100%.

[0021] In some embodiments, a FcRn antagonist composition having a relative potency of about 70% to about 135% is identified as a FcRn antagonist composition suitable for clinical use. In other embodiments, a FcRn antagonist composition having a relative potency of about 85% to about 120% is identified as a FcRn antagonist composition suitable for clinical use. For example, the FcRn antagonist may be identified as suitable for use in the treatment of an antibody-mediated autoimmune disease. In some embodiments, the antibody-mediated autoimmune disease is myasthenia gravis (MG), optionally generalized myasthenia gravis (gMG).

[0022] In some embodiments, the assay is repeated with an internal-assay control (IAC) in place of the test FcRn antagonist composition. Advantageously, repeating the assay with an IAC can be used to quality control the assay itself.

[0023] In some embodiments, the FcRn antagonist composition comprises or consists of a population of FcRn antagonist molecules.

[0024] In some embodiments, the FcRn antagonist molecules comprise or consist of a variant Fc region or FcRn binding fragment thereof that binds to FcRn with increased affinity relative to a wild-type Fc region of IgG.

[0025] In some embodiments, the variant Fc region comprises or consists of a first Fc domain and a second Fc domain which form a homodimer or heterodimer.

[0026] In some embodiments, the first Fc domain and / or the second Fc domain comprise amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. For example, the first Fc domain and / or second Fc domain may comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

[0027] In some embodiments, the first Fc domain and the second Fc domain each have an amino acid sequence represented by SEQ ID NO: 1 . Optionally, the first Fc domain and / or the second Fc domain comprise or consist of an amino acid sequence independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0028] In some embodiments, the population of FcRn antagonist molecules is heterogeneous.

[0029] In some embodiments, the population of FcRn antagonist molecules comprises or consists of efgartigimod.

[0030] In some embodiments, the population of FcRn antagonist molecules comprises or consists of a biosimilar of efgartigimod.

[0031] In some embodiments of any aspect, the reference standard is a FcRn antagonist composition comprising or consisting of efgartigimod. In some embodiments, the reference standard is a FcRn antagonist composition comprising or consisting of a biosimilar of efgartigimod.

[0032] In some embodiments, the method is carried out to confirm the identity of an efgartigimod alfa composition or of an efgartigimod biosimilar composition. The method may also or alternatively be carried out to assess the biological activity of an efgartigimod composition or of an efgartigimod biosimilar composition (for example, obtained from a particular manufacturing lot). The method may also or alternatively be carried out to assess the stability of an efgartigimod composition or of an efgartigimod biosimilar composition (for example, obtained from a particular manufacturing lot).

[0033] In some embodiments, in step a), the FcRn antagonist composition is combined with human IgG prior to the addition of FcRn. Without wishing to be bound by theory, this order of mixing is expected to achieve a more sensitive assay.

[0034] In some embodiments, the FcRn antagonist composition is serially diluted such that multiple samples in the concentration range 0.001 pg / mL to 20 pg / mL, optionally in the concentration range 0.004 pg / mL to 15 pg / mL, are incubated with FcRn in the presence of human IgG. In some embodiments, the FcRn antagonist composition is serially diluted such that multiple samples in the concentration range 0.010 pg / mL to 15 pg / mL are incubated with FcRn in the presence of human IgG.

[0035] In some embodiments, in step a), the concentration of human IgG is about 10.0 pg / mL or about 20.0 pg / mL. In some embodiments, in step a), the concentration of FcRn is about 0.5 pg / mL or about 1 .0 pg / mL. In some embodiments, in step a), the incubating is for at least 1 hour at ambient temperature.

[0036] In some embodiments, in step b), the FcRn - human IgG complexes are separated from any unbound human IgG by first immobilising the FcRn and secondly, removing any unbound human IgG in a wash step. Various methods of immobilisation are well known by a person skilled in the art. In some embodiments, the FcRn is biotinylated and is immobilised by contacting the FcRn with a substrate having a biotin-binding protein thereon, optionally streptavidin or neutravidin.

[0037] In some embodiments, step c) involves detecting FcRn - human IgG complexes using an antihuman IgG antibody. In some embodiments, the competition binding assay is a competition enzyme-linked immunosorbent assay (ELISA).

[0038] In some embodiments, the method comprises the steps of: ai) adding human IgG, preferably human lgG3, to multiple samples of the FcRn antagonist composition, each sample having a different concentration of the FcRn antagonist composition; aii) incubating the samples with biotinylated FcRn; bi) applying the samples to a substrate coated with a biotin-binding protein, optionally a neutravidin-coated ELISA plate, so as to immobilise the biotinylated FcRn; bii) washing the substrate one or more times to remove unbound human IgG, optionally wherein the wash buffer comprises PBS pH 6.0 + 0.05% Tween20; c) detecting the quantity of immobilised FcRn-human IgG complexes using an enzyme- linked anti-human IgG antibody and an enzyme substrate.

[0039] In some embodiments, in step c), the enzyme-linked anti-human IgG antibody is a peroxidase- linked antibody and / or the enzyme substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0040] In some embodiments, step c) comprises the sub-steps: ci) adding the peroxidase-linked antibody to the immobilised FcRn-IgG complexes and optionally, incubating for 1 hour at ambient temperature; cii) one or more washes with a wash buffer, optionally wherein the wash buffer comprises PBS pH 6.0 + 0.05% Tween20; ciii) the addition of TMB and optionally, incubating the FcRn-IgG-anti-IgG antibody complexes for 15 min at ambient temperature; civ) quenching the reaction by adding sulfuric acid, optionally 0.5M sulfuric acid; and cv) reading the absorbance at 450 nm with subtraction of the reference wavelength at 620 nm.

[0041] In a related second aspect, the invention provides a method of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of an IgG Fc region to FcRn using a competition binding assay, the assay comprising the steps of: a) incubating the FcRn antagonist composition with FcRn in the presence of an IgG Fc region such that FcRn antagonist and IgG Fc region compete for binding to FcRn; b) separating any FcRn - IgG Fc region complexes from any unbound IgG Fc region; and c) detecting the quantity of FcRn - IgG Fc region complexes. In some embodiments of this related aspect, the IgG Fc region with which the FcRn antagonist competes is a wild-type IgG Fc region. In some embodiments, the IgG Fc region is a human IgG Fc region, optionally a wild-type human IgG Fc region.

[0042] Embodiments described above in relation to the first aspect of the invention are equally applicable to the related second aspect.

[0043] In a further aspect, the invention provides a kit for carrying out methods in accordance with the first aspect of the invention, the kit comprising: a) human IgG or an IgG Fc region; b) FcRn; and c) means to detect FcRn-human IgG complexes or FcRn-IgG Fc region complexes.

[0044] In some embodiments, the kit comprises: a) the human IgG is human lgG3; b) the FcRn is biotinylated; and / or c) the means to detect the FcRn-human IgG complexes or the FcRn-IgG Fc region complexes comprises an enzyme-linked anti-human IgG antibody.

[0045] In some embodiments, the enzyme-linked anti-human IgG antibody is a peroxidase-linked antibody.

[0046] In some embodiments, the kit further comprises an enzyme substrate, optionally a chromogenic substrate, optionally 3,3',5,5'-tetramethylbenzidine (TMB).

[0047] In some embodiments, the kit further comprises an ELISA plate, optionally an ELISA plate coated with streptavidin or neutravidin.

[0048] In a further aspect, the invention provides a method of manufacturing a FcRn antagonist composition, comprising the method of determining the potency of a FcRn antagonist composition in accordance with the first aspect of the invention.

[0049] BRIEF DESCRIPTION OF THE FIGURES

[0050] FIG. 1 is a dose response curve showing the binding affinity of IgG for binding to biotinylated FcRn, captured on a neutravidin coated ELISA plate at pH 6.0. A higher signal to noise ratio was observed for hlgG3 compared to hlgG1 , lgG2 and lgG4. FIG. 2 depicts a schematic of an exemplary human lgG3 competitive ELISA of the present invention. Efgartigimod samples are pre-incubated with biotinylated FcRn and human lgG3 antibody. During the pre-incubation step, efgartigimod competes with the human lgG3 antibody for binding to biotinylated FcRn. After the pre-incubation step, the sample mixture is applied to an ELISA plate coated with neutravidin, which will capture and immobilise the biotinylated FcRn. The amount of human lgG3 antibody bound to FcRn is detected by the addition of antihuman IgG (Fab specific)-HRP linked antibody and a chromogenic substrate.

[0051] FIG. 3 depicts a typical ELISA plate set-up for use with the competitive ELISA of the present invention. The ELISA plate consists of duplicate titration series for: the reference standard (e.g. efgartigimod reference batch) sample (rows A and B); the Internal Assay Control (IAC) sample (rows C and D); and two FcRn antagonist composition samples (e.g. from two efgartigimod batches).

[0052] FIG. 4 Competition ELISA Response Curves and IC50 Values for different efgartigimod batches (batches 4-7) (upper panel) and different ARGX-113 Variants (20% and 60% oxidized efgartigimod) (lower panel).

[0053] FIG. 5 Competition ELISA IC50 Values with their 95% Cl for efgartigimod batches (batches 4- 7) and efgartigimod containing different levels of oxidized variants (20%, 60%) compared to reference standard P3179589ARS.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] A. Definitions

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art in the technical field of the invention. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0057] The term “human neonatal Fc receptor”, “human FcRn”, “hFcRn” or “FcRn” as used herein refers to the human cell surface receptor protein encoded by the FCGRT gene. The amino acid sequence of human FcRn is set forth in RefSeq NP_004098. In addition, the definition covers all isoforms and allelic variants of human FcRn. FcRn contributes to humoral immunity by recycling IgG and extending its serum half-life. Mechanistically, IgG is first taken up into FcRn- expressing cells through pinocytosis. The FcRn binds to the Fc region of IgG in early endosomes and traffics the IgG away from lysosomal degradation and to the extracellular space. A similar mechanism also permits IgG transport across cellular barriers (such as the epithelium in the gut or lung). Although not measured in the methods described herein, FcRn also recycles albumin in a similar fashion. Albumin interacts with FcRn at a site distinct from IgG, and therefore, binding of albumin and IgG may occur concurrently.

[0058] The term “FcRn antagonist” is used broadly herein to describe any innovator, generic, or biosimilar agent or molecule that specifically binds to FcRn. It encompasses peptides, small molecules, antibody fragments or antibodies. The method of the present invention tests the ability of a FcRn antagonist composition to inhibit (fully or partially) the binding of human IgG to FcRn. As explained elsewhere herein, FcRn antagonists with such capability have therapeutic potential for treating IgG-mediated autoimmune disease by accelerating the clearance and degradation of circulating IgG autoantibodies.

[0059] The term “FcRn antagonist composition” is used herein to mean a population of FcRn antagonist molecules, for example obtained from a particular manufacturing lot or batch. In some embodiments, the FcRn antagonist compositions described herein comprise or consist of a population of FcRn antagonist molecules wherein the FcRn antagonist molecules are variant Fc regions or FcRn binding fragments thereof. The variant Fc regions or FcRn binding fragments thereof may have increased binding affinity for FcRn relative to a wild-type Fc region of IgG. The population of FcRn antagonist molecules within the FcRn antagonist composition may be homogeneous. Alternatively, the population of FcRn antagonist molecules may be heterogeneous, for example comprising a sub-population of identical FcRn antagonist molecules, as well as FcRn antagonist molecules that differ with respect to amino acid sequence, glycosylation, oxidation status etc, as described elsewhere herein.

[0060] The term “human IgG” is used broadly herein to refer to any immunoglobulin or antibody molecule comprising the native Fc region of a human IgG (i.e., IgG 1 , lgG2, lgG3 or lgG4). Therefore, the term encompasses human, humanised and chimeric antibodies comprising the native Fc region of a human IgG. For example, “human lgG3” encompasses human, humanised and chimeric antibodies comprising the native Fc region of human lgG3. Moreover, the term encompasses but is not limited to recombinant, monoclonal and polyclonal human IgG antibodies.

[0061] The term “potency” as used herein is a measure of the FcRn antagonist composition’s biological activity. The potency of a FcRn antagonist composition may be reported in terms of the amount or concentration required to elicit the relevant biological effect. In the methods of the present invention, the potency of a FcRn antagonist composition in inhibiting the binding of human IgG to FcRn is specifically tested. The potency of a FcRn antagonist composition may be defined by its half maximal inhibitory concentration (IC50 value), i.e., the concentration required to inhibit the relevant biological effect (e.g., binding of human IgG to FcRn) by 50%. The lower the IC50, the greater the potency of the FcRn antagonist composition.

[0062] A “competition binding assay” or “competitive binding assay” as used herein, is an assay based on the competition between two binding molecules for binding to a target protein. A range of concentrations of one binding molecule is typically tested in the presence of a single concentration of the other competing binding molecule. In the methods of the present invention, a competition binding assay is used based on the competition between a test FcRn antagonist composition and human IgG for binding to FcRn. Typically, a range of concentrations of the FcRn antagonist composition is tested for competition with a single concentration of human IgG for binding to FcRn.

[0063] As used herein a “titration series” refers to two or more samples (e.g., of the FcRn antagonist composition), each sample having a different concentration (e.g., of the FcRn antagonist composition).

[0064] A “dose response curve” as used herein refers to a graph plotting the concentration of a drug (e.g., a FcRn antagonist composition) versus a relevant biological activity (e.g., binding of IgG to FcRn).

[0065] A “competition enzyme-linked immunosorbent assay (ELISA)” is used broadly herein to refer to any competition ELISA (also known as a competitive / inhibition ELISA), wherein a test binding molecule competes with a reference antibody for binding to a limited amount of antigen, and wherein the reference antibody is either enzyme-linked itself or is detected by an enzyme-linked antibody. For example, the present invention encompasses a competition ELISA wherein a test FcRn antagonist composition competes with human IgG (the reference antibody) for binding to a limited amount of FcRn (antigen), and wherein the human IgG is detected by an enzyme- linked anti-human IgG antibody (e.g., a Fab-specific anti-human IgG antibody). ELISAs include, but are not limited to, those based on colorimetry, chemiluminescence and fluorometry.

[0066] The term “antibody” is used herein in the broadest sense to refer to any immunoglobulin-like molecule or assembly comprising heavy and light chains, as well as “VHH antibodies” or “heavy-chain only antibodies”. The five immunoglobulin classes (IgG, IgM, IgA, IgD or IgE) are encompassed, particularly IgG (comprising two light chains and two heavy chains joined by interchain disulfide bonds). Moreover, the term encompasses but is not limited to recombinant, monoclonal, polyclonal, monospecific and multispecific (e.g., bispecific or tri-specific) antibodies, as well as single domain antibodies (sdAb). Antibodies may be produced by hybridomas, B cells or genetically engineered host cells. As used herein, the term “antigen binding fragment” refers to a polypeptide fragment of an antibody or immunoglobulin that specifically binds to antigen or competes with intact antibody (i e. , with the intact antibody from which they were derived) for antigen binding. Antigen binding fragments include, for example, a light chain variable domain (VL), a heavy chain variable domain (VH), a VHH, a single chain antibody (scFv), sc(Fv)2, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, an Fd fragment, an Fv fragment, disulphide linked Fv, a one-armed (monovalent) antibody, a diabody, triabody, tetrabody or minibody or any antigen binding molecule formed by combination, assembly or conjugation of such antigen binding fragments. Fragments can be obtained, e.g., via chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.

[0067] As used herein, the terms “variable region” and “variable domain" are used interchangeably and refer to VH and / or VL domains, or portions thereof, that confer antibody specificity for a target antigen. The variability is concentrated in three "hypervariable loops" (HVs) in each of the VL domain (L1 , L2 and L3) and the VH domain (H1 , H2 and H3) which form part of the antigen binding site. The hypervariable loops L1 , L2, L3, H1 , H2 and H3 comprise a "complementarity determining region" or "CDR" (CDRL1 , CDRL2, CDRL3, CDRH1 , CDRH2, CDRH3) which form the antibody binding site as the antibody assumes its 3D configuration in an aqueous environment. Thus, anti-FcRn antibodies or antigen binding fragments comprise CDRs specific for human FcRn. Similarly Fab-specific anti-hlgG antibodies or antigen binding fragments comprise CDRs specific for the Fab region of human IgG.

[0068] As used herein, the term “Fc region” refers to the portion of any mammalian immunoglobulin (such as human IgG) that is formed by the Fc domains of its two heavy chains. The term encompasses both native and variant Fc regions. The native Fc region of IgG is homodimeric and can be obtained, for example, by papain digestion.

[0069] The term "Fc domain" is used broadly herein to refer to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a portion of the hinge region (e.g., upper, middle, and / or lower hinge region), a CH2 domain, and a CH3 domain.

[0070] As used herein, the term "hinge region" refers to the portion of a heavy chain immunoglobulin molecule that joins the CH1 domain to the CH2 domain. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol. 161 : 4083 (1998)). As used herein, the term "CH1 domain" refers to the first (most amino terminal) constant region domain of an immunoglobulin heavy chain that extends from about positions 118-215 (EU numbering). The CH1 domain is adjacent to the VH domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule and does not form a part of the Fc region.

[0071] As used herein, the term "CH2 domain" refers to the portion of a heavy chain immunoglobulin molecule that extends from about positions 231 -340 (EU numbering).

[0072] As used herein, the term "CH3 domain" includes the portion of a heavy chain immunoglobulin molecule that extends approximately 110 residues from C-terminus of the CH2 domain, e.g., from about position 341-446 (EU numbering).

[0073] As used herein, the term “EU numbering” or "EU position" refers to the amino acid position in the EU numbering convention for the Fc region described in Edelman, G.M. et al., Proc. Natl. Acad. Sci. USA, 63: 78-85 (1969) and Kabat et al., in "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, 5th edition, 1991.

[0074] The term “clinical use” as used herein refers to any use of a product for the treatment, prevention or diagnosis of any disease or condition in a human patient, and more broadly, uses of a product for the care of, or testing of, a human patient having any disease or condition. The term encompasses investigational clinical trials, as well as marketed uses of an authorised product.

[0075] The term “biosimilar” as used herein refers to a biological molecule that is highly similar (particularly, in terms of active properties) to another that has already been approved for clinical use (for example, efgartigimod).

[0076] The term “generic” as used herein refers to a small-molecule that has the same active ingredient, dosage, safety, effectiveness as a brand-name drug that has been approved for use.

[0077] The term “innovator” as used herein refers to the first drug containing specific active ingredients) to obtain marketing authorisation.

[0078] A “reference standard” as used herein typically refers to a particular FcRn antagonist composition (e.g., a gold-standard preparation of an innovator, generic or biosimilar composition) against which the potency of the test FcRn antagonist composition is compared. The term may also refer to a pre-determined potency (for example, a pre-determined IC50) against which a test FcRn antagonist composition is compared. For the methods of the present invention, the reference standard may be a FcRn antagonist composition comprising a majority population of identical FcRn antagonist molecules corresponding to the FcRn antagonist molecules of the composition under test. For embodiments wherein the composition under test is heterogeneous, i.e., comprises a sub-population of identical FcRn antagonist molecules as well as FcRn antagonist molecules that differ with respect to amino acid sequence, glycosylation, oxidation status etc, the majority population of the reference standard may correspond to the FcRn antagonist molecules of the sub-population of the composition under test. The reference standard FcRn antagonist composition may comprise a majority of FcRn antagonist molecules approved for clinical use e.g., efgartigimod, or a corresponding biosimilar thereof. Alternatively, or additionally, the reference standard may have a potency (e.g., an I C50) which is known to meet the requirements for clinical use.

[0079] The term “variant Fc region” is used broadly herein to refer to any Fc region comprising one or more amino acid substitutions, deletions, additions, linkage of additional moieties, and / or post- translational modifications (e.g., alteration of glycans) relative to a native Fc region. Variant Fc regions typically comprise one or more point-mutations relative to a native Fc region, for example a wild-type human IgG Fc region. The variant Fc regions described herein typically exhibit increased binding affinity for FcRn as compared with wild-type IgG, particularly human wild-type IgG, particularly human wild-type lgG1. In the methods of the present invention, variant Fc regions can be tested for their ability to outcompete the native Fc region of IgGs for binding to FcRn.

[0080] A “variant Fc region” encompasses homodimeric Fc regions wherein each of the constituent Fc domains is the same, as well as heterodimeric Fc regions wherein each of the constituent Fc domains are different. Examples of such heterodimeric Fc regions include, without limitation, Fc regions made using the “knobs and holes” technology as described in, for example, US 8216805, which is incorporated by reference herein in its entirety. The term further encompasses single chain Fc regions where the constituent Fc domains are linked together by a linker moiety, as described in, for example, US20090252729A1 and US20110081345A1 , which are each incorporated by reference herein in their entirety.

[0081] As used herein, the term “FcRn binding fragment” refers to a portion of an Fc region that is sufficient to confer FcRn binding. The ability of an Fc-region fragment to bind to FcRn can be determined using any binding assay recognised in the art e.g., Biacore™ surface plasmon resonance assays. “Efgartigimod” (also known as “efgartigimod alfa” or“ARGX-1 13”) is an FcRn antagonist consisting of a variant Fc fragment of the human lgG1 Fc region. Efgartigimod has been modified with ABDEG™ (antibody that enhances IgG degradation) technology (Vaccaro et al., 2005 Nat. Biotechnol. 23(10):1283-8) to increase its affinity for FcRn compared to wild-type IgG at both physiological and acidic pH, resulting in constitutive inhibition of FcRn-mediated recycling of IgG. The amino acid sequence of the Fc domains of efgartigimod is set out in SEQ ID NO: 2 (see Table 1). Efgartigimod is sold under the brand name “Vyvgart” and is FDA- and EMA- approved for the treatment of generalized myasthenia gravis (gMG) in adults who are anti-acetylcholine receptor (AChR) antibody positive. Efgartigimod has been shown to reduce overall levels of IgG, including the abnormal anti-AChR antibodies that are present in myasthenia gravis.

[0082] An “antibody-mediated autoimmune disease” or “IgG-mediated autoimmune disease” as used herein refers to the broad class of diseases characterized by the presence in a subject's serum of autoimmune or autoreactive IgG antibodies. Example IgG-mediated autoimmune diseases include allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, Alzheimer’s disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac spruedermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn’s disease, dermatomyositis, dilated cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves’ disease, Guillain-Barre syndrome (GBS), Goodpasture syndrome, graft-versus-host disease (GVHD), Hashimoto’s thyroiditis, hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura or immune thrombocytopenia (e.g. primary ITP), IgA neuropathy, IgM polyneuropathies, immune mediated thrombocytopenia juvenile arthritis, Kawasaki’s disease, lichen planus, lichen sclerosus, lupus erythematosus, Meniere’s disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes mellitus, multifocal motor neuropathy (MMN), myasthenia gravis (e.g. generalized MG or gMG), myositis, paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris (PV), pemphigus foliaceus (PF), pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynaud’s phenomenon, Reiter’s syndrome, rheumatoid arthritis (RA), sarcoidosis, scleroderma, Sjdgren"s syndrome, solid organ transplant rejection, stiff-person syndrome, systemic lupus erythematosus (SLE), Takayasu’s arteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenia purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitides, vitiligo, and Wegener’s granulomatosis.

[0083] An “internal assay control” or IAC as used herein refers to a preparation or sample of a FcRn antagonist composition (such as efgartigimod or a corresponding biosimilar thereof) used to assess the accuracy and sensitivity of the competition binding assay (e.g., ELISA) itself. Two or more (e.g., three) data points on the resulting dose response curve of the IAC can be used as an intra-assay control check, to ensure consistency of measurement between assays performed for example at different manufacturing sites.

[0084] The terms “ambient temperature” and “room temperature” are used interchangeably herein, according to their usual definition known in the art. The ambient temperature may be a temperature of about 15°C to about 25°C.

[0085] B. Methods of determining the potency of FcRn antagonist compositions

[0086] The present invention relates to methods of measuring the potency of human neonatal Fc receptor (FcRn) antagonist compositions in outcompeting human IgG for binding to FcRn. By using a competition binding assay, the methods model the in vivo biological mode-of-action of FcRn antagonists in blocking recycling of endogenous IgG via FcRn, and thereby enhancing lysosomal degradation of IgG. This is particularly advantageous to determine the efficacy and safety of FcRn antagonists for treating IgG-mediated autoimmune disease, wherein accelerated clearance of autoreactive IgG is desired.

[0087] Thus, in a first aspect, the invention provides a method of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of human IgG to FcRn using a competition binding assay, the assay comprising the steps of: a) incubating the FcRn antagonist composition with FcRn in the presence of human IgG such that FcRn antagonist and human IgG compete for binding to FcRn; b) separating any FcRn - human IgG complexes from any unbound human IgG; and c) detecting the quantity of FcRn - human IgG complexes. In a related second aspect, the invention provides a method of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of an IgG Fc region to FcRn using a competition binding assay, the assay comprising the steps of: a) incubating the FcRn antagonist composition with FcRn in the presence of an IgG Fc region such that FcRn antagonist and IgG Fc region compete for binding to FcRn; b) separating any FcRn - IgG Fc region complexes from any unbound IgG Fc region; and c) detecting the quantity of FcRn - IgG Fc region complexes.

[0088] In this related aspect, the human IgG with which the FcRn antagonist competes in accordance with the first aspect of the invention is replaced with any suitable IgG Fc region, as defined elsewhere herein.

[0089] As defined elsewhere herein, the test FcRn antagonist composition comprises or consists of a population of FcRn antagonist molecules that specifically bind to FcRn. The competition binding assay tests the ability of the FcRn antagonist composition to block or reduce the binding of human IgG to FcRn, and therefore indirectly tests the biological activity of the FcRn antagonist composition in enhancing IgG degradation and clearance.

[0090] The greater the “potency” of the FcRn antagonist composition, the lower the concentration that may be required to inhibit binding of human IgG to FcRn, and thus, the lower the concentration that may be required to achieve a therapeutic benefit. Accordingly, the methods described herein can be used to check that any particular FcRn antagonist composition will have the desired level of potency before release for clinical use.

[0091] The FcRn antagonist compositions for testing in accordance with the methods described herein may comprise or consist of FcRn antagonist molecules that are biological products e.g. antibodies or antibody fragments as described in more detail herein below. Given the complexities associated with the production of biological products, the manufacture of FcRn antagonist compositions has the potential to lead to differences in quality or identity between different manufacturing lots or batches. The potency of the FcRn antagonist compositions, as measured by the methods of the invention, also acts as an indirect measure of the quality or identity of the FcRn antagonist composition. For example, the presence of different FcRn antagonist isoforms or variants within the composition may be detected by differences in potency.

[0092] All embodiments of the methods described herein below apply equally to all aspects of the invention. In some embodiments, steps a) - c) of the assay are be carried out at a pH of between about 5 to about 8, optionally between about 5.5 to about 7.5, optionally between about 6.0 to about

[0093] 7.4. This models the in vivo competition or inhibition of endogenous IgG-FcRn interactions.

[0094] In some embodiments, steps a) - c) of the assay are carried out at a pH of about 5.5 to about

[0095] 6.5, optionally about pH 6.0. This typically reflects the competition or inhibition of endogenous IgG-FcRn interactions in the early endosome, and therefore the ability of FcRn antagonists to inhibit IgG recycling to the cell surface.

[0096] Steps a) - c) of the assay may alternatively, or additionally, be carried out at a pH of between about 7.3 to about 7.4, optionally about 7.4. This typically reflects the competition of endogenous IgG-FcRn interactions at the cell surface, and therefore models the in vivo inhibition of receptor-mediated (rather than fluid-phase) IgG uptake.

[0097] Human IgG (hlqG)

[0098] As defined elsewhere herein, human IgG comprises a native Fc region of human IgG and will therefore compete for binding to FcRn in the methods of the invention. The human IgG may be a human, humanised or chimeric antibody comprising a native Fc region of human IgG.

[0099] The human IgG may be human lgG1 , lgG2, lgG3 or lgG4. In another words, the human IgG may comprise the native Fc region of human IgG 1 , lgG2, lgG3 or lgG4.

[0100] In some embodiments, the human IgG is wild-type IgG. Advantageously, full-length human wildtype IgG can be used to model the binding of endogenous IgG to FcRn.

[0101] In preferred embodiments, the human IgG is human lgG3, preferably wild-type human lgG3. Surprisingly, the present inventors have found that human lgG3 has a bigger assay window than human IgG 1 , lgG2, lgG4 (see FIG. 1), and therefore is more sensitive to detecting small differences in potency between test compositions or manufacturing lots. In addition, lgG3 is highly relevant in the context of treating myasthenia gravis wherein depletion of anti-AChR autoantibodies of this IgG subtype is desired.

[0102] IgG Fc region

[0103] In a related second aspect of the invention, the human IgG is replaced with any suitable IgG Fc region. It will be appreciated that human IgG may be replaced with an IgG Fc region in any of the embodiments described herein. The IgG Fc region will compete for binding to FcRn in the methods of the invention. In some embodiments the IgG Fc region is an Fc region of human IgG 1 , lgG2, lgG3 or lgG4. In preferred embodiments, the IgG Fc region is a wild-type Fc region, preferably a wild-type human IgG Fc region. In some embodiments, the IgG Fc region is an Fc region of human lgG3, preferably a human wild type hlgG3 Fc region.

[0104] It will also be appreciated that human IgG may be replaced with an IgG Fc region comprising a non-native Fc region. For example, the non-native IgG Fc region may comprise amino acid substitutions, deletions and / or additions which do not impact binding to FcRn. As such, the non- native IgG Fc region may compete for binding to FcRn in the same manner as human IgG.

[0105] It follows that, in some embodiments, the IgG Fc region is a modified Fc region, for example a modified Fc region of human IgG 1 , lgG2, lgG3 or lgG4. In some embodiments, the IgG Fc region is a modified Fc region of human lgG3.

[0106] The FcRn antagonist composition

[0107] The methods of the present invention are carried out so as to determine the potency of a FcRn antagonist composition, also referred to herein as a “test” FcRn antagonist composition. As explained elsewhere herein, the potency of different FcRn antagonist compositions can differ depending on manufacturing batch or lot for example.

[0108] The methods of the present invention are particularly applicable in situations where one is aiming to manufacture FcRn antagonist compositions comprising or consisting of essentially the same FcRn antagonist molecules as in previous compositions; however, the reality is that the end product may vary from lot-to-lot (for example, comprising modifications or variants) such that testing of individual FcRn antagonist compositions is required to determine the potency of any particular composition.

[0109] FcRn antagonist compositions for testing in accordance with the methods described herein comprise or consist of a population of “FcRn antagonist molecules”, otherwise referred to herein as “FcRn antagonists”. The population of FcRn antagonists within the composition may be homogeneous i.e. , all FcRn antagonists are identical. It is more likely however, that the FcRn antagonist composition tested in accordance with the methods described herein is heterogeneous with respect to the FcRn antagonists contained therein. In such embodiments, the composition may comprise a sub-population of FcRn antagonists that are identical, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% of FcRn antagonists that are identical. The remaining FcRn antagonist molecules within such heterogeneous compositions may be modified or variant forms of the FcRn antagonist form of said sub-population, for example variants differing with respect to amino acid sequence, glycosylation and / or oxidation status. The presence of such modified or variant forms within the FcRn antagonist composition may affect the potency determined in accordance with the methods described herein.

[0110] The FcRn antagonist compositions may comprise or consist of any FcRn antagonists, but typically the FcRn antagonists are suitable for treating IgG-mediated autoimmune disease. In some embodiments, the FcRn antagonist compositions comprise or consist of FcRn antagonists that are biological products (such as efgartigimod or a corresponding biosimilar thereof).

[0111] In some embodiments, the FcRn antagonists may be peptides, antibodies or antibody fragments capable of specifically binding to FcRn.

[0112] In some embodiments, the FcRn antagonists are anti-FcRn antibodies or antigen binding fragments thereof, particularly anti-FcRn antibodies or antigen binding fragments thereof having one or more variable regions with binding specificity for FcRn.

[0113] In some embodiments, the FcRn antagonists comprise or consist of variant Fc regions or FcRn binding fragments thereof, wherein “variant Fc regions” are defined elsewhere herein as Fc regions differing in sequence and / or post-translational modifications relative to a native or wildtype Fc region. The variant Fc regions or FcRn binding fragments thereof, as contemplated herein, may bind to human FcRn with increased affinity relative to the wild-type Fc region of IgG. The variant Fc regions or FcRn binding fragments thereof may have increased binding affinity for FcRn at acidic pH (e.g., pH of between about 5.5 to about 6.5, or about 6.0) and / or increased binding affinity for FcRn at neutral pH (e.g., pH of between about 7.3-7.4 or of about 7.4) compared to a wild-type Fc region of IgG. In some embodiments, the variant Fc region or FcRn binding fragment exhibits pH-independent binding affinity for FcRn. In some embodiments, the variant Fc region or FcRn binding fragment exhibits pH-dependent binding affinity for FcRn. Binding affinity may be measured by any suitable method known in the art, for example Biacore™ surface plasmon resonance binding assays.

[0114] The variant Fc region may be derived from any immunoglobulin (IgM, IgG, IgD, IgA and IgE), but preferably the variant Fc region is a variant IgG Fc region. The variant IgG Fc region may be derived from any IgG isotype (IgG 1 , lgG2, lgG3 and lgG4). In certain embodiments, the variant Fc region is a variant IgG 1 Fc region. In preferred embodiments, the variant Fc region is a variant human IgG Fc region, such as a variant human IgG 1 Fc region. In some embodiments, the variant Fc region is a chimeric Fc region comprising portions of more than one different Fc region. Suitable examples of chimeric Fc regions are set forth in US20110243966A1 , which is herein incorporated by reference in its entirety.

[0115] For embodiments wherein the FcRn antagonists comprise variant Fc regions or FcRn binding fragments thereof, the FcRn antagonist may not be a full-length antibody. In certain embodiments, the FcRn antagonist that comprises a variant Fc region or FcRn binding fragment thereof does not comprise an antibody variable region. In certain embodiments, the FcRn antagonist that comprises a variant Fc region or FcRn binding fragment thereof does not comprise a CH1 domain. In particular embodiments, the FcRn antagonist consists of a variant Fc region or FcRn binding fragment thereof, wherein the variant Fc region is a variant human IgG Fc region. In some embodiments, the variant Fc region is a variant human IgG 1 Fc region.

[0116] In some embodiments, the variant Fc region is derived from residues 220-447 (EU numbering) of human lgG1 . An Fc region (e.g., variant Fc region) can be truncated to produce a FcRn- binding fragment thereof.

[0117] The variant Fc region or FcRn-binding fragment thereof may comprise or consist of a first Fc domain and a second Fc domain which form a homodimer or heterodimer (i.e. , the first and second Fc domains may be the same or different from each other).

[0118] In some embodiments, the first Fc domain and / or the second Fc domain comprise amino acids Y, T, E, K, and F at positions 252, 254, 256, 433, and 434 (EU numbering), respectively. The M252Y / S254T Z T256E / H433K / N434F substitutions are ABDEG™ mutations, which increase the affinity for FcRn at both acidic pH (pH 6.0) and neutral pH (pH 7.4). Variant Fc regions having the ABDEG™ mutations have been shown to result in a very rapid lowering of endogenous IgG molecules. The first Fc domain and / or second Fc domain may comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436 (EU numbering), respectively. The “Y” residue at position 456 is the wild-type residue found in the human IgG 1 Fc region and, in addition to the ABDEG™ mutations, forms the complete “ABDEG™ signature”, which increases binding affinity for FcRn.

[0119] Non-limiting examples of Fc domains that may be present in the variant Fc regions tested are shown in Table 1 below. Table 1: Amino acid sequences of non-limiting examples of Fc domains

[0120] In embodiments wherein the FcRn antagonists comprise or consist of variant Fc regions, the variant Fc regions may comprise or consist of a first Fc domain and a second Fc domain each having an amino acid sequence represented by SEQ ID NO: 1 . In some embodiments, the first Fc domain and / or the second Fc domain comprise or consist of an amino acid sequence independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. Optionally, the first Fc domain and the second Fc domain comprise or consist of an amino acid sequence independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0121] In certain embodiments, the first and second Fc domains each comprise, or optionally consist of, the amino acid sequence set forth in SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 represents the Fc domains of the FcRn antagonist efgartigimod alfa (ARGX-113).

[0122] In some embodiments, the first and second Fc domains each comprise or consist of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the first and second Fc domains each comprise or consist of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the first and second Fc domains each comprise or consist of the amino acid sequence set forth in SEQ ID NO: 5.

[0123] As explained above, the FcRn antagonist composition tested in accordance with the methods described herein may be a heterogeneous population of FcRn antagonist molecules. For embodiments wherein the FcRn antagonists comprise or consist of variant Fc regions, the composition may comprise a sub-population of variant Fc regions that are identical, whilst the remainder of the variant Fc regions molecules may differ, for example with respect to amino acid sequence (for example, may comprise substitutions, deletions and / or additions) with respect to post-translational modifications (e.g. glycosylation or linkage of additional moieties), and / or with respect to electrostatic charge, isomerization, deamidation and / or oxidation of amino acid residues. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% of the FcRn antagonist molecules (e.g. variant Fc regions) within the test FcRn antagonist composition are identical with respect to amino acid sequence, with respect to post-translational modifications (e.g. glycosylation) and / or with respect to electrostatic charge, isomerization, deamidation and / or oxidation of amino acid residues.

[0124] In some embodiments, the FcRn antagonist composition tested in accordance with the methods described herein comprises or consists of a sub-population of identical variant Fc regions having first and second Fc domains consisting of the amino acid sequence of SEQ ID NO: 2, as well as variant Fc regions having first and / or second Fc domains consisting of an amino acid sequence as represented by SEQ ID NO:1 but that is not SEQ ID NO:2. In some embodiments, the FcRn antagonist composition tested may comprise or consist of a sub-population of identical variant Fc regions having first and second Fc domains consisting of the amino acid sequence of SEQ ID NO:2, as well as variant Fc regions having first and / or second Fc domains consisting of the amino acid sequence of SEQ ID NO:2 but which differ from the variant Fc regions of the sub-population with respect to glycosylation, electrostatic charge, isomerization, deamidation and / or oxidation of amino acid residues.

[0125] In some embodiments, the FcRn antagonist composition tested in accordance with the methods described herein comprises or consists of a population of FcRn antagonist molecules that are variant Fc regions as described in US provisional application 63 / 383599, the contents of which are incorporated herein in their entirety.

[0126] In some embodiments, the FcRn antagonist composition tested in accordance with the methods described herein comprises or consists of efgartigimod. In other words, in some embodiments, the test FcRn antagonist composition is an efgartigimod composition.

[0127] As described elsewhere herein, the efgartigimod composition may comprise modified or variant forms of efgartigimod. For example, a portion of the FcRn antagonist molecules of the test efgartigimod composition may comprise a N-linked glycan at EU position 297 of one or both of the Fc domains. In some embodiments, a portion of the efgartigimod molecules comprise a fucosylated N-linked glycan at EU position 297 of one or both of the Fc domains.

[0128] In some embodiments, the FcRn antagonist composition tested in accordance with the methods described herein comprises or consists of a biosimilar of efgartigimod. Similar to an efgartigimod composition, a biosimilar composition may comprise modified or variant forms.

[0129] In some embodiments, the FcRn antagonist comprises or consists of a variant Fc region or FcRn binding fragment linked to a half-life extending molecule. In certain embodiments, the half-life extending molecule is polyethylene glycol (PEG) or human serum albumin (HSA).

[0130] The FcRn antagonist compositions described above (e.g., efgartigimod or a corresponding biosimilar thereof) are tested in accordance with the methods of the invention wherein a competition binding assay is used to determine the potency of the composition in inhibiting the binding of human IgG to FcRn. The competition binding assay comprises three steps: a) incubating the FcRn antagonist composition with FcRn in the presence of human IgG such that FcRn antagonist and human IgG compete for binding to FcRn; b) separating any FcRn - human IgG complexes from any unbound human IgG; and c) detecting the quantity of FcRn - human IgG complexes.

[0131] Each of these steps will be described in more detail herein below. Step a) Incubation of the FcRn antagonist composition with FcRn and human IgG

[0132] In step a) of the method, the FcRn antagonist composition is incubated with FcRn in the presence of human IgG such that FcRn antagonist and human IgG compete for binding to the FcRn.

[0133] Serial dilutions of the FcRn antagonist composition may be prepared before step a) is carried out. The testing of multiple concentrations of the FcRn antagonist composition allows for the generation of a dose response curve of the FcRn antagonist composition inhibiting the binding of human IgG to FcRn.

[0134] Thus, in some embodiments, the FcRn antagonist composition is serially diluted to obtain two or more samples having different concentrations of the FcRn antagonist composition, optionally in the presence of human IgG (e.g., human lgG3). In other words, a titration series of the FcRn antagonist composition may be prepared, optionally in the presence of human IgG (e.g., human lgG3). In certain embodiments, serial dilutions of 1 :2.5 or 1 :2.25 are performed, for example to obtain a titration series as set out in Table 3 or Table 4 of Example 1 .

[0135] In some embodiments, the FcRn antagonist composition is serially diluted to obtain multiple samples in the concentration range of about 0.008 pg / mL to about 30 pg / mL FcRn antagonist composition (e.g., by performing dilution steps of 1 / 2.5), optionally in the presence of about 40.0 pg / mL or about 20.0 pg / mL human IgG (e.g., human lgG3). For example, a titration series of FcRn antagonist composition of about 0.020 pg / mL to about 30 pg / mL FcRn antagonist composition may be prepared (e.g., by performing dilution steps of 1 / 2.25), optionally in the presence of about 40.0 pg / mL or about 20.0 pg / mL human IgG (e.g., human lgG3).

[0136] In some embodiments, the FcRn antagonist composition, or titration series thereof, is combined with human IgG (e.g., human lgG3) prior to the addition of FcRn in step a). Without wishing to be bound by theory, this order of mixing (known as a “true competition assay”) is expected to achieve a more sensitive assay.

[0137] Thus, step a) may comprise adding FcRn to the FcRn antagonist composition or titration-series thereof, preferably pre-mixed with the human IgG (e.g., human lgG3). In some embodiments, the FcRn is biotinylated FcRn. In certain embodiments, about 2.0 pg / mL FcRn (e.g., biotinylated FcRn) is added to samples comprising about 40.0 pg / mL human IgG (e.g., human lgG3) at about a 1 :1 ratio. In certain embodiments, about 1.0 pg / mL FcRn (e.g., biotinylated FcRn) is added to samples comprising about 20.0 pg / mL human IgG (e.g., human lgG3) at about a 1 :1 ratio. Accordingly, in some embodiments, step a) results in a final concentration of:

[0138] (i) about 1 .0 pg / mL biotinylated FcRn; about 20.0 pg / mL hulgG3; and about 0.001 pg / mL to about 20 pg / mL (or about 0.004 pg / mL to about 15 pg / mL) titration series of FcRn antagonist composition; or

[0139] (ii) about 0.5 pg / mL biotinylated FcRn; about 10.0 pg / mL hulgG3; and 0.001 pg / mL to about 20 pg / mL (or about 0.004 pg / mL to about 15 pg / mL) titration series of FcRn antagonist composition.

[0140] In some embodiments, step a) of the assay is carried out at a pH of about 5 to about 8, optionally between about 5.5 to about 7.5, optionally between about 6.0 to about 7.4. In some embodiments, step a) may be carried out a pH of about 7.3 to about 7.4, optionally about 7.4. In certain embodiments, step a) is carried out at a pH of about 5.5 to about 6.5, optionally about pH 6.0. For example, step a) may comprise incubation with an Assay Buffer at pH 6.0, optionally wherein the Assay Buffer comprises 1xPBS pH 6.0 + 0.1 % casein.

[0141] In some embodiments, the incubation period in step a) is for at least 1 hour at ambient temperature, optionally without shaking.

[0142] In some embodiments, the assay further comprises preparation of a control sample containing no FcRn antagonist - to control for positive binding between FcRn and human IgG. For example, the control sample may comprise about 1 .0 pg / mL biotinylated FcRn and about 20.0 pg / mL hlgG3; or about 0.5 pg / mL biotinylated FcRn and about 10.0 pg / mL hlgG3. Optionally, the control sample is incubated for at least 1 hour at ambient temperature, optionally without shaking.

[0143] Step b) Separation of the FcRn - human IgG complexes

[0144] In step b) of the method, any FcRn -human IgG complexes are separated from any unbound human IgG. Separation of FcRn-human IgG complexes from any unbound human IgG may be achieved by any suitable means known to those skilled in the art.

[0145] In some embodiments, in step b), the FcRn - human IgG complexes are separated from any unbound human IgG by immobilising the FcRn onto a substrate. The unbound human IgG may be removed in a subsequent wash step.

[0146] As noted above, the FcRn may be biotinylated FcRn. Thus, in some embodiments, in step b), biotinylated FcRn is immobilised with a substrate having a biotin-binding protein thereon, such as streptavidin, avidin or neutravidin. In some embodiments, the substrate is an immunoassay plate (e.g., ELISA plate), optionally coated with streptavidin or neutravidin. In certain embodiments, the immunoassay plate (e.g., ELISA plate) is coated with neutravidin.

[0147] Alternatively, the FcRn may comprise any fusion tag known in the art. In some embodiments, the FcRn comprises a GST tag or His tag. Thus, in step b), FcRn comprising a GST tag may be immobilised with a substrate (e.g., an immunoassay plate) coated with glutathione.

[0148] Alternatively, in step b), FcRn comprising a His tag may be immobilised with a substrate (e.g., ELISA plate) coated with nickel or copper.

[0149] For embodiments wherein the FcRn - human IgG complexes are separated via immobilisation of the complexes on a substrate, samples from step a) are applied to the substrate (e.g., neutravidin-coated ELISA plate), so as to immobilise the FcRn (e.g., biotinylated FcRn). Optionally, 100 pL of each sample from step a) is transferred to a neutravidin-coated ELISA plate.

[0150] In some embodiments, in step b), the FcRn (e.g., biotinylated FcRn) is incubated with the substrate (e.g., the neutravidin-coated ELISA plate) for 1 hour at room temperature, optionally while shaking at 450 rpm.

[0151] In some embodiments, step b) comprises washing the substrate one or more times to remove unbound human IgG (e.g., human lgG3), optionally two times, three times, four times or five times. In certain embodiments, the substrate (e.g., the neutravidin-coated ELISA plate) is washed five times.

[0152] In some embodiments, step b) of the assay is carried out at a pH of about 5 to about 8, optionally between about 5.5 to about 7.5, optionally between about 6.0 to about 7.4. In some embodiments, step b) is carried out a pH of about 7.3 to about 7.4, optionally about 7.4.

[0153] In certain embodiments, step b) is carried out at a pH of about 5.5 to about 6.5, optionally about pH 6.0. For example, the wash buffer may comprise PBS pH 6.0 + 0.05% Tween20. In some embodiments, the biotin binding protein (e.g., neutravidin) is coated onto the substrate (e.g., ELISA plate) in a solution comprising PBS pH 6.0. In some embodiments, a blocking buffer is used to prepare the substrate (e.g., ELISA plate), wherein the blocking buffer comprises PBS pH 6.0.

[0154] Step c) Detecting the quantity of FcRn - human IgG complexes In step c) of the method, the quantity of FcRn-human IgG complexes is detected.

[0155] In some embodiments, step c) involves detecting FcRn - human IgG complexes by direct labelling of the human IgG (e.g., human lgG3). Thus, in some embodiments, the human IgG (e.g., human lgG3) is conjugated with a detectable moiety, optionally detectable by colorimetry, fluorescence, chemiluminescence or radioactivity.

[0156] Numerous labels are known for use in competition binding assays or immunoassays and are suitable for use with the methods of the present invention. The detectable moiety may comprise, for example, a chromophore, a fluorophore, a chemiluminescent label or a radioisotope.

[0157] In other embodiments, step c) involves detecting FcRn - human IgG complexes using an antihuman IgG antibody. In some embodiments, the anti-human IgG antibody specifically binds to the Fab region of human IgG. Since the Fc region of the human IgG is bound to the immobilised FcRn, an antibody that specifically binds to the Fab region can simultaneously bind to the human IgG.

[0158] The anti-human IgG antibody (e.g., Fab-specific anti-human IgG antibody) may be conjugated with a detectable moiety. The detectable moiety may be any detectable moiety known in the art or described herein. As such, the FcRn - human IgG complexes may be indirectly detected by labelling of the anti-human IgG antibody.

[0159] In certain embodiments, the competition binding assay is a competition enzyme-linked immunosorbent assay (ELISA). For such embodiments, step c) may involve detecting the quantity of FcRn-human IgG complexes using an enzyme-linked anti-human IgG antibody (e.g., Fab-specific anti-human IgG antibody) and an enzyme substrate.

[0160] The enzyme of the enzyme-linked anti-human IgG antibody may be selected from any one of peroxidase (e.g., horseradish peroxidase or alkaline phosphatase), p-galactosidase, acetylcholinesterase, catalase or luciferase.

[0161] In certain embodiments, the enzyme-linked anti-human IgG antibody is a peroxidase-linked antibody, optionally wherein the peroxidase is horseradish peroxidase (HRP) and alkaline phosphatase (AP). In certain embodiments, the enzyme-linked anti-human IgG antibody is an HRP-linked antibody. In some embodiments, the enzyme substrate is a chromogenic substrate, a fluorescent substrate or a chemiluminescent substrate. In some embodiments, the enzyme substrate is a chromogenic substrate selected from one of: 3,3',5,5'-tetramethylbenzidine (TMB); p- Nitrophenyl Phosphate (PNPP); 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]- diammonium salt (ABTS) or o-phenylenediamine dihydrochloride (OPD).

[0162] In certain embodiments, the enzyme substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0163] In some embodiments, step c) comprises the sub-step ci) of adding an anti-human IgG antibody (e.g., Fab-specific anti-human IgG antibody) to the immobilised FcRn-IgG complexes, optionally wherein the anti-human IgG antibody is peroxidase-linked (e.g., HRP-linked). The anti-human IgG antibody may be incubated with the immobilised FcRn-IgG complexes at ambient temperature. In some embodiments, the incubation is for 1 hour, optionally while shaking at 450 rpm.

[0164] In some embodiments, step c) further comprises the sub-step cii) of one or more washes with a wash buffer to remove unbound anti-human IgG antibody (e.g., HRP-linked anti-human IgG antibody). In some embodiments, the one or more washes comprise two washes, three washes, four washes or five washes, optionally five washes.

[0165] In some embodiments, step c) comprises the sub-step ciii) of adding an enzyme substrate (e.g., TMB) to visualize the bound human IgG (e.g., human lgG3-FcRn complexes). In some embodiments, sub-step ciii) involves incubating the FcRn-IgG-anti-IgG antibody complexes with the enzyme substate (e.g., TMB) at ambient temperature. In some embodiments, the incubation is for 15 minutes, optionally while shaking at 450 rpm.

[0166] In some embodiments, step c) of the assay (or steps ci)-ciii)) is carried out at a pH of about 5 to about 8, optionally between about 5.5 to about 7.5, optionally between about 6.0 to about 7.4.

[0167] In some embodiments, step c) (e.g., steps ci)-ciii)) is carried out a pH of about 7.3 to about 7.4, optionally about 7.4.

[0168] In certain embodiments, step c) of the assay (or steps ci)-ciii)) is carried out at a pH of about 5.5 to about 6.5, optionally about pH 6.0. For example, the wash buffer in step cii) comprise PBS pH 6.0 + 0.05% Tween20.

[0169] In some embodiments, step c) comprises the sub-step civ) of quenching the reaction, for example by adding sulfuric acid, optionally 0.5M sulfuric acid. In some embodiments, step c) further comprises the sub-step cv) of detecting the colorimetry, fluorescence or chemiluminescence. For example, colorimetry may be detected with an absorbance plate reader; fluorescence may be detected with a fluorometer, and chemiluminescence may be detected with a luminometer plate reader.

[0170] In some embodiments, the sub-step cv) comprises reading the absorbance at 450 nm, preferably with subtraction of the reference wavelength at 620 nm. This wavelength is suitable to detect 3,3',5,5'-tetramethylbenzidine (TMB).

[0171] Assay results and uses

[0172] The results of the competition binding assay may be used to generate a dose response curve of the FcRn antagonist composition inhibiting the binding of human IgG to FcRn. For example, different concentrations of the FcRn antagonist composition tested in the assay may be plotted against the OD signal obtained at 450 nm (with 620 nm subtraction).

[0173] The results of the assay may be used to calculate a half maximal inhibitory concentration (IC50) of the FcRn antagonist composition for inhibiting the binding of human IgG to FcRn.

[0174] Dose response curves are generated by plotting the corrected optical density (OD) of each sample (Y-axis, linear scale) against logio concentration (X-axis, logarithmic scale), using a 4- parameter logistic curve fitting. The four parameters of the response curves of the sample are fitted using the formula: y=D+(A-D) / 1+(x / C)Bwhere parameter A is the upper asymptote, B is the slope of the curve, C is the IC50 value and D is the lower asymptote. R2for the fitted response curves are calculated.

[0175] The potency determined for a FcRn antagonist composition tested in accordance with the methods described herein may be compared with a reference standard. The reference standard may be a pre-determined level of potency, for example, a pre-determined IC50. If the potency results for the FcRn antagonist composition are compared with the reference standard and found to match or fall within a defined window about the pre-determined level of potency (e.g., pre-determined IC50), the FcRn antagonist composition may be deemed suitable for clinical use.

[0176] In some embodiments, the reference standard may be a particular FcRn antagonist composition incorporated into the methods described herein. For example, assay steps a) - c) may be repeated with a reference standard in place of the test FcRn antagonist composition. The results of the assay may be compared with results obtained with a reference standard.

[0177] In some embodiments, the reference standard has a known potency (e.g. IC50), preferably wherein the potency is known to be suitable for clinical use. Thus, the potency measured with the reference standard may provide a measure of the potency required for clinical use.

[0178] For embodiments wherein the FcRn antagonist composition comprises or consists of a variant Fc region as defined elsewhere herein, the reference standard may comprise or consist of corresponding variant Fc regions and have a known potency.

[0179] For embodiments wherein the FcRn antagonist composition comprises or consists of a variant Fc region as defined elsewhere herein, the reference standard may be a preparation of efgartigimod of known potency. Alternatively, the reference standard may be a preparation of a biosimilar of efgartigimod, the preparation having a known potency.

[0180] The reference standard maybe a FcRn antagonist composition comprising or consisting of a majority population of identical FcRn antagonist molecules (e.g., variant Fc regions) corresponding to the FcRn antagonist molecules within (a sub-population of) the FcRn antagonist composition under test. Preferably, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of the FcRn antagonist molecules within the reference standard are identical, optionally with respect to amino acid sequence, with respect to post-translational modifications (e.g., glycosylation) and / or with respect to electrostatic charge, isomerization, deamidation and / or oxidation of amino acid residues.

[0181] In certain embodiments, the reference standard is a FcRn antagonist composition comprising a majority population of identical variant Fc regions as defined elsewhere. Preferably the majority population of variant Fc regions having Fc domains consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3.

[0182] For example, the majority population of identical variant Fc regions may comprise efgartigimod or a biosimilar of efgartigimod. Preferably, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of the FcRn antagonist molecules are variant Fc regions (e.g., efgartigimod alfa molecules or a corresponding biosimilar thereof) that are identical with respect to amino acid sequence, with respect to post-translational modifications (e.g., glycosylation) and / or with respect to electrostatic charge, isomerization, deamidation and / or oxidation of amino acid residues. The reference standard may comprise less than 1%, less than 2%, less than 3%, less than 5% or less than 10% of FcRn antagonist sequence variants (e.g., variants with substitutions, additions or truncations). The reference standard may comprise less than 1 %, less than 2%, less than 3%, less than 5% or less than 10% of FcRn antagonist glycosylation variants. The reference standard may comprise less than 1 %, less than 2%, less than 3%, less than 5% or less than 10% of FcRn antagonist charge variants. The reference standard may comprise less than 1 %, less than 2%, less than 3%, less than 5% or less than 10% of FcRn antagonist isomers. The reference standard may comprise less than 1%, less than 2%, less than 3%, less than 5% or less than 10% of FcRn antagonist deamidation variants. The reference standard may comprise less than 1%, less than 2%, less than 3%, less than 5% or less than 10% of FcRn antagonist oxidation variants. It will be appreciated that, in embodiments wherein the population of identical FcRn antagonist molecules consists of efgartigimod molecules, any isomers or variants (i.e., sequence, glycosylation, charge or deamidation variants) are efgartigimod isomers or variants. For embodiments wherein the identical FcRn antagonist molecules consist of a biosimilar of efgartigimod, any isomers or variants (i.e., sequence, glycosylation, charge or deamidation variants) are variants or isomers of the biosimilar of efgartigimod.

[0183] In preferred embodiments, the reference standard comprises less than 1 %, less than 2%, less than 3%, less than 5% or less than 10% of FcRn antagonist molecules (e.g., efgartigimod) having a free cysteine residue (i.e., non-disulphide bonded). It is preferable that FcRn antagonists do not comprise free cysteine residues to improve manufacturability.

[0184] In some embodiments, a relative potency of the FcRn antagonist composition is calculated as (IC50 of the reference standard I IC50 of the FcRn antagonist composition) x 100%.

[0185] For embodiments wherein the reference standard provides a measure of the potency required for clinical use, a FcRn antagonist composition having a relative potency of about 70% to about 135%, is identified as a FcRn antagonist composition suitable for clinical use. In some embodiments, a FcRn antagonist composition having a relative potency of about 75% to about 130%, or about 80% to about 125%, is identified as a FcRn antagonist composition suitable for clinical use. In certain embodiments, a FcRn antagonist composition having a relative potency of about 85% to about 120% is identified as a FcRn antagonist composition suitable for clinical use. For example, the FcRn antagonist composition may be identified as suitable for clinical use in the treatment of an antibody-mediated autoimmune disease.

[0186] In some embodiments, the antibody-mediated autoimmune disease is selected from the group consisting of allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, Alzheimer’s disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet’s disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac spruedermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn’s disease, dermatomyositis, dilated cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves’ disease, Guillain-Barre syndrome (GBS), Goodpasture syndrome, graft-versus-host disease (GVHD), Hashimoto’s thyroiditis, hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura or immune thrombocytopenia (e.g. primary ITP), IgA neuropathy, IgM polyneuropathies, immune mediated thrombocytopenia juvenile arthritis, Kawasaki’s disease, lichen planus, lichen sclerosus, lupus erythematosus, Meniere’s disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes mellitus, multifocal motor neuropathy (MMN), myasthenia gravis (e.g. generalized MG or gMG), myositis, paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris (PV), pemphigus foliaceus (PF), pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynaud’s phenomenon, Reiter’s syndrome, rheumatoid arthritis (RA), sarcoidosis, scleroderma, Sjogren’s syndrome, solid organ transplant rejection, stiff-person syndrome, systemic lupus erythematosus (SLE), Takayasu’s arteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenia purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitides, vitiligo, and Wegener's granulomatosis.

[0187] For example, the antibody-mediated autoimmune disease may be myasthenia gravis (e.g., generalized MG or gMG), immune thrombocytopenia (e.g. primary ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), pemphigus foliaceus (PF) or pemphigus vulgaris (PV). Available data from clinical trials shows that blocking FcRn-IgG interactions results in amelioration of these diseases, alongside reductions on circulating pathogenic IgGs.

[0188] As the skilled person will appreciate, a FcRn antagonist composition will be released as suitable for clinical use if it exhibits inter alia acceptable physiochemical properties, an acceptable safety profile and acceptable biological activity. All of these parameters are indicated by the relative potency.

[0189] It follows, that the methods described herein may be carried out to assess the identity of the test FcRn antagonist composition, for example to confirm the identity of an efgartigimod composition or of a corresponding biosimilar composition.

[0190] The methods of the invention have the potential to detect isoforms or variants (such as amino acid sequence variants or oxidised variants) within a test efgartigimod composition, or within a corresponding biosimilar composition, with altered potency. Accordingly, the identity of efgartigimod alfa compositions, or corresponding biosimilar compositions, can be checked, for example across different manufacturing lots.

[0191] The methods described herein may also or alternatively be carried out to assess the stability of the test FcRn antagonist composition, for example a test efgartigimod composition or corresponding biosimilar composition (e.g., obtained from a particular manufacturing lot). The methods of the invention can detect non-stable product, such as oxidized forms of the variant IgG Fc regions. The relative potency is indicative of stability because non-stable compositions (e.g. compositions having a relatively high proportion of oxidized variants) will have a reduced potency.

[0192] In some embodiments, the assay is repeated with an internal-assay control (IAC) in place of the test FcRn antagonist composition. Advantageously, repeating the assay with an IAC can be used to quality control the assay itself. The resulting IAC curve contains (e.g., three) crucial datapoints in its slope which should match each other in inter-assay checks; this allows for checking the correctness of the assay as such.

[0193] Similar to the reference standard, the internal-assay control is typically a FcRn antagonist composition comprising a population of FcRn antagonist molecules corresponding to the FcRn antagonist molecules within (a sub-population of) the FcRn antagonist composition under test but wherein the potency of the IAC is already known and can be used to quality control the assay. The potency of the IAC, when present, may serve as a quality control forthe assay when the potency falls within a pre-defined range. C. Kits

[0194] In a further aspect, the invention provides a kit for carrying out the methods of determining the potency of a human neonatal Fc receptor (FcRn) antagonist of the invention, the kit comprising: a) human IgG or an IgG Fc region, optionally wherein the human IgG is human lgG3; b) FcRn, optionally wherein the FcRn is biotinylated FcRn; and c) optionally, means to detect FcRn-human IgG complexes or FcRn-IgG Fc region complexes.

[0195] As described elsewhere herein, the FcRn may be conjugated to a fusion tag such as a GST-tag or His-tag. The human IgG (e g., human lgG3) may be conjugated to a detectable moiety described herein. Alternatively, the means to detect the FcRn-human IgG complexes or the FcRn-IgG Fc region complexes may comprises an anti-human IgG antibody. For embodiments wherein an anti-human IgG antibody is used to detect an- FcRn-human IgG complex, the antihuman IgG antibody may be Fab specific. In some embodiments, the anti-human IgG antibody is conjugated to a detectable moiety described herein. In alternative embodiments, the antihuman IgG antibody is an enzyme-linked antibody.

[0196] In certain embodiments, the kit comprises: a) human lgG3 or a human lgG3 Fc region; b) biotinylated FcRn; and c) an enzyme-linked anti-human IgG antibody, preferably wherein the anti-human IgG antibody is Fab specific.

[0197] The enzyme-linked anti-human IgG antibody may comprise an anti-human IgG antibody conjugated to any enzyme described elsewhere herein. In some embodiments, the enzyme- linked anti-human IgG antibody is a peroxidase-linked anti-human IgG antibody, such as HRP- linked anti-human IgG antibody.

[0198] In some embodiments, the kit further comprises an enzyme substrate, optionally a chromogenic substrate. The enzyme substrate may be any enzyme substrate described elsewhere herein. In some embodiments, the enzyme substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0199] In some embodiments, the kit further comprises a substrate for immobilising the FcRn, optionally wherein the substrate is an immunoassay plate, e.g., an ELISA plate. In certain embodiments where the FcRn is biotinylated FcRn, the substrate may be an immunoassay plate, e.g., an ELISA plate coated with streptavidin or neutravidin, optionally neutravidin. In embodiments wherein the FcRn is conjugated to a GST-tag, the ELISA plate may be coated with glutathione. In embodiments wherein the FcRn is conjugated to a His tag, the ELISA plate may be coated with nickel or copper.

[0200] The kits provided herein may, optionally, comprise instructions for use e.g., instructions for carrying out the competition binding assay of the methods of the first aspect of the invention.

[0201] D. Methods of manufacturing

[0202] In a further aspect, the invention provides methods of manufacturing a FcRn antagonist composition (e.g., efgartigimod or a corresponding biosimilar thereof), comprising a method of determining the potency of a FcRn antagonist composition in inhibiting the binding of human IgG to FcRn. The method for determining potency may be any method described herein in accordance with the first or second aspects of the invention.

[0203] In the methods of manufacturing contemplated herein, the method of determining the potency of the FcRn antagonist composition may be preceded by any one or more of the following steps: expression of the FcRn antagonist molecules in a mammalian cell line, e.g. a Chinese

[0204] Hamster Ovary (CHO) cell line; harvesting of the FcRn antagonist; purification of the FcRn antagonist; virus reduction / inactivation; concentration and diafiltration.

[0205] In some embodiments, the method of manufacturing further comprises one or more steps of measuring the binding affinity of the FcRn antagonist composition for FcRn, optionally using a Biacore™ assay. Typically, the KD or dissociation constant of the FcRn antagonist composition for FcRn is measured.

[0206] In some embodiments, the binding affinity or Koof the FcRn antagonist composition for FcRn is measured at a pH of about 5 to about 8, optionally between about 5.5 to about 7.5. In some embodiments, the binding affinity or Ko of the FcRn antagonist composition for FcRn is measured at a pH of about a pH of about 5.5 to about 6.5, optionally about pH 6.0. This reflects the binding of the FcRn antagonist for FcRn in the early endosome.

[0207] In some embodiments, the binding affinity or Koof the FcRn antagonist composition for FcRn is alternatively, or additionally, measured at a pH of between about 7.3 to about 7.4, optionally about 7.4. This reflects the physiological pH, and therefore reflects the ability of FcRn antagonist to be internalized into FcRn-expressing cells via receptor-mediated (rather than fluidphase) uptake.

[0208] Thus, the step of determining the potency of a FcRn antagonist composition in inhibiting the binding of human IgG to FcRn according to the methods of the invention may act as a second step of quality control.

[0209] Incorporation by Reference

[0210] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0211] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods, uses and the like within the scope of these claims and their equivalents be covered thereby.

[0212] EXAMPLES

[0213] The invention will be further understood with reference to the following non-limiting examples.

[0214] Example 1 - Competition ELISA

[0215] The following example describes the development of an analytical method for testing the relative potency of efgartigimod batches. This method is based on the competitive binding of efgartigimod and human lgG3 to human FcRn at pH 6.0. The principle is depicted in FIG. 2. a) Pre-incubation

[0216] A titration series of efgartigimod samples was incubated with biotinylated human FcRn and human lgG3.

[0217] First, human lgG3 solutions were prepared consisting of 40.0 pg / mL wild-type human lgG3 (U- Protein Express BV, 53E2-lgG3, batch A) or 20.0 pg / mL wild-type human lgG3 (U-Protein Express BV, 53E2-lgG3, batch B) in Assay Buffer (1xPBS pH 6.0 + 0.1 % casein). An intermediate stock of 1 .0 mg / mL of efgartigimod test sample was prepared in PBS. The intermediate stock was used to prepare a sample (S1) containing 30.0 pg / mL efgartigimod in a hulgG3 dilution according to Table 2.

[0218] Table 2: Preparation of sample S1 in hlgG3 dilution

[0219] Sample S1 was then used as the starting concentration for the preparation of a dilution series of efgartigimod samples in human lgG3 dilution with Assay Buffer. Dilution steps of 1 :2.5 or 1 :2.25 were performed according to Table 3 or Table 4, respectively. The selection of dilution curve (1 :2.5 or 1 :2.25) was based on the read-out of the curve, to obtain an optimal distribution of the Optical Density (OD) values over the four-parameter curve. Each dilution series was prepared in duplicate.

[0220] Table 3: Preparation of efgartigimod sample dilution series in 1 :2.5 in hlgG3 dilution

[0221] Table 4: Preparation of efgartigimod sample dilution series in 1 :2.25 in hlgG3 dilution

[0222]

[0223] Next, biotinylated human FcRn solutions were prepared consisting of

[0224] (i) 2.0 pg / mL biotinylated human FcRn (Immunitrack; Cat N°: ITF01) in Assay Buffer when using hlgG3 batch A, or

[0225] (ii) 1 .0 pg / mL biotinylated human FcRn (Immunitrack; Cat N°: ITF01) in Assay Buffer when using hlgG3 batch B (see example provided in Table 5).

[0226] Table 5: Preparation of biotinylated human FcRn solutions

[0227] In a following step, the efgartigimod sample titration series in hlgG3 samples were mixed with biotinylated human FcRn in Assay Buffer at a 1 :1 ratio.

[0228] In the experiments where hlgG3 batch A was used, the final concentrations of the three components in the pre-incubation mixture were: 1.0 pg / mL biotinylated human FcRn; 20.0 pg / mL hlgG3; and a titration series of efgartigimod (from 15 — 0.004 pg / mL or from 15 - 0.010 pg / mL, depending on the dilution scheme).

[0229] In the experiments where hlgG3 batch B was used, the final concentrations were: 0.5 pg / mL biotinylated human FcRn; 10.0 pg / mL hulgG3; and titration series of efgartigimod (from 15 - 0.004 pg / mL or from 15 — 0.010 pg / mL, depending on the dilution scheme). The BLANK sample only contained 1 .0 pg / mL biotinylated human FcRn and 20.0 pg / mL hlgG3; no efgartigimod.

[0230] A titration series of an efgartigimod reference standard mixed with hlgG3 and FcRn was prepared in the same manner as the efgartigimod samples described above. The efgartigimod reference standard was an efgartigimod reference batch (batch 1).

[0231] A titration series of an efgartigimod Internal Assay Control (IAC) mixed with hlgG3 and FcRn, was also prepared in the same manner as the efgartigimod samples described above.

[0232] The IAC samples, used to quality control the assay, were an efgartigimod IAC batch (batch 2 or batch 3).

[0233] The samples were prepared and analysed in duplicate in a 96-well plate. The efgartigimod sample / hlgG3 / FcRn samples were incubated for 1 h at room temperature without shaking. b) ELISA

[0234] After the incubation step, the samples were transferred to an ELISA plate. An example of a typical ELISA plate set-up is given in FIG. 3.

[0235] Prior to transfer, the ELISA plate was prepared by loading 100 pL of Coating Solution (1 pg / mL Neutravidin biotin-binding protein (Thermo Scientific; Cat N°: 31000) in 1x PBS pH 6.0) into each well, covering the ELISA plate with a sealing tape and incubating overnight (14-18 h) at 2°C - 8°C. After incubation, the ELISA plate was washed 3 times with sufficient volume of Wash Buffer (PBS pH 6.0 + 0.05% v / v Tween20) per well (250-300 pL). Then, 250 pL Blocking Buffer (1 % w / v casein solution in 1xPBS pH 6.0) was added to each well, before sealing the ELISA plate and incubating for 2 h at room temperature while shaking at 450 rpm. Excess Blocking Buffer was discarded by inverting the ELISA plate over a sink and by tapping the plate on absorbent paper towel. The ELISA plate was again washed 3 times with sufficient volume of Wash Buffer per well (250-300 pL).

[0236] Next, 100 pL of each sample from the pre-incubation step was transferred to the ELISA plate. The ELISA plate was sealed and incubated for 1 h at room temperature while shaking at 450 rpm, before washing 5 times with sufficient volume of Wash Buffer per well (250-300 pL). c) Quantification

[0237] To detect bound human lgG3, 100 pL of Detection antibody Goat Anti-Human IgG (Fab specific)-peroxidase (Sigma; Cat N°: A0293) was added to each well of the ELISA plate. The ELISA plate was sealed and incubated for 1 h at room temperature while shaking at 450 rpm, before washing 5 times with sufficient volume of Wash Buffer per well (250-300 pL).

[0238] After removal of the Wash Buffer, 100 pL of chromogenic substrate, tetramethylbenzidine (Calbiochem, Cat N°: CL07-1000ML), was transferred to each well of the ELISA plate to visualize bound human lgG3. The ELISA plate was covered and protected from light (e.g., with aluminium foil), and incubated for around 15 min (depending on substrate) at room temperature while shaking at 450 rpm.

[0239] Immediately after the 15 min colour development, the reaction was quenched by adding 100 pL per well of 0.5 M H2SO4 and briefly shaking the plate. Then the absorbance at 450 nm (reference wavelength set at 620 nm) was read immediately after quenching the reaction using a microplate absorbance reader. d) Analysis

[0240] The different concentrations of efgartigimod samples (x-axis) were plotted versus Optical Density (OD) signal obtained at 450 nm with 620 nm subtraction (y-axis), using the GraphPad Prism software. In more detail, the efgartigimod sample concentrations are transformed using the function logX, and the resulting logX values were analysed with the function "non-linear regression (curve fit)". The "log (agonist) vs. response-variable slope (four parameters)" was selected using "ordinary fit". The software calculates the IC50, R-square, top, bottom and slope of the curve, and plots a four-parameter curve. The Graphpad Prism software also calculates the 95% confidence intervals of best-fit parameters.

[0241] The relative potency of efgartigimod test samples can be determined by comparing the IC50 of an efgartigimod test sample with the IC50 value of the reference standard, according to the following formula: 100

[0242] A potency of between 70-135%, more preferably between 85-120%, is acceptable for a efgartigimod product or batch to be released for clinical use (and indicates that the efgartigimod product is of suitable efficacy and safety).

[0243] Example 2 - Use of the Competition ELISA to measure the potency of efgartiqimod samples The assay described in Example 1 above was used to measure the potency of the following efgartigimod samples: efgartigimod batches 4-7; efgartigimod samples having different levels of oxidation - 20% or 60%.

[0244] Samples were compared against a reference standard (efgartigimod batch P3179589ARS).

[0245] The ELISA curves are shown in FIG. 4. A good fit of the data was obtained with the four- parameter model. The obtained IC50 values and their corresponding 95% confidence intervals are presented in FIG. 5.

[0246] The percentage blockage of hlgG3 binding relative to the reference standard and the percentage oxidized species present in each sample is presented in Table 6.

[0247] Table 6: Competition ELISA testing results as % blockage relative to the reference standard c> Oxidized variants as determined by CEX analysis.

[0248] (2)Calculated as IC50 (reference) / IC50 (sample).

[0249] - Not tested.

[0250] Conclusions

[0251] The Competition ELISA described in the Examples above is a suitable in vitro model to monitor the primary mechanism of action of FcRn antagonists such as efgartigimod i.e., constitutive blockage of FcRn through competitive binding of FcRn with endogenous IgGs. The assay is particularly suitable to control lot-to-lot quality / consistency of efgartigimod product preparations or batches. As demonstrated herein, efgartigimod compositions with reduced potency (e.g. as a result of a high percentage of oxidized variants) can be detected and rejected if not meeting the acceptance criteria for clinical use.

Claims

CLAIMS1 . A method of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of human IgG to FcRn using a competition binding assay, the assay comprising the steps of: a) incubating the FcRn antagonist composition with FcRn in the presence of human IgG such that FcRn antagonist and human IgG compete for binding to FcRn; b) separating any FcRn - human IgG complexes from any unbound human IgG; and c) detecting the quantity of FcRn - human IgG complexes.

2. The method of claim 1 , wherein the human IgG is human lgG3.

3. The method of claim 1 or claim 2, wherein steps a) - c) are carried out at a pH of about 5 to about 8.

4. The method of claim 1 or claim 2, wherein steps a) - c) are carried out at a pH of about 5.5 to about 6.5.

5. The method of claim 1 or claim 2, wherein steps a) - c) are carried out at a pH of about 6.0.

6. The method of any one of claims 1-5, wherein the FcRn antagonist composition is serially diluted such that two or more samples having different concentrations of the FcRn antagonist composition are incubated with FcRn in the presence of human IgG.

7. The method of claim 6, wherein results of the assay are used to generate a dose response curve of the FcRn antagonist composition inhibiting the binding of human IgG to FcRn.

8. The method of claim 6 or claim 7, wherein results of the assay are used to calculate a half maximal inhibitory concentration (IC50) of the FcRn antagonist composition.

9. The method of any one of claims 1-8, wherein results of the assay are compared with a reference standard.

10. The method of claim 9, wherein the assay is repeated with the reference standard in place of the test FcRn antagonist composition.

11. The method of claim 9 or claim 10, wherein a relative potency of the FcRn antagonist composition is calculated as:(IC50 of the reference standard / IC50 of the FcRn antagonist composition) x 100%.

12. The method of claim 11 , wherein a FcRn antagonist composition having a relative potency of about 70% to about 135% is identified as a FcRn antagonist composition suitable for clinical use, optionally suitable for use in the treatment of an antibody-mediated autoimmune disease.

13. The method of claim 11 , wherein a FcRn antagonist composition having a relative potency of about 85% to about 120% is identified as a FcRn antagonist composition suitable for clinical use, optionally suitable for use in the treatment of an antibody-mediated autoimmune disease.

14. The method of claim 12 or claim 13, wherein the antibody-mediated autoimmune disease is myasthenia gravis (MG), optionally generalized myasthenia gravis (gMG).

15. The method of any one of claims 1-14, wherein the assay is repeated with an internal-assay control in place of the test FcRn antagonist composition.

16. The method of any one of claims 1-15, wherein the FcRn antagonist composition comprises or consists of a population of FcRn antagonist molecules.

17. The method of claim 16, wherein the FcRn antagonist molecules comprise or consist of a variant Fc region or FcRn binding fragment thereof that binds to FcRn with increased affinity relative to the wild-type Fc region of IgG.

18. The method of claim 17, wherein the variant Fc region comprises or consists of a first Fc domain and a second Fc domain which form a homodimer or heterodimer.

19. The method of claim 18, wherein the first Fc domain and / or the second Fc domain comprise amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively.

20. The method of claim 18, wherein the first Fc domain and / or second Fc domain comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.21 . The method of any one of claims 18-20, wherein the first Fc domain and the second Fc domain each have an amino acid sequence represented by SEQ ID NO: 1 .

22. The method of any one of claims 18-21 , wherein the first Fc domain and / or the second Fc domain comprise or consist of an amino acid sequence independently selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

23. The method of any one of claims 16-22, wherein the population of FcRn antagonist molecules is heterogeneous.

24. The method of any one of claims 16-23, wherein the population of FcRn antagonist molecules comprises or consists of efgartigimod.

25. The method of any one of claims 16-23, wherein the population of FcRn antagonist molecules comprises or consists of a biosimilar of efgartigimod.

26. A method of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of human IgG to FcRn using a competition binding assay, the assay comprising the steps of: a) incubating the FcRn antagonist composition with FcRn in the presence of human IgG such that FcRn antagonist and human IgG compete for binding to FcRn; b) separating any FcRn - human IgG complexes from any unbound human IgG; and c) detecting the quantity of FcRn - human IgG complexes, wherein the FcRn antagonist composition comprises or consists of a biosimilar of efgartigimod, optionally wherein results of the assay are compared with a reference standard.

27. The method of any one of claims 9-26, wherein the reference standard is a FcRn antagonist composition comprising or consisting of efgartigimod.

28. The method of claim 25 or claim 26, wherein the reference standard is a FcRn antagonist composition comprising or consisting of a biosimilar of efgartigimod.

29. The method of claim 27 or 28, wherein the method is carried out to confirm the identity of an efgartigimod composition or of an efgartigimod biosimilar composition.

30. The method of claim 27 or 28, wherein the method is carried out to assess the stability of an efgartigimod composition or of an efgartigimod biosimilar composition.31 . The method of any one of claims 27-30, wherein the method is carried out to assess the biological activity of an efgartigimod composition or of an efgartigimod biosimilar composition.

32. The method of any one of claims 1-31 , wherein in step a), the FcRn antagonist composition is combined with human IgG prior to the addition of FcRn.

33. The method of any one of claims 1-32, wherein the FcRn antagonist composition is serially diluted such that multiple samples in the concentration range 0.001 pg / mL to 20 pg / mL, optionally in the concentration range 0.004 pg / mL to 15 pg / mL are incubated with FcRn in the presence of human IgG.

34. The method of any one of claims 1-33, wherein in step a), the concentration of human IgG is about 10.0 pg / mL.

35. The method of any one of claims 1-33, wherein in step a), the concentration of human IgG is about 20.0 pg / mL.

36. The method of any one of claims 1-35, wherein in step a), the concentration of FcRn is about 0.5 pg / mL.

37. The method of any one of claims 1-35, wherein in step a), the concentration of FcRn is about 1 .0 pg / mL.

38. The method of any one of claims 1-37, wherein in step a), the incubating is for at least 1 hour at ambient temperature.

39. The method of any one of claims 1-38, wherein in step b), the FcRn - human IgG complexes are separated from any unbound human IgG by first immobilising the FcRn and secondly, removing any unbound human IgG in a wash step.

40. The method of claim 39, wherein the FcRn is biotinylated and is immobilised by contacting the FcRn with a substrate having a biotin-binding protein thereon, optionally streptavidin or neutravidin.

41. The method of any one of claims 1-40, wherein step c) involves detecting FcRn - human IgG complexes using an anti-human IgG antibody.

42. The method of any one of claims 1-41 , wherein the competition binding assay is a competition enzyme-linked immunosorbent assay (ELISA).

43. The method of any one of claims 1-42, wherein the method comprises the steps of: ai) adding human IgG, preferably human lgG3, to multiple samples of the FcRn antagonist composition, each sample having a different concentration of the FcRn antagonist composition;aii) incubating the samples with biotinylated FcRn; bi) applying the samples to a substrate coated with a biotin-binding protein, optionally a neutravidin-coated ELISA plate, so as to immobilise the biotinylated FcRn; bii) washing the substrate one or more times to remove unbound human IgG, optionally wherein the wash buffer comprises PBS pH 6.0 + 0.05% Tween20; c) detecting the quantity of immobilised FcRn-human IgG complexes using an enzyme- linked anti-human IgG antibody and an enzyme substrate.

44. The method of claim 42 or 43, wherein in step c), the enzyme-linked anti-human IgG antibody is a peroxidase-linked antibody and / or the enzyme substrate is 3, 3', 5,5'- tetramethylbenzidine (TMB).

45. The method of claim 44, wherein step c) comprises the sub-steps: ci) adding the peroxidase-linked antibody to the immobilised FcRn-IgG complexes and optionally, incubating for 1 hour at ambient temperature; cii) one or more washes with a wash buffer, optionally wherein the wash buffer comprises PBS pH 6.0 + 0.05% Tween20; ciii) the addition of TMB and optionally, incubating the FcRn-IgG-anti-IgG antibody complexes for 15 min at ambient temperature; civ) quenching the reaction by adding sulfuric acid, optionally 0.5M sulfuric acid; and cv) reading the absorbance at 450 nm with subtraction of the reference wavelength at 620 nm.

46. A method of determining the potency of a human neonatal Fc receptor (FcRn) antagonist composition in inhibiting the binding of an IgG Fc region to FcRn using a competition binding assay, the assay comprising the steps of: a) incubating the FcRn antagonist composition with FcRn in the presence of an IgG Fc region such that FcRn antagonist and IgG Fc region compete for binding to FcRn; b) separating any FcRn - IgG Fc region complexes from any unbound IgG Fc region; and c) detecting the quantity of FcRn - IgG Fc region complexes.

47. The method of claim 46, wherein the IgG Fc region is wild-type.

48. The method of claim 46 or claim 47, wherein the IgG Fc region is a human IgG Fc region.

49. A kit for carrying out the method of any one of claims 1 -48, the kit comprising a) human IgG or an IgG Fc region; b) FcRn; andc) means to detect FcRn-human IgG complexes or FcRn-IgG Fc region complexes.

50. The kit of claim 49, wherein a) the human IgG is human lgG3; b) the FcRn is biotinylated; and / or c) the means to detect the FcRn-human IgG complexes or the FcRn-IgG Fc region complexes comprises an enzyme-linked anti-human IgG antibody.51 . The kit of claim 50, wherein the enzyme-linked anti-human IgG antibody is a peroxidase- linked antibody.

52. The kit of claim 50 or 51 , additionally comprising an enzyme substrate, optionally a chromogenic substrate, optionally TMB.

53. The kit of any one of claims 50-52, additionally comprising an ELISA plate, optionally an ELISA plate coated with streptavidin or neutravidin.

54. A method of manufacturing a FcRn antagonist composition, comprising the method of any one of claims 1-48.

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