Monovalent molecules binding to IGA and methods of use

Abstract

The present invention relates to monovalent antigen-binding molecules that bind to IgA, such as one-armed antibodies, and their use in the treatment of disorders. The monovalent antigen-binding molecules comprise an antigen-binding domain that binds to IgA, and a variant Fc region or FcRn binding fragment thereof that binds to the human neonatal Fc receptor (hFcRn) with increased affinity relative to a wild-type Fc region. The monovalent antigen-binding molecules of the invention bind IgA and have utility in the treatment of IgA-mediated disorders.

Description

[0001] MONOVALENT MOLECULES BINDING TO IGA AND METHODS OF USE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to monovalent antigen-binding molecules that bind to IgA, such as one-armed antibodies, and their use in the treatment of disorders. The monovalent antigen-binding molecules comprise an antigen-binding domain that binds to IgA, and a variant Fc region or FcRn binding fragment thereof that binds to the human neonatal Fc receptor (hFcRn) with increased affinity relative to a wild-type Fc region. The monovalent antigen-binding molecules may comprise a variant Fc region incorporating, for example, ABDEG™ or NHance® technology, wherein the variant Fc region binds to human FcRn (i) with increased affinity relative to a wild-type Fc domain; and (ii) in a pH-dependent manner. The monovalent antigen-binding molecules of the invention bind IgA and have utility in the treatment of IgA-mediated disorders.

[0004] BACKGROUND TO THE INVENTION

[0005] Immunoglobulin A (IgA) is the most prevalent antibody class produced in humans. The daily production rate of IgA is around 66 mg / kg, which is higher than all other antibody isotypes combined. Given this abundance, it has been postulated that IgA plays a significant role in immune defence (Breedveld and van Egmond, 2019; Aleyd, Heineke, and van Egmond, 2015).

[0006] In the mucosal membranes, the level of IgA is greater than all other types of antibody combined and IgA plays an important role in passive immunity in such areas. The role of IgA in the mucosal areas, such as the intestinal lumen, is relatively well-characterised. Dimeric IgA in the form of secretory IgA (slgA) is secreted by epithelial cells into extracellular secretions (such as mucus). slgA in extracellular secretions is a first line of immune defence acting as a barrier against pathogens and commensals by preventing colonisation and penetration of the mucosal epithelium so as to avoid infection and antigen leakage into the systemic circulation. IgA is also present in the serum, typically in a monomeric form. Serum IgA is capable of binding to receptors such as FcaRI (also known as CD89), which mediates effector functions in order to initiate an inflammatory response. FcaRI is a member of the Fc receptor immunoglobulin superfamily and is expressed on cells from the myeloid lineage (including monocytes, macrophages, Kupffer cells, eosinophils, neutrophils, and certain subsets of dendritic cells) as well as on platelets (Monteiro, Kubagawa, and Cooper, 1990; Hostoffer, Krukovets, and Berger, 1993; Qian et al., 2008). Once FcaRI binds to IgA immune complexes (i.e. opsonized pathogens), there is cross-linking and an induction of pro-inflammatory responses.

[0007] The presence of excessive IgA immune complexes, IgA autoantibodies or serum IgA is thought to lead to uncontrolled and disproportionate immune cell activation, which in turn, can lead to severe tissue damage in autoimmune diseases.

[0008] SUMMARY OF THE INVENTION

[0009] There is a need to develop agents that target IgA since elevated levels of IgA and IgA immune complexes are implicated in disease. As of the filing date of this application, there are no approved products that modulate IgA that have been proven useful to treat IgA- mediated disease. The present invention addresses this need by providing monovalent antigen-binding molecules that bind to IgA, otherwise referred to as monovalent IgA- binding molecules.

[0010] The present invention provides monovalent antigen-binding molecules that are particularly suited to the treatment of IgA-mediated disorders. Advantageously, the monovalent antigen-binding molecules are able to reduce the levels of serum IgA, prevent IgA binding to its receptors (such as FcaRI) and also displace receptor (e.g., FcaRI)-bound IgA.

[0011] In further detail, the present application exemplifies monovalent antigen-binding molecules of the invention and their ability to reduce serum IgA levels in vivo. As reported herein, the monovalent antigen-binding molecules of the invention bind to IgA and the resulting complex can be internalised via neonatal Fc receptor (FcRn)-mediated cellular uptake. Once internalised, the serum IgA molecules are subjected to lysosomal degradation thereby reducing serum IgA levels. The monovalent antigen-binding molecules are recycled back to the extracellular space. The recycling of the monovalent antigen-binding molecules allows the process of serum IgA capture and degradation to be repeated by the same monovalent antigen-binding molecule. This process is referred to herein as “IgA sweeping”.

[0012] Surprisingly, the reduction of IgA observed with the monovalent antigen-binding molecules of the invention (e.g., one-armed modified antibodies) is greater as compared with bivalent IgA-binding molecules (e.g., conventional two-armed antibodies). The monovalent antigenbinding molecules of the invention improve both the speed of serum IgA reduction as well as the amount of serum IgA removal as compared to bivalent IgA-binding counterparts.

[0013] This finding is unexpected because whilst a monovalent antigen-binding molecule can only bind a single IgA antigen at any given time, a bivalent IgA-binding molecule can bind two IgA antigens simultaneously. It is therefore surprising that the monovalent antigen-binding molecules of the invention reduce serum IgA to a greater extent than bivalent IgA-binding molecules.

[0014] The results reported herein demonstrate that FcRn receptor occupancy is higher with monovalent antigen-binding molecules of the invention as compared with bivalent IgA- binding molecules. Without wishing to be bound by theory, it is believed that monovalent antigen-binding molecules reduce steric hinderance as compared to bivalent IgA-binding molecules and thereby improve access to the FcRn receptor. The improved accessibility of a monovalent antigen-binding molecule-serum IgA complex to FcRn results in enhanced IgA sweeping.

[0015] Taking into account the above, the monovalent antigen-binding molecules of the invention exhibit improved IgA sweeping and are advantageous for use in the treatment of IgA- mediated diseases.

[0016] In a first aspect, the present invention provides a monovalent antigen-binding molecule comprising:

[0017] - an antigen-binding domain that binds to IgA; and - a variant Fc region or a FcRn binding fragment thereof, that binds to human FcRn with increased affinity relative to a wild-type Fc region; and wherein the variant Fc region comprises a first Fc domain and a second Fc domain.

[0018] In certain embodiments, the monovalent antigen-binding molecule exhibits lower IgA binding affinity at an acidic pH than at a neutral pH. In certain embodiments, the human IgA binding affinity at pH 6 is reduced by at least 25%, preferably at least 50%, as compared with the human IgA binding affinity at pH 7.4. In certain embodiments, the human IgA binding affinity at pH 5 is reduced by at least 75%, preferably 100%, as compared with the human IgA binding affinity at pH 7.4.

[0019] In certain embodiments, the monovalent antigen-binding molecule inhibits binding of IgA to an IgA receptor. In certain embodiments, the IgA receptor is FcaRI (CD89) or CD71 .

[0020] In certain embodiments, the monovalent antigen-binding molecule displaces IgA from an IgA receptor. In certain embodiments, the monovalent antigen-binding molecule displaces IgA from a FcaRI (CD89) receptor.

[0021] In certain embodiments, the monovalent antigen-binding molecule inhibits or reduces IgA immune complex formation.

[0022] In certain embodiments, the antigen-binding domain is selected from: a Fab; an Fv; a scFv; and a VHH domain. In preferred embodiments, the antigen-binding domain is a Fab.

[0023] In certain embodiments, the antigen-binding domain is attached to the N-terminus of either the first Fc domain or the second Fc domain.

[0024] In certain embodiments, the antigen-binding domain is a Fab and the C-terminus of the Fab heavy chain is attached to the N-terminus of either the first Fc domain or the second Fc domain via an IgG hinge region.

[0025] In certain embodiment, the variant Fc region or FcRn binding fragment thereof binds to FcRn with increased affinity relative to a wild-type IgG Fc region. In certain embodiments, the variant Fc region or FcRn binding fragment thereof binds to human FcRn with increased affinity relative to a wild-type human IgG Fc region, preferably a wild-type human IgG 1 Fc region. In certain embodiments, the variant Fc region or FcRn binding fragment thereof binds to human FcRn with increased affinity at pH 6.0 and pH 7.4. In certain embodiments, the binding affinity of the variant Fc region or FcRn binding fragment thereof for human FcRn at pH 6.0 is increased by at least 20x, preferably at least 30x, relative to a wild-type human IgG 1 Fc region. In certain embodiments, the binding affinity of the variant Fc region or FcRn binding fragment thereof for human FcRn at pH 6.0 is stronger than KD 15 nM. In certain embodiments, the binding affinity of the variant Fc region or FcRn binding fragment thereof for human FcRn at pH 7.4 is stronger than KD 320 nM.

[0026] In certain embodiments, the variant Fc region or FcRn binding fragment thereof comprises at least one amino acid substitution as compared with the corresponding wild-type Fc region, wherein the at least one amino acid substitution confers increased binding affinity relative to a wild-type Fc region. In certain embodiments, the variant Fc region or FcRn binding fragment thereof is a variant human Fc region or FcRn binding fragment thereof. In certain embodiments, the variant Fc region or FcRn binding fragment thereof is a variant IgG Fc region or FcRn binding fragment thereof. In preferred embodiments, the variant Fc region or FcRn binding fragment thereof is a variant IgG 1 Fc region or FcRn binding fragment thereof.

[0027] In certain embodiments, the variant Fc region or FcRn binding fragment thereof comprises the amino acids:

[0028] (i) Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively; or

[0029] (ii) Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively.

[0030] In certain embodiments, the first Fc domain or second Fc domain comprises the amino acids:

[0031] (i) Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively; or

[0032] (ii) Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively.

[0033] In certain embodiments, the first Fc domain and the second Fc domain comprise the amino acids:

[0034] (i) Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively; or

[0035] (ii) Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively.

[0036] In certain embodiments, the variant Fc region or the FcRn binding fragment thereof further comprises at least one additional amino acid substitution as compared with the corresponding wild-type Fc region; and wherein the at least one additional substitution reduces or eliminates Fc effector function.

[0037] In certain embodiments, the first Fc domain and / or second Fc domain comprise the amino acids:

[0038] (i) A and A at EU positions 234, 235; and optionally

[0039] (ii) G at EU position 329.

[0040] In certain embodiments, the first Fc domain and the second Fc domain comprise the amino acids A, A, G, Y, T, E, K, F and Y at EU positions 234, 235, 329, 252, 254, 256, 433, 434 and 436, respectively.

[0041] In certain embodiments, the variant Fc region or the FcRn binding fragment thereof further comprises at least one additional amino acid substitution as compared with the corresponding wild-type Fc region; and wherein the at least one substitution promotes dimerisation between the first Fc domain and the second Fc domain.

[0042] In certain embodiments, the first Fc domain and the second Fc domain comprise knob-into- holes amino acid substitutions. In certain embodiments, the first Fc domain comprises the amino acid W at EU position 366; and the second Fc domain comprises the amino acids S, A and V at EU positions 366, 368 and 407, respectively.

[0043] In preferred embodiments: the first Fc domain comprises the amino acids A, A, G, Y, T, E, W, K, F and Y at EU positions 234, 235, 329, 252, 254, 256, 366, 433, 434 and 436, respectively; and the second Fc domain comprises the amino acids A, A, G, Y, T, E, K, F, Y, S, A and V at EU positions 234, 235, 329, 252, 254, 256, 433, 434, 436, 366, 368 and 407, respectively. The antigen-binding domain may preferably be a Fab that is attached to the first Fc domain.

[0044] In further preferred embodiments, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 and / or the second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34.

[0045] In certain embodiments, the first Fc domain and the second Fc domain do not comprise an N-linked glycan at EU position 297. In certain embodiments, the first Fc domain and second Fc domain comprise an afucosylated N-linked glycan at EU position 297. In certain embodiments, the first Fc domain and second Fc domain comprise an N-linked glycan having a bisecting GIcNac at EU position 297 of the Fc domains.

[0046] In certain embodiments, the monovalent antigen-binding molecule is a humanised or germlined variant of non-human antibodies, such as for example camelid-derived antibodies.

[0047] In certain embodiments, the antigen-binding molecule is a modified IgG antibody having only one Fab arm. The modified IgG antibody may preferably be a modified lgG1 antibody.

[0048] In certain embodiments, the monovalent antigen-binding molecule binds to membranebound IgA or free IgA.

[0049] Further provided herein are isolated polynucleotides or polynucleotides, which encode the monovalent antigen-binding molecules. Also provided herein are expression vectors comprising said isolated polynucleotides or polynucleotides that are operably linked to regulatory sequences which permit expression of the monovalent antigen-binding molecules. Also provided are host cells or cell-free expression systems containing the expression vectors. Further provided are methods of producing recombinant monovalent antigen-binding molecules, the methods comprising culturing the host cells or cell free expression systems under conditions which permit expression of the monovalent antigenbinding molecule and recovering the expressed monovalent antigen-binding molecule.

[0050] In a further aspect, the present invention provides a pharmaceutical composition comprising a monovalent antigen-binding molecule of the invention and at least one pharmaceutically acceptable carrier or excipient.

[0051] In still further aspects, the present invention provides a monovalent antigen-binding molecule according to the invention or a pharmaceutical composition of the invention for use as a medicament.

[0052] Also provided are, methods of treating a disorder in a subject, wherein the method comprises administering to a patient in need thereof a therapeutically effective amount of a monovalent antigen-binding molecule of the invention or a pharmaceutical composition of the invention. The disorder may preferably be an IgA-mediated disorder. In certain embodiments, the disorder is an IgA autoantibody-mediated disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows a log-linear plot of the average h Ig A percentage-time profiles in serum upon IP administration of the test item (A) and a log-linear plot of the average anti-lgA concentration-time profiles in serum upon IP administration of the test item (B). The number of a1 KI mice included per group in the analysis is indicated with (n=x) and data is depicted as mean ± SD.

[0054] Figure 2 shows a log-linear plot of individual cynomolgus monkey serum anti-lgA concentration-time profiles upon IV administration of (A) Clone F-hlgG1 LALA HN (G1 ), (B) Clone F-h IgG 1 LALA ABDEG (G2) and (C) Clone F- h IgG 1 HN (G4) antibodies. 3 monkeys were analysed in each group. Potential PK outliers are indicated with *.

[0055] Figure 3 shows linear plots of individual cynomolgus monkey serum IgA concentration-time profiles upon IV administration of (A) Clone F-hlgG1 LALA HN (G1), (B) Clone F-hlgG1 LALA ABDEG (G2) and (C) Clone F-h IgG 1 HN (G4) antibodies. 3 monkeys were analysed in each group. Potential PD outliers are indicated with *.

[0056] Figure 4 shows linear plots of the absolute drop in individual cynomolgus monkey serum IgA concentration-time profiles upon IV administration of (A) Clone F-hlgG1 LALA HN (G1 ), (B) Clone F-hlgG1 LALA ABDEG (G2) and (C) Clone F-hlgG1 HN (G4) antibodies. 3 monkeys were analysed per group.

[0057] Figure 5 shows linear plots of individual cynomolgus monkey serum IgG concentrationtime profiles upon IV administration of (A) Clone F-hlgG1 LALA HN (G1 ), (B) Clone F- hlgG1 LALA ABDEG (G2) and (C) Clone F-hlgG1 HN (G4) antibodies. 3 monkeys were analysed per group. Potential outliers are indicated with *.

[0058] Figure 6 shows a diagrammatic representation of the different Fc engineering strategies implemented on Clone A to investigate IgA sweeping efficacy. Mutations are indicated with stars and listed below the molecules.

[0059] Figure 7 shows the results of a competition assay to assess aspecific binding of the different Clone A-hlgG1 Fc variants to RBL-WT cells using flow cytometry. MFI signals (anti-lg A-PE signal) of conditions with no anti-lg A (background signal; left bar) and high anti-lgA concentration (83.3 nM for two-armed variants, 125 nM for one-armed variants LALA ABDEG and LALA HN pl4 and 62.5 nM for LALA HN pl2 one-armed; right bar).

[0060] Figure 8 shows FcRn occupancy of Clone A-hlgG1 Fc variants (LALA ABDEG two-armed and one-armed), ARGX-113 and ARGXdG on U937 cells.

[0061] Figure 9 shows the results of an IgA internalization assay for the Clone A- h IgG 1 Fc variants in HEK-FcRn cells (A) or HEK-WT cells (B) using flow cytometry. Full bars depict the anti-lgA antibody alone and striped bars depict lgA:anti- IgA immune complexes (1 :1 ratio).

[0062] Figure 10 shows an FcRn degradation assay in HEK-FcRn-GFP cells using flow cytometry. Full bars depict anti-lgA antibody alone, striped bars depict anti-lgA immune complexes (1 :1 ratio). Immunovant (IMVT) anti-FcRn antibody was added as a positive control. Data depicted as mean MFI of FcRn#GFP signal (A) and relative amount of FcRn (% MFI) compared to the untreated control (B).

[0063] (* indicates excluded outlier datapoint).

[0064] Figure 11 shows log-linear plots of the average concentration-time profiles of human Mota- IgA (A) and anti-lgA antibody serum levels (B) upon IP administration of IgA (on DO and D2, 20mg / kg) and the test item (on DO, 10 mg / kg). The number of mice included per group in the analysis is indicated with n=x (for the PK profile) and n=x / y (for the PD profile, x being the number of mice included after the first human Mota-IgA injection and y the number of mice included after the second human Mota-IgA injection). Data is plotted as mean ± SD.

[0065] Figure 12 shows log-linear plots of the average concentration-time profiles of human Mota- IgA (A) and anti-lgA antibody serum levels (B) upon IP administration of IgA (on DO and D2, 20mg / kg) and the test item (on DO, 10 mg / kg). The number of mice included per group in the analysis is indicated with n=x (for the PK profile) and n=x / y (for the PD profile, x being the number of mice included after the first human Mota-IgA injection and y the number of mice included after the second human Mota-IgA injection). Data is plotted as mean ± SD. DETAILED DESCRIPTION

[0066] A. Definitions

[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art in the technical field of the invention.

[0068] “Antibody” - As used herein, the term “antibody” is intended to encompass full-length antibodies and variants thereof, including but not limited to bivalent antibodies, humanised antibodies, germlined antibodies (see definitions below). The term “antibody” is typically used herein to refer to immunoglobulin polypeptides having a combination of two heavy and two light chains wherein the polypeptide has significant specific immunoreactive activity to an antigen of interest (herein IgA). For antibodies of the IgG class, the antibodies comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000- 70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region. The light chains of an antibody are classified as either kappa or lambda (K,X). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C- terminus at the bottom of each chain.

[0069] An antibody typically comprises two antigen-binding domains that are each capable of binding an antigen. This means that a single antibody can simultaneously bind two antigen molecules and therefore antibodies are typically referred to as being “bivalent” for an antigen. The antigen-binding domains are located at the tips of the forked ends of the Y configuration. The forked ends of a “Y” configuration immunoglobin polypeptide are also referred to in the art as the “arms” of an antibody molecule. Therefore, an antibody can be referred to as having two arms. Each of the two arms typically comprise a single antigenbinding domain (i.e. a Fab fragment).

[0070] Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, j , a, 8, e) with some subclasses among them (e.g., y1-y4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgD or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , etc. are well characterized and are known to confer functional specialization. The term “antibody” as used herein encompasses antibodies from any class or subclass of antibody.

[0071] “Monovalent antigen-binding molecule” - As used herein the term “monovalent antigenbinding molecule” (or monovalent antigen binding molecule) refers to an antigen-binding molecule having a single valency for its respective antigen i.e. IgA. The monovalent antigen-binding molecules described herein are capable of binding only a single antigen (i.e. IgA) at any given time. By way of example, a monovalent antigen-binding molecule may be a modified antibody, particularly a modified IgG antibody, with one antigen-binding arm (such as a single Fab region). In other words, the term monovalent antigen-binding molecule encompasses one-armed modified antibodies, particularly one-armed modified IgG antibodies. Figure 6 includes diagrams that show the difference in structure between bivalent antigen-binding molecules (or conventional heterotetrameric two-armed antibodies) and exemplary monovalent antigen-binding molecules of the invention (i.e. one- armed modified antibodies).

[0072] “Antigen binding domain” - As used herein “antigen binding domain” refers to any polypeptide domain that binds to an antigen (i.e. IgA herein). The term “antigen binding domain” as used herein is intended to encompass polypeptides derived from antibodies, such as Fab fragments, F(ab')2 fragments, single-chain Fvs (scFv), VH domains (VH), VL domains (VL), VHH domains and antigen binding fragments of the above. The term also encompasses synthetic antigen-binding polypeptides or antibody mimetic polypeptides such as, for example, anticalins and DARPins.

[0073] “Variable region” or “variable domain” - The terms "variable region" and "variable domain" are used herein interchangeably and are intended to have equivalent meaning. The term "variable" refers to the fact that certain portions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody or monovalent antigen-binding molecule for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VL domain and the VH domain which form part of the antigen binding site. The first, second and third hypervariable loops of the VLambda light chain domain are referred to herein as L1 (A), L2(A) and L3(A) and may be defined as comprising residues 24-33 (L1 (A), consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(A), consisting of 3 residues) and 90-96 (L3(A), consisting of 5 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VKappa light chain domain are referred to herein as L1 (K), L2(K) and L3(K) and may be defined as comprising residues 25-33 (L1 (K), consisting of 6, 7, 8, 11 , 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VH domain are referred to herein as H1 , H2 and H3 and may be defined as comprising residues 25-33 (H1 , consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91 -105 (H3, highly variable in length) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

[0074] Unless otherwise indicated, the terms L1 , L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both Vkappa and Vlambda isotypes. The terms H1 , H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including y, E, 5, a or p.

[0075] The hypervariable loops L1 , L2, L3, H1 , H2 and H3 may each comprise part of a "complementarity determining region" or "CDR", as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous, since the hypervariable loops (HVs) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983) and the limits of the HVs and the CDRs may be different in some VH and VL domains.

[0076] The CDRs of the VL and VH domains can typically be defined as comprising the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable domain, and residues 31 -35 or 31 -35b (HCDR1 ), 50-65 (HCDR2) and 95- 102 (HCDR3) in the heavy chain variable domain; (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Thus, the HVs may be comprised within the corresponding CDRs and references herein to the "hypervariable loops" of VH and VL domains should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise indicated.

[0077] The more highly conserved portions of variable domains are called the framework region (FR), as defined below. The variable domains of native heavy and light chains each comprise four FRs (FR1 , FR2, FR3 and FR4, respectively), largely adopting a p-sheet configuration, connected by the three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies. Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high sequence variability, five of the six loops adopt just a small repertoire of main-chain conformations, called “canonical structures”. These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the ability to assume unusual main-chain conformations.

[0078] “CDR” - As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen binding sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat etal., Sequences of protein of immunological interest. (1991 ), and by Chothia et al., J. Mol. Biol. 196:901 -917 (1987) and by MacCallum etal., J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison. Preferably, the term “CDR” is a CDR as defined by Kabat based on sequence comparisons.

[0079] Table 1 : CDR definitions

[0080] 1Residue numbering follows the nomenclature of Kabat et al., supra2Residue numbering follows the nomenclature of Chothia et al., supra3Residue numbering follows the nomenclature of MacCallum etal., supra

[0081] “Framework region” - The term “framework region” or “FR region” as used herein, includes the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g., using the Kabat definition of CDRs). Therefore, a variable region framework is between about 100-120 amino acids in length but includes only those amino acids outside of the CDRs. For the specific example of a heavy chain variable domain and for the CDRs as defined by Kabat etal., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1-30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acids 103 to the end of the variable region. The framework regions for the light chain are similarly separated by each of the light chain variable region CDRs. Similarly, using the definition of CDRs by Chothia et al. or McCallum etal. the framework region boundaries are separated by the respective CDR termini as described above. In preferred embodiments the CDRs are as defined by Kabat. In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding site (i.e antigen binding domain) as the antibody assumes its three- dimensional configuration in an aqueous environment. The remainder of the heavy and light variable domains show less inter-molecular variability in amino acid sequence and are termed the framework regions. The framework regions largely adopt a p-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the p-sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding site (i.e. antigen binding domain) formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The position of CDRs can be readily identified by one of ordinary skill in the art.

[0082] “Constant region” - As used herein, the term “constant region” refers to the portion of an antibody molecule or monovalent antigen-binding molecule outside of the variable domains or variable regions. Immunoglobulin light chains have a single domain “constant region”, typically referred to as the “CL” or “CL1 domain”. This domain lies C terminal to the VL domain. Immunoglobulin heavy chains differ in their constant region depending on the class of immunoglobulin (y, | , a, 8, e). Heavy chains y, a and 8 have a constant region consisting of three immunoglobulin domains (referred to as CH1 , CH2 and CH3) with a flexible hinge region separating the CH1 and CH2 domains. Heavy chains p and £ have a constant region consisting of four domains (CH1-CH4). The constant domains of the heavy chain are positioned C terminal to the VH domain.

[0083] The numbering of the amino acids in the heavy and light immunoglobulin chains run from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Different numbering schemes are used to define the constant domains of the immunoglobulin heavy and light chains. In accordance with the Ell numbering scheme, the heavy chain constant domains of an IgG molecule are identified as follows: CH1 - amino acid residues 118-215; CH2 - amino acid residues 231-340; CH3 - amino acid residues 341-446. In accordance with the Kabat numbering scheme, the heavy chain constant domains of an IgG molecule are identified as follows: CH1 - amino acid residues 114-223; CH2 - amino acid residues 244-360; CH3 - amino acid residues 361 -477.

[0084] “Fc domain” - As used herein, the “Fc domain” defines the portion of the constant region of an immunoglobulin heavy chain including the CH2 and CH3 domains. It typically defines 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. The Fc domain typically includes some residues from the hinge region. Accordingly, a complete Fc domain typically comprises at least a portion of a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, and a CH3 domain.

[0085] The “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux K.H. et a / . J. Immunol. 161 :4083-90 1998). Antigen-binding molecules of the invention comprising a “fully human” hinge region may contain one of the hinge region sequences shown in Table 2 below.

[0086] Table 2: Human hinge sequences

[0087] “Variant Fc domain” - As used herein, the term "variant Fc domain" refers to an Fc domain with one or more alterations relative to a wild-type Fc domain, for example an Fc domain with one or more alterations relative to the Fc domain of a naturally-occurring or “wild-type” human IgG. Alterations can include amino acid substitutions, additions and / or deletions, linkage of additional moieties, and / or alteration of the native glycans.

[0088] “Fc region” - As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains of the two heavy chains. A native or wild-type Fc region is typically homodimeric.

[0089] “Variant Fc region” - As used herein the term “variant Fc region” refers to an Fc region comprising a first Fc domain and a second Fc domain wherein at least one of the Fc domains has one or more alterations relative to the wild-type domains of a wild-type Fc region. For example, a variant Fc region as described herein may have one or more alterations relative to the Fc region of a naturally-occurring human IgG, particularly human IgG 1 . The term “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 is different. For heterodimeric Fc regions, one or both of the Fc domains may be variant Fc domains.

[0090] “FcRn binding fragment” - As used herein the term “FcRn binding fragment” refers to a portion of an Fc domain or Fc region that is sufficient to confer FcRn receptor binding.

[0091] “Specificity”- The monovalent antigen-binding molecules and modified antibodies described herein bind to a particular target antigen - IgA. It is preferred that the monovalent antigen-binding molecules and modified antibodies “specifically bind” to their target antigen, wherein the term “specifically bind” refers to the ability of any monovalent antigen-binding molecule or antibody to preferentially immunoreact with a given target i.e. IgA. The monovalent antigen-binding molecules of the present invention are monospecific and contain one binding site (i.e. antigen binding domain), which specifically binds a particular target antigen (i.e. IgA).

[0092] “Modified antibody” - As used herein, the term “modified antibody” includes synthetic forms of antibodies which are altered such that they are not naturally occurring. Examples include but are not limited to antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); heavy chain molecules joined to scFv molecules and the like. scFv molecules are known in the art and are described, e.g., in US patent 5,892,019.

[0093] The term “modified antibody”, particularly as used in the context of the present invention, refers to antibodies that are monovalent, particularly one-armed modified antibodies having a single antigen-binding arm.

[0094] Modified antibodies in accordance with the present invention may comprise any suitable antigen-binding domain as defined elsewhere herein linked to a variant Fc domain or FcRn binding fragment thereof as defined elsewhere herein.

[0095] “Humanising substitutions” - As used herein, the term “humanising substitutions” refers to amino acid substitutions in which the amino acid residue present at a particular position in the VH or VL domain of an antibody or monovalent antigen-binding molecule is replaced with an amino acid residue which occurs at an equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be a VH or VL domain encoded by the human germline. Humanising substitutions may be made in the framework regions and / or the CDRs of the antibodies, defined herein.

[0096] “Humanised variants” - As used herein the term “humanised variant” or “humanised antibody” refers to a variant antibody or monovalent antigen-binding molecule which contains one or more “humanising substitutions” compared to a reference antibody sequence, wherein a portion of the reference antibody (e.g. the VH domain and / or the VL domain or parts thereof containing at least one CDR) has an amino acid derived from a non-human species, and the “humanising substitutions” occur within the amino acid sequence derived from a non-human species.

[0097] “Germlined variants” - The term “germlined variant” or “germlined antibody” is used herein to refer specifically to “humanised variants” in which the “humanising substitutions” result in replacement of one or more amino acid residues present at (a) particular position(s) in the VH or VL domain of an antibody with an amino acid residue which occurs at an equivalent position in a reference human VH or VL domain encoded by the human germline. It is typical that for any given “germlined variant”, the replacement amino acid residues substituted into the germlined variant are taken exclusively, or predominantly, from a single human germline-encoded VH or VL domain. The terms “humanised variant” and “germlined variant” are often used interchangeably. Introduction of one or more “humanising substitutions” into a camelid-derived (e.g. llama derived) VH or VL domain results in production of a “humanised variant” of the camelid (llama)-derived VH or VL domain. If the amino acid residues substituted in are derived predominantly or exclusively from a single human germline-encoded VH or VL domain sequence, then the result may be a “human germlined variant” of the camelid (llama)-derived VH or VL domain.

[0098] “Affinity variants” - As used herein, the term “affinity variant” refers to a variant monovalent antigen-binding molecule or variant antibody which exhibits one or more changes in amino acid sequence compared to a reference monovalent antigen-binding molecule / antibody, wherein the affinity variant exhibits an altered affinity for the target antigen in comparison to the reference. For example, affinity variants will exhibit a changed affinity for a target, for example IgA, as compared to a reference monovalent IgA- binding molecule or reference IgA antibody. Preferably, the affinity variant will exhibit improved affinity for the target antigen, as compared to the reference. Affinity variants typically exhibit one or more changes in amino acid sequence in the CDRs, as compared to the reference monovalent antigen binding molecule / antibody. Such substitutions may result in replacement of the original amino acid present at a given position in the CDRs with a different amino acid residue, which may be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue. The amino acid substitutions may be conservative or non-conservative.

[0099] “Engineered” - As used herein the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques). Preferably, the monovalent antigen-binding molecules of the invention are engineered, including for example, humanized variants which have been engineered to improve one or more properties, such as antigen binding, stability / half-life or effector function.

[0100] “FcRn” - As used herein, the term "FcRn" refers to a neonatal Fc receptor. Exemplary FcRn molecules include human FcRn encoded by the FCAR gene as set forth in RefSeq NM 002000. “CD89” - As used herein, the term “CD89” refers to a FcaRI Fc receptor that binds to the constant region of IgA. The receptor exhibits a relatively low affinity of ~ 106M-1for monomeric and dimeric forms of IgA. However, it is able to bind IgA immune complexes with high avidity that results in cross-linking. Cross-linking of FcaRI by IgA immune complexes (or IgA-opsonized pathogens) induces a variety of processes, including phagocytosis, antibody-dependent cellular cytotoxicity, superoxide generation, release of inflammatory mediators, and cytokines as well as antigen presentation. Exemplary CD89 molecules include human CD89 as set forth in RefSeq: NM 000569, NM_133269, NM_133271 , NM_133272 and NM_133273.

[0101] “N-linked glycan” - As used herein the term “N-linked glycan” refers to the N-linked glycan attached to the nitrogen (N) in the side chain of asparagine in the sequence (i.e., Asn-X- Ser or Asn-X-Thr sequence, where X is any amino acid except proline) present in the CH2 domain of an Fc region. Such N-glycans are fully described in, for example, Drickamer K and Taylor ME (2006) Introduction to Glycobiology, 2nd ed., incorporated herein by reference in its entirety.

[0102] “Afucosylated” - As used herein the term “afucosylated” refers to an N-linked glycan which lacks a core fucose molecule as described in US Pat No. 8067232, incorporated herein by reference in its entirety.

[0103] “Bisecting GIcNAc” - As used herein the term “bisecting GIcNAc” refers to an N-linked glycan having an N-acetylglucosamine (GIcNAc) molecule linked to a core mannose molecule, as described in US Pat. No. 8021856, incorporated herein by reference in its entirety.

[0104] “IgA” - As used herein, the term “IgA” refers to “immunoglobulin A” molecules or “class A immunoglobulins”. IgA is the most abundant immunoglobin class at mucosal surfaces and the second most prevalent class in human serum. At mucosal surfaces, dimeric forms of IgA predominate (such as secreted IgA (slgA)) whereas in human serum the monomeric form is the most prevalent form of IgA. There are two known isotypes of IgA - Ig A1 and lgA2. The two isotypes are distinguished from one another by the size of their hinge regions and the number of glycosylation sites. lgA1 contains a 13 amino acid hinge region with many O-linked glycosylation sites whereas lgA2 does not contain this region and also has two additional N-linked carbohydrate chains. The distribution of these isotypes differs in the mucosal areas and serum. Serum IgA is mostly comprised of lgA1 (around 90%). In contrast, mucosal IgA consist of both isotypes and the ratio of the isotypes differs according to the specific location of the mucosal area (Cerutti 2008; Breedveld and van Egmond 2019; de Sousa-Pereira and Woof 2019). Both lgA1 and lgA2 can also be present as membrane-bound forms, such as for example on B cells. As explained in greater detail elsewhere, serum IgA, IgA autoantibodies and IgA-immune complexes have been implicated in various autoimmune disorders.

[0105] “Antibody-mediated disorder” - As used herein, the term “antibody-mediated disorder” refers to any disease or disorder caused or exacerbated by the presence of an antibody in a subject. An “IgA-mediated disorder” refers to a disease or disorder caused or exacerbated by the presence of IgA antibodies, including for example IgA-immune complexes.

[0106] “Treat, treating and treatment” - As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, for example, a subject having an antibody- mediated disease or disorder (e.g. autoimmune disease) or predisposed to having such a disease or disorder, an antigen-binding molecule in accordance with the present invention, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0107] “Subject” - As used herein, the term “subject” refers to any human or non-human animal. In certain embodiments, the term “subject” refers to any human or non-human mammal. In preferred embodiments, the subject is a human. In certain embodiments the subject is an adult human. As used herein, an “adult human” is a human who is at least 18 years of age.

[0108] B. Monovalent antiqen-bindinq molecules

[0109] In a first aspect, the present invention provides a monovalent antigen-binding molecule comprising: - an antigen-binding domain that binds to IgA; and

[0110] - a variant Fc region or a FcRn binding fragment thereof, that binds to human FcRn with increased affinity relative to a wild-type Fc region; and wherein the variant Fc region comprises a first Fc domain and a second Fc domain.

[0111] The monovalent antigen-binding molecules of the invention are characterized in that they possess a single antigen valency. More specifically, the monovalent antigen-binding molecules have a single antigen-binding domain wherein the antigen-binding domain binds to IgA. The monovalent antigen-binding molecule described herein are also referred to as monovalent IgA-binding molecules.

[0112] Embodiments pertaining to structural and functional features of the monovalent antigenbinding molecules of the invention are set forth below.

[0113] (i) pH-dependent antipen bindinp

[0114] The monovalent antigen-binding molecules described herein may exhibit pH-dependent antigen binding i.e., pH-dependent binding to IgA. As used herein, “pH-dependent binding” means that the antigen-binding molecules exhibit lower IgA binding affinity at an acidic pH than at a neutral pH. pH-dependent IgA binding is advantageous since it results in enhanced IgA sweeping from serum.

[0115] In more detail, once a monovalent antigen-binding molecule is bound to IgA and internalised by FcRn, the complex enters the endosomal compartment. Whilst serum and cellular pH is typically neutral, the pH of the endosomal compartments is slightly acidic. Monovalent antigen-binding molecules that are able to dissociate from IgA in the early endosome (i.e. at an acidic pH) can be recycled back to the cell surface. In contrast, monovalent antigen-binding molecules that bind with high affinity to IgA in the endosomal compartments would typically be trafficked with IgA to the lysosomes for degradation. pH-dependent monovalent antigen-binding molecules in accordance with the present invention have the potential to eliminate serum IgA (including IgA immune complexes) by binding and internalising IgA. Once internalised, the lower IgA binding affinity in the acidic endosomal compartment will facilitate release of the IgA by the monovalent antigen-binding molecules such that the IgA is trafficked to the lysosomes for degradation. The free monovalent antigen-binding molecules can be recycled to the cell surface such that they can mediate binding, internalisation and degradation of further IgA molecules. In this way, whilst a monovalent antigen-binding molecule of the invention is capable of binding only a single IgA molecule at any given time, the molecule is capable of binding a plurality of IgA molecules at different points in time via the recycling method described above.

[0116] The pH-dependent IgA binding activity may be an intrinsic property of the monovalent antigen-binding molecules of the invention i.e., the molecules may have been selected for this property. Alternatively or in addition, the monovalent antigen-binding molecules described herein may be engineered so as to exhibit pH-dependent IgA binding. Methods of engineering pH-dependent antigen binding activity in antibody molecules are described in, for example, EP2275443, which is incorporated herein by reference. Methods of engineering pH-dependent antigen binding in antibody molecules are also described in WO2018 / 206748, which is incorporated herein by reference. Such methods described therein apply also to the monovalent antigen-binding molecules of the invention. The monovalent antigen-binding molecules described herein may be modified by any technique so as to achieve pH-dependent binding. For example, the monovalent antigen-binding molecules may be modified in accordance with the methods described in EP2275443 or WO2018 / 206748 such that they exhibit pH-dependent IgA binding.

[0117] For pH-dependent embodiments of the monovalent antigen-binding molecules described herein, the IgA binding activity is lower at endosomal pH as compared to the IgA binding activity at serum pH. The endosomal pH is typically acidic pH whereas the serum pH is typically neutral pH. Accordingly, the monovalent antigen-binding molecules may exhibit pH-dependent IgA binding such that their IgA binding activity is lower at acidic pH as compared to the IgA binding activity at neutral pH. Endosomal pH or “acidic pH” may be pH of from about pH 4.0 to about pH 6.5, preferably from about pH 5.5 to about pH 6.5, preferably from about pH 5.5 to about pH 6.0, preferably pH 5.5, pH 5.6, pH 5.7 or pH 5.8. Serum pH or “neutral pH” may be pH of from about pH 6.9 to about pH 8.0, preferably from about pH 7.0 to about pH 8.0, preferably from about pH 7.0 to about pH 7.4, preferably pH 7.0 or pH 7.4.

[0118] In certain embodiments, the monovalent antigen-binding molecules exhibit pH-dependent binding such that the IgA-binding affinity at pH 5.5 is lower as compared with the IgA- binding affinity at pH 7.4. The pH-dependent monovalent antigen-binding molecules may be characterised in that the dissociation constant (KD) for the monovalent antigen-binding molecule-lgA interaction at acidic pH or pH 5.5 is higher than the dissociation constant (KD) for the monovalent IgA binding molecule-lgA interaction at neutral pH or pH 7.4. In certain embodiments, the monovalent antigen-binding molecules exhibit pH-dependent binding such that the ratio of KD for IgA at pH 5.5 and KD for IgA at pH 7.4 (KD(pH5.5) / KD(pH7.4)) is at least 1 .5, at least 2, at least 4, at least 6, at least 8, at least 10 or at least 12.

[0119] In certain embodiments, the monovalent antigen-binding molecules exhibit pH-dependent binding such that the human IgA-binding affinity at pH 6 is lower as compared with the human IgA binding affinity at pH 7.4. In certain embodiments, the monovalent antigenbinding molecules exhibit pH-dependent binding such that the human IgA-binding affinity at pH 6 is reduced by at least 25% as compared with the human IgA binding affinity at pH 7.4. In preferred embodiments, the monovalent antigen-binding molecules exhibit pH- dependent binding such that the human IgA-binding affinity at pH 6 is reduced by at least 50% as compared with the human IgA binding affinity at pH 7.4.

[0120] In other embodiments, the monovalent antigen-binding molecules exhibit pH-dependent binding such that the human IgA-binding affinity at pH 5 is lower as compared with the human IgA binding affinity at pH 7.4. In certain embodiments, the monovalent antigenbinding molecules exhibit pH-dependent binding such that the human IgA-binding affinity at pH 5 is reduced by at least 75% as compared with the human IgA binding affinity at pH 7.4. In preferred embodiments, the monovalent antigen-binding molecules exhibit pH- dependent binding such that the human IgA-binding affinity at pH 5 is reduced by at least 100% as compared with the human IgA binding affinity at pH 7.4.

[0121] In some embodiments, the monovalent antigen-binding molecules are capable of binding to IgA at a neutral pH and are not capable of binding to IgA at an acidic pH. In said embodiments, acidic pH is about pH 5.0, about pH 5.5 or about pH 6.0. In said embodiments, neutral pH is about pH 7.4.

[0122] The pH-dependent IgA-binding activity of a monovalent antigen-binding molecule may be engineered by modifying the molecule so as to impair IgA binding affinity at acidic pH and / or to increase IgA binding affinity at neutral pH. The monovalent antigen-binding molecule may be modified by substituting at least one amino acid of the antigen-binding domain with histidine, or by inserting at least one histidine into the antigen-binding domain. Such histidine mutation (substitution or insertion) sites are not particularly limited, and any site is acceptable as long as the IgA binding activity at endosomal pH (for example pH 5.5) is lower than that at serum pH (for example pH 7.4) as compared to before the mutation or insertion.

[0123] The antigen-binding domain of the monovalent antigen-binding molecules described herein will typically comprise a variable region or domain as defined elsewhere herein. In certain embodiments, the monovalent antigen-binding molecules may be engineered so as to exhibit pH-dependent IgA binding by the introduction of one or more substitutions into a variable region or domain of the antigen-binding domain. In preferred embodiments, the monovalent antigen-binding molecules are engineered so as to exhibit pH-dependent IgA binding by introducing one or more substitutions into one or more CDRs of a variable region or domain of the antigen-binding domain. The substitutions may introduce one or more His residues into one or more sites of a variable region or domain, preferably the heavy chain and / or light chain CDRs so as to confer pH-dependent IgA binding.

[0124] For embodiments of the invention wherein the antigen-binding domain comprises a variable region comprising three heavy chain CDR sequences and three light chain CDR sequences, the six CDRs combined may consist of a total of 1 -10 His substitutions, optionally 1 -5 His substitutions, optionally 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 His substitutions. The monovalent antigen-binding molecules may be engineered in accordance with the methods described in WO2018 / 206748, incorporated herein by reference. Non-histidine substitutions may also be incorporated into variable domains, particularly the CDRs, of the pH-dependent monovalent antigen-binding molecules described herein.

[0125] In preferred embodiments, the exemplary monovalent antigen-binding molecules having the particular CDR, VH and / or VL domain sequences recited elsewhere herein (see section vi below) are engineered such that they exhibit pH-dependent IgA binding. For example, the CDR sequences of the exemplary monovalent antigen-binding molecules described herein may be modified by the introduction of one or more histidine substitutions so as to produce monovalent antigen-binding molecules exhibiting pH-dependent IgA binding and / or enhanced pH-dependent IgA binding. Calcium-dependent antigen binding can be used as an alternative to pH-dependent antigen binding to facilitate antigen sweeping. Calcium ion concentration is known to be lower in endosomes as compared to the serum. Therefore, a molecule that exhibits calciumdependent IgA binding can be used to achieve enhanced IgA sweeping in an analogous manner to a molecule with pH-dependent IgA binding. As such, in certain embodiments the monovalent antigen-binding molecules described herein exhibit calcium-dependent IgA binding. In further embodiments, the monovalent antigen-binding molecules described herein exhibit lower IgA binding affinity at lower calcium ion concentrations as compared to IgA binding affinity at higher calcium ion concentrations.

[0126] (II) Monovalent IcjA-bindincj molecules

[0127] The monovalent antigen-binding molecules of the invention are also referred to herein as “monovalent IgA binding molecules” and “monovalent IgA-binding molecules” since the single antigen they bind is IgA. As described elsewhere herein, monovalent IgA-binding molecules of the invention are advantageous in that they exhibit improved IgA sweeping as compared with the corresponding bivalent IgA-binding molecules.

[0128] The monovalent IgA-binding molecules of the present invention may adopt the format of any suitable antigen-binding molecule displaying immunoreactivity for IgA, provided that they comprise an antigen-binding domain that binds to IgA and a variant Fc region or FcRn binding fragment thereof as defined herein.

[0129] The antigen-binding domain may be any domain that exhibits binding specificity for IgA. In certain embodiments, said antigen-binding domain comprises or consists of a Fab, a VH- VL domain pairing, a scFv fragment, a VHH domain, a disulf ide-linked Fv (sdFv), a singlechain Fv (scFv), or antigen-binding fragments of the above. In certain embodiments, said antigen-binding domain comprises or consists of a Fab, an Fv, a scFv or a VHH domain. In certain embodiments, the antigen-binding domain is a Fab. The monovalent IgA-binding molecules as described herein preferably comprise a single Fab (i.e. one Fab arm).

[0130] The monovalent IgA-binding molecules of the present invention encompass modified forms of conventional heterotetrameric antibodies i.e. anti-lg A antibodies modified to be monovalent such that they are capable of binding to only one IgA antigen at any given time. Such modified anti-lg A antibodies are also referred to herein as monovalent anti- IgA antibodies or monovalent IgA antibodies.

[0131] Monovalent antigen-binding molecules, particularly monovalent anti-lg A antibodies, of the invention are intended for human therapeutic use. It follows that monovalent anti- IgA antibodies of the invention will typically be modified forms of human IgA, IgD, IgE, IgG or IgM antibodies, preferably IgG antibodies in which case they can belong to any of the four sub-classes IgG 1 , lgG2a and b, lgG3 or lgG4. In preferred embodiments, the monovalent anti-lg A antibodies of the invention are modified IgG antibodies, optionally of the IgG 1 subclass. In further preferred embodiments, the monovalent anti- IgA antibodies of the invention are modified IgG antibodies having only one Fab arm, optionally modified IgG 1 antibodies having only one Fab arm. The monovalent anti- IgA antibodies of the invention preferably derive from monoclonal antibodies since monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0132] The monovalent anti- IgA antibodies described herein may exhibit high human homology. Such monovalent anti-lgA antibodies may comprise VH and VL domains of native nonhuman antibodies which exhibit sufficiently high % sequence identity to human germline sequences. In certain embodiments, the monovalent anti-lgA antibodies are humanised or germlined variants of non-human antibodies.

[0133] The monovalent IgA-binding molecules described herein may bind to IgA so as to inhibit the binding of IgA to an IgA receptor. In some embodiments, the monovalent IgA binding molecules can bind to all naturally occurring forms and isotypes of IgA. In other embodiments, the IgA is selected from: monomeric IgA, dimeric IgA, and multimeric IgA. In some embodiments, the IgA is secretory IgA (slgA). In further embodiments, the IgA is membrane bound IgA. In other embodiments, the IgA is free IgA. In other embodiments, the IgA is lgA1. In another embodiment, the IgA is lgA2. In preferred embodiments, the IgA that is bound is human IgA. In most preferred embodiments, the IgA that is bound is human serum IgA.

[0134] In certain embodiments, the monovalent IgA-binding molecules of the invention prevent IgA binding to IgA receptors such as FcaRI (CD89) and / or CD71 (TfR1 ). The monovalent IgA binding molecules of the invention may prevent IgA binding to FcaRI (CD89). The monovalent IgA-binding molecules may target aggregated autoantibodies and / or autoantibodies complexed with self-antigens (i.e. immune complexes). Such autoantibodies typically bind to activating Fc receptors, causing numerous autoimmune diseases (which occur in part because of immunologically mediated inflammation against self-tissues) (see e.g., Clarkson et al., A / E / W314(9), 1236-1239 (2013);

[0135] US20040010124A1 ; US20040047862A1 ; and US2004 / 0265321 A1 , incorporated herein by reference in their entirety). Therefore, in certain embodiments, the monovalent IgA-binding molecules inhibit or reduce the formation of IgA aggregates and / or IgA immune complexes. An IgA immune complex includes by way of non-limiting example, IgA-IgG complexes, IgA- antigen complexes, IgA-pathogen complexes and IgA-FcaRI (CD89) complexes.

[0136] In certain embodiments, the monovalent IgA-binding molecules of the invention are also capable of displacing IgA that is already associated (i.e. bound) with IgA receptors, such as FcaRI (CD89) and / or CD71 (TfR1 ). In some embodiments, the monovalent IgA-binding molecules displace IgA from FcaRI (CD89) receptors. In some embodiments, the monovalent IgA-binding molecules prevent binding of IgA to FcaRI (CD89) receptor and also displace IgA bound to FcaRI (CD89) receptor.

[0137] (Hi) Variant Fc regions and FcRn binding fragments thereof

[0138] The present invention provides monovalent antigen-binding molecules comprising a variant Fc region or FcRn binding fragment thereof. This variant Fc region or FcRn binding fragment thereof is characterised by the ability to bind to the human neonatal Fc receptor, FcRn, with increased affinity relative to a wild-type Fc region. Put another way, the binding affinity between the variant Fc region or FcRn binding fragment thereof and human FcRn is higher as compared with the binding affinity between a wild-type Fc region and human FcRn.

[0139] The variant Fc region or FcRn binding fragment thereof binds to human FcRn with increased affinity relative to a wild-type Fc region. In certain embodiments, the wild-type Fc region against which the binding affinity of the variant Fc region is compared may be the wild-type Fc region from which the variant Fc region derives. As described above, a variant Fc region in the context of the present invention refers to an Fc region with one or more alterations relative to a wild-type Fc region, for example the Fc region of a naturally- occurring or “wild-type” human IgG. Alterations can include amino acid substitutions, additions and / or deletions, linkage of additional moieties, and / or alteration of the native glycans. If the naturally-occurring or wild-type Fc region from which the variant Fc domain derives is a human IgG 1 Fc region, the variant Fc region may bind to human FcRn with higher affinity than the wild-type human IgG 1 Fc region.

[0140] The increased affinity for human FcRn exhibited by the variant Fc region or FcRn binding fragment may be relative to a wild-type Fc region that is not necessarily the Fc region from which the variant Fc region or FcRn binding fragment derives. For example, the variant Fc region or FcRn binding fragment thereof may bind to human FcRn with increased affinity relative to a wild-type human IgG Fc region. The wild-type human IgG may be an IgG 1 , lgG2, lgG3 or lgG4.

[0141] In preferred embodiments, the variant Fc region or FcRn binding fragment thereof binds to human FcRn with increased affinity relative to a wild-type human lgG1 Fc region or a wildtype human lgG3 Fc region. In a preferred embodiment, the variant Fc region or FcRn binding fragment thereof binds to human FcRn with increased affinity relative to a wild-type human lgG1 Fc region.

[0142] The variant Fc regions or FcRn binding fragments described herein may bind to human FcRn with increased affinity at acidic pH, for example pH 6.0. Alternatively or in addition, the variant Fc regions or FcRn binding fragments described herein may bind to human FcRn with increased affinity at neutral pH, for example pH 7.4. In preferred embodiments, the variant Fc regions or FcRn binding fragments bind to human FcRn with increased affinity at both pH 6.0 and pH 7.4. In certain embodiments, the variant Fc regions and / or FcRn binding fragments bind to human FcRn with reduced pH-dependence as compared with a wild-type Fc region, particularly a wild-type human IgG 1 Fc region. For embodiments where the variant Fc region or FcRn binding fragment binds to human FcRn with reduced pH-dependence, it is still preferred that the binding affinity is increased at pH 6.0 and pH 7.4.

[0143] As explained herein, the binding affinity between the variant Fc regions or FcRn binding fragments described herein and human FcRn is increased such that the monovalent antigen-binding molecules of the present invention compete with endogenous IgGs for binding to human FcRn.

[0144] As reported in Vaccaro et al. (Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nature Biotechnology (2005) 23(10): 1283-1288), Ulrichts et al. (Neonatal Fc receptor antagonist efgartigimod safely and sustainably reduces IgGs in humans. J. Clinical Investigation. (2018) 128(10): 4372-4386), and also reported herein, a variant Fc region comprising Fc domains having ABDEG™ mutations (M252Y / S254T / T256E / H433K / N434F) can bind to human FcRn with increased affinity. Vaccaro et al. (incorporated herein by reference) reports a binding affinity for human FcRn at pH 6.0 for the variant ABDEG™ Fc region of KD15.5 nM as compared with a binding affinity of KD370 nM for wild-type human IgG 1 (as measured by surface plasmon resonance analysis). Thus, in certain embodiments, the variant Fc region or FcRn binding fragments described herein bind to human FcRn at pH 6.0 with an affinity that is increased by at least 20x as compared with a wild-type human IgG 1 Fc region. In certain embodiments, the variant Fc region or FcRn binding fragments described herein bind to human FcRn at pH 6.0 with an affinity that is increased by at least 25x, preferably at least 30x, as compared with a wild-type human lgG1 Fc region. The binding affinity of the variant Fc region or FcRn binding fragment may be compared with the binding affinity of the wild-type human IgG 1 Fc region when the affinity of the Fc region (or fragment) is tested in the context of a full-length IgG molecule.

[0145] As reported in Ulrichts et al., supra., the FcRn antagonist, efgartigimod, has equilibrium dissociation constants (KD) for human FcRn of 14.2 nM and 320 nM at pH 6.0 and pH 7.4, respectively. Thus, in certain embodiments, the variant Fc region or FcRn binding fragments described herein bind to human FcRn at pH 6.0 with a binding affinity stronger than KD 15 nM. Alternatively or in addition, the variant Fc region or FcRn binding fragments described herein may bind to human FcRn at pH 7.4 with a binding affinity stronger than KD 320 nM.

[0146] As noted elsewhere, the variant Fc region of the monovalent antigen-binding molecules of the invention comprises a first Fc domain and a second Fc domain. The first Fc domain and / or the second Fc domain may comprise one or more alterations relative to a wild-type Fc domain. Fc domains having one or more alterations relative to a wild-type Fc domain are referred to herein as variant Fc domains. It is these alterations to the first and / or second Fc domains of the variant Fc region that confer the increased affinity to human FcRn relative to a wild-type Fc region.

[0147] In certain embodiments, the variant Fc domains or FcRn binding fragments comprise at least one amino acid substitution relative to a wild-type Fc domain. The variant Fc domains or FcRn binding fragments may comprise, in certain embodiments, at least two, at least three, at least four or at least five amino acid substitutions relative to a wild-type Fc domain.

[0148] In certain embodiments, both the first and second Fc domains are variant Fc domains. In such embodiments, the first Fc domain comprises at least one amino acid substitution relative to a wild-type Fc domain and the second Fc domain comprises at least one amino acid substitution relative to a wild-type Fc domain. In some embodiments, the first variant Fc domain and the second variant Fc domain are identical.

[0149] The number of alterations in the variant Fc domains may be limited relative to the corresponding wild-type Fc domain. For example, the total number of amino acid substitutions in a variant Fc domain may be limited relative to the corresponding wild-type Fc domain. In certain embodiments, a variant Fc domain consists of no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 15, no more than 20 alterations as compared with the corresponding wild-type Fc domain. The alterations may be selected from amino acid substitutions, additions and / or deletions, linkage of additional moieties, and / or alteration of the native glycans. In certain embodiments, a variant Fc domain consists of no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 15, no more than 20 amino acid substitutions as compared with the corresponding wild-type Fc domain.

[0150] In certain embodiments, a variant Fc domain comprises or consists of at least one amino acid substitution but no more than 20 amino acid substitutions in total. In certain embodiments, a variant Fc domain comprises or consists of at least two amino acid substitutions but no more than 20 amino acid substitutions in total. In certain embodiments, a variant Fc domain comprises or consists of at least one amino acid substitution but no more than 10 amino acid substitutions in total. In certain embodiments, a variant Fc domain comprises or consists of at least two amino acid substitutions but no more than 10 amino acid substitutions in total. In certain embodiments, a variant Fc domain comprises or consists of at least one amino acid substitution but no more than 5 amino acid substitutions in total. In certain embodiments, a variant Fc domain comprises or consists of at least two amino acid substitutions but no more than 5 amino acid substitutions in total.

[0151] The wild-type Fc region from which the variant Fc region derives may be an IgG Fc region. In such embodiments, the variant Fc region is a variant IgG Fc region. In preferred embodiments, the variant Fc region is a variant IgG 1 Fc region i.e. the variant Fc region possesses one or more alterations relative to a wild-type IgG 1 region.

[0152] Since the monovalent antigen-binding molecules of the present invention may be for use in human patients, the variant Fc regions or FcRn binding fragments thereof will preferably be variant forms of human Fc regions i.e. the variant Fc regions or FcRn binding fragments thereof will be variant human Fc regions or FcRn binding fragments thereof. The variant Fc region may be a human variant IgG region, for example a human variant IgG region selected from IgG 1 , lgG2, lgG3 or lgG4. In particularly preferred embodiments, the variant Fc region is a variant IgG 1 Fc region or FcRn binding fragment thereof.

[0153] The variant Fc region or FcRn binding fragments thereof of the monovalent antigen-binding molecules of the invention may comprise any non-native amino acid residues, provided that the variant Fc region or FcRn binding fragment exhibits the requisite increased binding affinity for human FcRn. As used herein, the term “non-native amino acid” means an amino acid that does not occur naturally, i.e. in the wild-type sequence, at the position at which it is located in the variant Fc region or FcRn binding fragment thereof.

[0154] Antibodies having a variant Fc region and exhibiting increased binding affinity for human FcRn have been reported in the literature. These variant Fc regions have been reported as having various non-native amino acids at specific positions within the Fc region. The variant Fc regions and FcRn binding fragments of the monovalent antigen-binding molecules described herein may comprise any of the non-native amino acids and / or amino acid substitutions described in the literature as capable of increasing Fc region binding affinity for human FcRn. The variant Fc region and FcRn binding fragments of the monovalent antigen-binding molecules described herein may also comprise any combinations of non-native amino acids and / or amino acid substitutions described in the literature as capable of increasing Fc region binding affinity for FcRn. Non-limiting examples of amino acid substitutions that may be included in the variant Fc regions or FcRn binding fragments described herein are reported in Yeung et al. (Engineering Human IgG 1 Affinity to Human Neonatal Fc Receptor: Impact of Affinity Improvement on Pharmacokinetics in Primates. J. Immunol. (2009) 182: 7663-7671), and also International patent application no. WO2011 / 122011 , the entire contents of which are incorporated herein by reference.

[0155] In certain embodiments, the first Fc domain and / or second Fc domain of the variant Fc regions described herein comprise a combination of amino acids selected from the following:

[0156] (i) Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively; or

[0157] (ii) Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively; or

[0158] (iii) K and F at EU positions 433 and 434, respectively.

[0159] In further embodiments, the first Fc domain and / or second Fc domain of the variant Fc regions described herein comprise a combination of amino acids selected from the following:

[0160] (i) Y, P and Y at EU positions 252, 308 and 434, respectively;

[0161] (ii) Y, T and E at EU positions 252, 254 and 256, respectively;

[0162] (iii) Q and L at EU positions 250 and 428, respectively;

[0163] (iv) P and A at EU positions 308 and 434, respectively;

[0164] (v) P and Y at EU positions 308 and 434, respectively; or

[0165] (vi) Y, E and Y at EU positions 252, 286 and 434, respectively.

[0166] As indicated above, the positions are defined in accordance with EU numbering. EU numbering refers to the 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.

[0167] In certain embodiments, the first Fc domain and / or second Fc domain of the variant Fc regions described herein comprise at least one amino acid selected from the following: 237M; 238A; 239K; 248I; 250A; 250F; 250I; 250M; 250Q; 250S; 250V; 250W; 250Y; 252F; 252W; 252Y; 254T; 255E; 256D; 256E; 256Q; 257A; 257G; 257I; 257L; 257M; 257N; 257S; 257T; 257V; 258H; 265A; 270F; 286A; 286E; 289H; 297A; 298G; 303A; 305A; 307A; 307D; 307F; 307G; 307H; 307I; 307K; 307L; 307M; 307N; 307P; 307Q; 307R; 307S; 307V;

[0168] 307W; 307Y; 308A; 308F; 308I; 308L; 308M; 308P; 308Q; 308T; 309A; 309D; 309E; 309P; 309R; 311 A; 31 1 H; 31 11; 312A; 312H; 314K; 314R; 315A; 315H; 317A; 325G; 332V; 334L; 360H; 376A; 378V; 380A; 382A; 384A; 385D; 385H; 386P; 387E; 389A; 389S; 424A; 428A; 428D; 428F; 428G; 428H; 428I; 428K; 428L; 428N; 428P; 428Q; 428S; 428T; 428V; 428W; 428Y; 433K; 434A; 434F; 434H; 434S; 434W; 434Y; 436H; 436I and 436F, wherein the positions are defined in accordance with Ell numbering. EU numbering refers to the 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. The variant Fc domains or FcRn binding fragments described herein may comprise 2, 3, 4 or 5 amino acids selected from the following: 237M; 238A; 239K; 248I; 250A; 250F; 250I; 250M; 250Q; 250S; 250V; 250W; 250Y; 252F; 252W; 252Y; 254T; 255E; 256D; 256E; 256Q; 257A; 257G; 257I; 257L; 257M; 257N; 257S; 257T; 257V; 258H; 265A; 270F; 286A; 286E; 289H; 297A; 298G; 303A; 305A; 307A; 307D; 307F; 307G; 307H; 307I; 307K; 307L; 307M; 307N; 307P; 307Q; 307R; 307S; 307V; 307W; 307Y; 308A; 308F; 308I; 308L; 308M; 308P; 308Q; 308T; 309A; 309D; 309E; 309P; 309R; 31 1 A; 311 H; 3111; 312A; 312H; 314K; 314R; 315A; 315H; 317A; 325G; 332V; 334L; 360H; 376A; 378V; 380A; 382A; 384A; 385D; 385H; 386P; 387E; 389A; 389S; 424A; 428A; 428D; 428F; 428G; 428H; 428I; 428K; 428L; 428N; 428P; 428Q; 428S; 428T; 428V; 428W; 428Y; 433K; 434A; 434F; 434H; 434S; 434W; 434Y; 436H; 436I and 436F, wherein the positions are defined in accordance with EU numbering and wherein any combinations are contemplated.

[0169] In preferred embodiments, the variant Fc region comprises a combination of amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively. It has been reported in the literature that said mutations to the wild-type IgG 1 backbone improve affinity for FcRn at both neutral and acidic pHs. In a further preferred embodiment, the variant Fc region comprises a combination of amino acids Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively. In certain embodiments, the first Fc domain and / or second Fc domain of the variant Fc region comprise the amino acids Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively. In further preferred embodiments, the variant Fc region comprises a combination of amino acids Y, P and Y at EU positions 252, 308 and 434, respectively. In other preferred embodiments, the variant Fc region comprises a combination of amino acids Y, T and E at EU positions 252, 254 and 256, respectively.

[0170] In an alternative embodiment, the first Fc domain and / or second Fc domain of the variant Fc region comprise a combination of amino acids K and F at EU positions 433 and 434, respectively. It has been reported in the literature that said mutations improve affinity for FcRn at acidic (endosomal) pHs. The enhanced affinity at endosomal pH, results in a prolonged half-life due to FcRn-mediated recycling.

[0171] In certain embodiments, the first Fc domain and / or second Fc domain comprise a combination of amino acid substitutions: M252Y, S254T, T256E, H433K and N434F, wherein the positions are defined in accordance with EU numbering. In certain embodiments, the first Fc domain or second Fc domain comprise a combination of amino acid substitutions: M252Y, S254T, T256E, H433K, N434F and a Y at position 436, wherein the positions are defined in accordance with EU numbering. In a preferred embodiment, both the first and second Fc domain each comprise a combination of amino acid substitutions: M252Y, S254T, T256E, H433K, N434F and a Y at position 436, wherein the positions are defined in accordance with EU numbering.

[0172] In further embodiments, the first Fc domain and / or second Fc domain of the variant Fc regions described herein comprise a combination of amino acid substitutions selected from the following:

[0173] (i) M252Y, V308P and N434Y;

[0174] (ii) M252Y, S254T and T256E;

[0175] (iii) T250Q and M428L;

[0176] (iv) V308P and N434A;

[0177] (v) V308P and N434Y; or

[0178] (vi) M252Y, N286E and N434Y, wherein the positions are defined in accordance with EU numbering.

[0179] In certain embodiments, the first Fc domain and / or second Fc domain of the variant Fc regions described herein comprise at least one amino acid substitution selected from: G237M; P238A; S239K; K248I; T250A; T250F; T250I; T250M; T250Q; T250S; T250V; T250W; T250Y; M252F; M252W; M252Y; S254T; R255E; T256D; T256E; T256Q; P257A; P257G; P257I; P257L; P257M; P257N; P257S; P257T; P257V; E258H; D265A; D270F; N286A; N286E; T289H; N297A; S298G; V303A; V305A; T307A; T307D; T307F; T307G; T307H; T307I; T307K; T307L; T307M; T307N; T307P; T307Q; T307R; T307S; T307V; T307W; T307Y; V308A; V308F; V308I; V308L; V308M; V308P; V308Q; V308T; V309A; V309D; V309E; V309P; V309R; Q311A; Q311 H; Q311 I; D312A; D312H; L314K; L314R;

[0180] N315A; N315H; K317A; N325G; I332V; K334L; K360H; D376A; A378V; E380A; E382A;

[0181] N384A; G385D; G385H; Q386P; P387E; N389A; N389S; S424A; M428A; M428D; M428F;

[0182] M428G; M428H; M428I; M428K; M428L; M428N; M428P; M428Q; M428S; M428T;

[0183] M428V; M428W; M428Y; H433K; N434A; N434F; N434H; N434S; N434W; N434Y; Y436H; Y436I and Y436F, wherein the positions are defined in accordance with Ell numbering.

[0184] The variant Fc domains or FcRn binding fragments described herein may comprise 2, 3, 4 or 5 amino acid substitutions selected from the following: G237M; P238A; S239K; K248I; T250A; T250F; T250I; T250M; T250Q; T250S; T250V; T250W; T250Y; M252F; M252W; M252Y; S254T; R255E; T256D; T256E; T256Q; P257A; P257G; P257I; P257L; P257M; P257N; P257S; P257T; P257V; E258H; D265A; D270F; N286A; N286E; T289H; N297A; S298G; V303A; V305A; T307A; T307D; T307F; T307G; T307H; T307I; T307K; T307L;

[0185] T307M; T307N; T307P; T307Q; T307R; T307S; T307V; T307W; T307Y; V308A; V308F; V308I; V308L; V308M; V308P; V308Q; V308T; V309A; V309D; V309E; V309P; V309R; Q311A; Q311 H; Q311 I; D312A; D312H; L314K; L314R; N315A; N315H; K317A; N325G; I332V; K334L; K360H; D376A; A378V; E380A; E382A; N384A; G385D; G385H; Q386P; P387E; N389A; N389S; S424A; M428A; M428D; M428F; M428G; M428H; M428I; M428K; M428L; M428N; M428P; M428Q; M428S; M428T; M428V; M428W; M428Y; H433K;

[0186] N434A; N434F; N434H; N434S; N434W; N434Y; Y436H; Y436I and Y436F, wherein the positions are defined in accordance with EU numbering, and wherein any combinations of substitutions are contemplated.

[0187] In further embodiments, the first Fc domain or second Fc domain comprise a combination of amino acid substitutions: H433K and N434F, wherein the positions are defined in accordance with EU numbering. In further embodiments, both the first and second Fc domain each comprise a combination of amino acid substitutions: H433K and N434F, wherein the positions are defined in accordance with EU numbering. The variant Fc regions of the monovalent antigen-binding molecules of the present invention may comprise further alterations, particularly amino acid substitutions in addition to the alterations that increase binding affinity for FcRn. These alterations, particularly amino acid substitutions, may further improve the properties of the monovalent antigenbinding molecules.

[0188] In particular, the variant Fc region may comprise at least one additional amino acid substitution that reduces or eliminates Fc effector function of the monovalent antigenbinding molecules. In some embodiments, the first Fc domain or second Fc domain comprises the amino acids A and A at EU positions 234 and 235, respectively. In further embodiments, the first Fc domain or second Fc domain comprises amino acid substitutions L234A and L235A, wherein the positions are defined in accordance with EU numbering. In some embodiments, both the first and second Fc domains each comprise the amino acids A and A at EU positions 234 and 235, respectively. In some embodiments, both the first and second Fc domains each comprise a combination of amino acid substitutions: L234A and L235A, wherein the positions are defined in accordance with EU numbering. The so- called “LALA” mutations are known to reduce Fc binding to Fey receptors.

[0189] In some embodiments, the first Fc domain or second Fc domain comprises the amino acids A, A and G at EU positions 234, 235 and 329, respectively. In further embodiments, the first Fc domain or second Fc domain comprises amino acid substitutions L234A, L235A and P329G, wherein the positions are defined in accordance with EU numbering. In some embodiments, both the first and second Fc domains each comprise the amino acids A, A and G at EU positions 234, 235 and 329, respectively. In some embodiments, both the first and second Fc domains each comprise a combination of amino acid substitutions: L234A, L235A and P329G, wherein the positions are defined in accordance with EU numbering. The so-called “LALAPG” mutations have been reported to completely block Fc binding to Fey receptors (Schlothauer et al., 2016).

[0190] The variant Fc regions of the monovalent antigen-binding molecules of the present invention may comprise further amino acid alterations, particularly amino acid substitutions, that increase the net surface charge and / or the isoelectric point of the molecules. In particular, it can be desirable to introduce amino acid substitutions to yield positive patches on the surface of the variant Fc region or FcRn binding fragment. Such modifications may enhance electrostatic attraction between the monovalent antigen-binding molecules that are described herein and negatively charged cell membranes. The resulting increase in electrostatic attractions between the monovalent antigen-binding molecules described herein and the cell membrane can also result in enhanced FcRn-mediated cellular uptake (described for example in WO2017217525, incorporated herein by reference). Therefore, in some embodiments, the variant Fc region comprises one or more amino acid substitutions that enhance electrostatic interactions of the monovalent antigen-binding molecules with a cell membrane.

[0191] Examples of amino acid substitutions that promote electrostatic interactions with a cell membrane are known in the literature. Any suitable means to promote electrostatic interactions may be adopted in the variant Fc region or FcRn binding fragments of the monovalent antigen-binding molecules of the present disclosure.

[0192] In some embodiments, the first Fc domain or second Fc domain comprises the amino acid K at Ell position 413. In further embodiments, the first Fc domain or second Fc domain comprises amino acid substitution D413K, wherein the position is defined in accordance with EU numbering. In some embodiments, both the first and second Fc domain each comprise the amino acid K at EU position 413. In further embodiments, both the first and second Fc domain each comprise amino acid substitution D413K, wherein the position is defined in accordance with EU numbering.

[0193] In some embodiments, the first Fc domain or second Fc domain comprises the amino acids R and K at EU positions 401 and 413, respectively. In further embodiments, the first Fc domain or second Fc domain comprises amino acid substitutions D401 R and D413K, wherein the positions are defined in accordance with EU numbering. In some embodiments, both the first and second Fc domain each comprise the amino acids R and K at EU positions 401 and 413, respectively. In further embodiments, both the first and second Fc domain each comprise amino acid substitutions D401 R and D413K, wherein the positions are defined in accordance with EU numbering.

[0194] In some embodiments, the first Fc domain or second Fc domain comprises the amino acids R, R and K at EU positions 311 , 400 and 413, respectively. In further embodiments, the first Fc domain or second Fc domain comprises amino acid substitutions Q311 R, S400R and D413K, wherein the positions are defined in accordance with Ell numbering. In some embodiments, both the first and second Fc domain each comprise the amino acids R, R and K at EU positions 311 , 400 and 413, respectively. In further embodiments, both the first and second Fc domain each comprise amino acid substitutions Q311 R, S400R and D413K, wherein the positions are defined in accordance with EU numbering.

[0195] In some embodiments, the first Fc domain or second Fc domain comprises the amino acids R and K at EU positions 311 and 413, respectively. In further embodiments, the first Fc domain or second Fc domain comprises amino acid substitutions Q311 R and D413K, wherein the positions are defined in accordance with EU numbering. In some embodiments, both the first and second Fc domain each comprise the amino acids R and K at EU positions 311 and 413, respectively. In further embodiments, both the first and second Fc domain each comprise amino acid substitutions Q311 R and D413K, wherein the positions are defined in accordance with EU numbering.

[0196] As noted above, the monovalent antigen-binding molecules of the invention comprise a single antigen binding domain. For embodiments in which the antigen binding domain is a Fab, the heavy chain of the Fab is covalently attached to only one Fc domain (or variant Fc domain) of the variant Fc region or FcRn binding fragment thereof. In other words, the molecule may be considered heterodimeric since one Fc domain (or variant Fc domain) is covalently attached to the heavy chain of a Fab and one Fc domain (or variant Fc domain) is not covalently attached to any Fab (nor any other antigen binding domain).

[0197] As such, in order to produce the monovalent antigen-binding molecules of the invention, heterodimerisation between a first Fc domain (or variant Fc domain) and a second Fc domain (or variant Fc domain) of the Fc region is required. In other words, heterodimerisation between a first Fc domain (or variant Fc domain) that is attached to the heavy chain of a Fab with a second Fc domain (or variant Fc domain) that is not attached to any antigen binding domain is required to arrive at the monovalent antigen-binding molecules of the invention. In view of this, in certain embodiments it is advantageous to promote heterodimerisation. Accordingly, the variant Fc region or FcRn binding fragments may comprise at least one alteration, particularly at least one amino acid substitution, that promotes dimerisation between a first Fc domain (or variant Fc domain) and a second Fc domain (or variant Fc domain). More specifically, the first and / or second Fc domains may further comprise alterations, for example one or more amino acid substitutions, to promote heterodimerisation.

[0198] Examples of amino acid substitutions that promote heterodimerisation are known in the literature and any suitable means to promote heterodimerisation may be adopted in the variant Fc region or FcRn binding fragments of the monovalent antigen-binding molecules of the present invention.

[0199] In some embodiments, the variant Fc region or FcRn binding fragments thereof comprise “knob into hole” substitutions, which are known in the literature (see for example WO 2006 / 028936 which is incorporated herein by reference). This technology promotes heterodimerisation by introducing different but complementary amino acid substitutions into the first Fc domain and the second Fc domain. In some embodiments, the first Fc domain comprises amino acid W at EU position 366 and the second Fc domain comprises amino acids S, A, and V at EU positions 366, 368, and 407, respectively. In other embodiments, the second Fc domain comprises amino acid W at EU position 366 and the first Fc domain, comprises amino acids S, A, and V at EU positions 366, 368, and 407, respectively.

[0200] In certain embodiments, the first Fc domain comprises an amino acid substitution T366W, and the second Fc domain comprises amino acid substitutions T366S, L368A, and Y407V, wherein the positions are defined in accordance with EU numbering.

[0201] In other embodiments, the second Fc domain comprises an amino acid substitution T366W, and the first Fc domain comprises amino acid substitutions T366S, L368A, and Y407V, wherein the positions are defined in accordance with EU numbering.

[0202] In some embodiments, the first Fc domain comprises the amino acids E and D at EU positions 370 and 409, respectively, and the second Fc domain comprises the amino acid K at EU positions 357 and 399. In some embodiments, the first Fc domain comprises the amino acids H and A at EU positions 364 and 405, respectively, and the second Fc domain comprises the amino acids T and F at EU positions 349 and 394, respectively. In some embodiments, the first Fc domain comprises amino acids V, Y, A, and V at EU positions 350, 351 , 405, and 407, respectively, and the second Fc domain comprises the amino acids V, L, L, and W at EU positions 350, 366, 392, and 394, respectively. In some embodiments, the first Fc domain comprises the amino acids D, M, and A at Ell positions 360, 399, and 407, respectively, and the second Fc domain comprises amino acids R, R, V, and V at EU positions 345, 347, 366, and 409, respectively.

[0203] In some embodiments, the first Fc domain comprises the amino acid D at EU positions 409 and 392, and the second Fc domain comprises amino acid K at EU positions 399 and 356. In some embodiments, the first Fc domain comprises the amino acids E, W, and C at EU positions 360, 409, and 349, respectively, and the second Fc domain comprises the amino acids R, V, T, and C at EU positions 347, 399, 405, and 354, respectively. In some embodiments, the first Fc domain comprises the amino acids E and W at EU positions 370 and 409, respectively, and the second Fc domain comprises the amino acids N, V, and T at EU positions 357, 399, and 405, respectively.

[0204] Taking the above into account, in preferred embodiments, the first Fc domain and the second Fc domain comprise different sets of amino acid substitutions. By way of nonlimiting example, the first Fc domain may comprise a “knob” mutation(s) and the second Fc domain may comprise a “hole” mutation(s) or vice versa. In a further preferred embodiment, the first and second Fc domains both comprise a set of the same amino acid substitutions such as ABDEG™ mutations (described in further detail below) as well as a set of different amino acid substitutions (such as knob-into-hole mutations).

[0205] In certain embodiments, the present invention provides monovalent antigen-binding molecules comprising variant Fc regions or FcRn binding fragments thereof incorporating ABDEG™ technology. As reported in Vaccaro etal. (Nat. Biotechnology (2005) 23(10):1283-8), ABDEG™ antibodies (meaning “antibodies that enhance IgG degradation”) comprise an engineered or variant Fc region. This engineered or variant Fc region can bind to the neonatal Fc receptor, FcRn, with higher affinity and reduced pH dependence as compared with the Fc region of wild-type antibodies.

[0206] ABDEG™ antibodies and FcRn antagonists incorporating ABDEG™ technology have been described for the treatment of antibody-mediated diseases such as autoimmune diseases (see W02006 / 130834 and WO2015 / 100299, incorporated herein by reference). The Fc domain amino acid “signature” of ABDEG™ is well-characterised. Therefore, in certain embodiments, the present invention provides monovalent antigen-binding molecules comprising a variant Fc region or FcRn binding fragment thereof, wherein the first Fc domain and / or second Fc domain comprises the amino acids Y, T, E, K and F at Ell positions 252, 254, 256, 433 and 434, respectively. Preferably, the first Fc domain and / or second Fc domain also comprises the amino acid Y at EU position 436. The amino acid Y is the native amino acid at EU position 436 in wild-type human IgG 1 Fc domain. This Fc domain amino acid signature is the ABDEG™ signature.

[0207] As described above, the variant Fc region of ABDEG™ monovalent antigen-binding molecules is engineered so as to increase the binding affinity for the Fc receptor FcRn, particularly human FcRn. The variant ABDEG™ Fc region or FcRn binding fragment thereof binds to FcRn with increased affinity relative to a wild-type Fc region. In such embodiments, the wild-type Fc region may be the wild-type Fc region from which the variant Fc region derives. For example, if the variant ABDEG™ Fc region is derived from a human IgG 1 Fc region, the variant Fc domain may bind to FcRn with higher affinity than the human lgG1 Fc domain.

[0208] In certain embodiments, the variant ABDEG™ Fc region or FcRn binding fragment thereof binds to FcRn, preferably human FcRn, with increased affinity relative to a wild-type IgG Fc region, preferably a wild-type human IgG Fc region. In a preferred embodiment, the variant ABDEG™ Fc region or FcRn binding fragment thereof binds to FcRn, preferably human FcRn, with increased affinity relative to a wild-type human IgG 1 Fc domain or a wild-type human lgG3 Fc domain.

[0209] The variant ABDEG™ Fc regions or FcRn binding fragments thereof of the monovalent antigen-binding molecules described herein may be variant Fc regions or FcRn binding fragments derived from any suitable wild-type immunoglobulin Fc region. In certain embodiments, the variant ABDEG™ Fc region or FcRn binding fragment thereof is a variant IgG Fc region or FcRn binding fragment thereof. The wild-type IgG region may be an IgG of any sub-class including IgG 1 , lgG2, lgG3 and lgG4, preferably IgG 1 . The wildtype IgG region is preferably human. In preferred embodiments, the variant ABDEG™ Fc region or FcRn binding fragment thereof is a variant IgG 1 Fc region or FcRn binding fragment thereof. In such embodiments, the variant ABDEG™ Fc region has the amino acid sequence of a wild-type IgG 1 region having first and / or second Fc domains comprising or consisting of the ABDEG™ amino acid signature described herein, specifically amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively. The wild-type IgG 1 region is preferably human.

[0210] In certain embodiments, the first and / or second Fc domains of the variant ABDEG™ Fc region or FcRn binding fragment thereof consist of no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 15, no more than 20 alterations as compared with the corresponding wild-type Fc domains. The alterations may be selected from amino acid substitutions, additions and / or deletions, linkage of additional moieties, and / or alteration of the native glycans. In certain embodiments, the first and / or second Fc domains of the variant ABDEG™ Fc region or FcRn binding fragment thereof consist of no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11 , no more than 12, no more than 15, no more than 20 amino acid substitutions as compared with the corresponding wild-type Fc domains.

[0211] In certain embodiments, the first and / or second Fc domains of the variant ABDEG™ Fc region or FcRn binding fragment thereof comprise or consist of at least five amino acid substitutions but no more than 20 amino acid substitutions in total. In certain embodiments, the first and / or second Fc domains of the variant ABDEG™ Fc region or FcRn binding fragment thereof comprise or consist of at least five amino acid substitutions but no more than 10 amino acid substitutions in total.

[0212] In certain embodiments, the first and / or second Fc domains of the variant Fc region or FcRn binding fragment is / are identical to the corresponding wild-type Fc domains but for the amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively. In certain embodiments, the first and / or second Fc domains of the variant Fc region or FcRn binding fragment is / are identical to the corresponding wild-type Fc domains but for the amino acids A, A, Y, T, E, K and F at EU positions 234, 235, 252, 254, 256, 433 and 434, respectively. In certain embodiments, the first and / or second Fc domains of the variant Fc region or FcRn binding fragment is / are identical to the corresponding wild-type Fc domains but for the amino acids A, A, G, Y, T, E, K and F at EU positions 234, 235, 329, 252, 254, 256, 433 and 434, respectively.

[0213] In further embodiments, the first Fc domain of the variant Fc region is identical to the corresponding wild-type Fc domain but for the amino acids A, A, G, Y, T, E, W, K and F at EU positions 234, 235, 329, 252, 254, 256, 366, 433 and 434, respectively; and the second Fc domain of the variant Fc region is identical to the corresponding wild-type Fc domain but for the amino acids A, A, G, Y, T, E, K, F, S, A and V at EU positions 234, 235, 329, 252, 254, 256, 433, 434, 366, 368 and 407, respectively. In preferred embodiments, the corresponding wild-type Fc domains are the Fc domains of the human IgG 1 Fc region.

[0214] Non-limiting examples of variant first and second Fc domains for inclusion in the monovalent antigen-binding molecules described herein are set forth in Table 3 below.

[0215] In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41 . In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 47. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 51 . In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 . In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 69. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 70. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71 . In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74. In certain embodiments, the first and / or second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75.

[0216] In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 52. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 68. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72. In certain embodiments, the first or second Fc domain is linked to a heavy chain CH1 domain and the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76.

[0217] In certain embodiments, the variant Fc region comprises:

[0218] - a first Fc domain that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 53, 54, 55, 61 , 62, 63, 69, 70 and 71 ; and

[0219] - a second Fc domain that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 57, 58, 59, 65, 66, 67, 73, 74 and 75.

[0220] In some embodiments, the variant Fc region comprises or consists of:

[0221] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53; and

[0222] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

[0223] In some embodiments, the variant Fc region comprises or consists of:

[0224] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53; and

[0225] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

[0226] In some embodiments, the variant Fc region comprises or consists of:

[0227] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53; and a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59.

[0228] In some embodiments, the variant Fc region comprises or consists of:

[0229] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54; and

[0230] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

[0231] In some embodiments, the variant Fc region comprises or consists of:

[0232] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54; and

[0233] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

[0234] In some embodiments, the variant Fc region comprises or consists of:

[0235] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54; and

[0236] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59.

[0237] In some embodiments, the variant Fc region comprises or consists of:

[0238] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55; and

[0239] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

[0240] In some embodiments, the variant Fc region comprises or consists of:

[0241] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55; and

[0242] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

[0243] In some embodiments, the variant Fc region comprises or consists of: - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55; and

[0244] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59.

[0245] In some embodiments, the variant Fc region comprises or consists of:

[0246] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 ; and

[0247] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

[0248] In some embodiments, the variant Fc region comprises or consists of:

[0249] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 ; and

[0250] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66.

[0251] In some embodiments, the variant Fc region comprises or consists of:

[0252] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 ; and

[0253] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67.

[0254] In some embodiments, the variant Fc region comprises or consists of:

[0255] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62; and

[0256] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66.

[0257] In some embodiments, the variant Fc region comprises or consists of:

[0258] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62; and

[0259] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the variant Fc region comprises or consists of:

[0260] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62; and

[0261] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67.

[0262] In some embodiments, the variant Fc region comprises or consists of:

[0263] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; and

[0264] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

[0265] In some embodiments, the variant Fc region comprises or consists of:

[0266] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; and

[0267] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66.

[0268] In some embodiments, the variant Fc region comprises or consists of:

[0269] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; and

[0270] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67.

[0271] In some embodiments, the variant Fc region comprises or consists of:

[0272] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 69; and

[0273] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73.

[0274] In some embodiments, the variant Fc region comprises or consists of:

[0275] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 69; and a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74.

[0276] In some embodiments, the variant Fc region comprises or consists of:

[0277] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 69; and

[0278] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75.

[0279] In some embodiments, the variant Fc region comprises or consists of:

[0280] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 70; and

[0281] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73.

[0282] In some embodiments, the variant Fc region comprises or consists of:

[0283] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 70; and

[0284] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74.

[0285] In some embodiments, the variant Fc region comprises or consists of:

[0286] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 70; and

[0287] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75.

[0288] In some embodiments, the variant Fc region comprises or consists of:

[0289] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71 ; and

[0290] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73.

[0291] In some embodiments, the variant Fc region comprises or consists of: - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71 ; and

[0292] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74.

[0293] In some embodiments, the variant Fc region comprises or consists of:

[0294] - a first Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71 ; and

[0295] - a second Fc domain that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75.

[0296] Table 3. Amino acid sequences of non-limiting examples of Fc domains and heavy chain constant regions incorporating Fc domains

[0297] For embodiments wherein the variant Fc region comprises one or more amino acid substitutions in addition to the ABDEG™ signature, the first Fc domain and / or second Fc domain may comprise the amino acids A, A at EU positions 234 and 235, respectively. In further embodiments, the first Fc domain and / or second Fc domain may comprise the amino acids A, A and G at EU positions 234, 235 and 329, respectively.

[0298] As noted above, in some embodiments, the monovalent antigen-binding molecules of the invention comprise a variant Fc region or FcRn binding fragments thereof comprising a first Fc domain and a second Fc domain, wherein at least one of the Fc domains is a variant Fc domain as described herein. In certain embodiments, both of the first Fc domain and the second Fc domain are variant Fc domains that comprise the amino acids Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436 respectively.

[0299] For heterodimeric embodiments, one or both of the Fc domains may be a variant Fc domain. In preferred embodiments, the variant Fc region comprises two different variant Fc domains that form a heterodimer. By way of example, in some embodiments, the first variant Fc domain comprises the ABDEG™ signature, L234A, L235A mutations and “knob” mutations; and the second variant Fc domain also comprises the ABDEG™ signature and L234A, L235A mutations but comprises “hole” mutations instead of the “knob” mutations. In further embodiments, the second variant Fc domain comprises the ABDEG™ signature, L234A, L235A mutations and “knob” mutations; and the first variant Fc domain also comprises the ABDEG™ signature and L234A, L235A mutations but comprises “hole” mutations instead of the “knob” mutations. In other embodiments, the first variant Fc domain comprises the ABDEG™ signature, L234A, L235A, P329G mutations and “knob” mutations; and the second variant Fc domain also comprises the ABDEG™ signature and L234A, L235A, P329G mutations but comprises “hole” mutations instead of the “knob” mutations. In further embodiments, the second variant Fc domain comprises the ABDEG™ signature, L234A, L235A, P329G mutations and “knob” mutations; and the first variant Fc domain also comprises the ABDEG™ signature and L234A, L235A, P329G mutations but comprises “hole” mutations instead of the “knob” mutations.

[0300] For embodiments wherein the variant Fc region comprises one or more amino acid substitutions in addition to the ABDEG™ signature, the first and / or second Fc domain of the variant Fc region may comprise one or more additional amino acid substitutions that have been reported to increase FcRn binding and thereby improve antibody pharmacokinetics. Such substitutions are reported in, for example, Zalevsky et al. (2010) Nat. Biotechnol. 28(2):157-9; Hinton et al. (2006) J Immunol. 176:346-356; Yeung et al. (2009) J Immunol. 182:7663-7671 ; Presta LG. (2008) Cum. Op. Immunol. 20:460-470; and Vaccaro et al. (2005) Nat. Biotechnol. 23(10):1283-88, the contents of which are incorporated herein in their entirety.

[0301] For embodiments wherein the variant Fc region comprises one or more amino acid substitutions in addition to the ABDEG™ signature, the first and / or second Fc domain of the variant Fc region may comprise a non-naturally occurring amino acid residue at one or more positions selected from the group consisting of 234, 235, 236, 239, 240, 241 , 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332, 333, and 334 as numbered by the EU index as set forth in Kabat. Optionally, the first and / or second Fc domain of the variant Fc region may comprise a non- naturally occurring amino acid residue at additional and / or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821 ; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752 and WO 05 / 040217, the contents of which are incorporated by reference herein in their entirety). In certain embodiments, the first and / or second Fc domain of the variant Fc region comprises at least one additional non-naturally occurring amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401 , 240A, 240T, 240M, 241 W, 241 L, 241 Y, 241 E, 241 R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A as numbered by the EU index as set forth in Kabat. Optionally, the first and / or second Fc domain may comprise additional and / or alternative non-naturally occurring amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821 ; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752 and WO 05 / 040217, the contents of which are incorporated by reference herein in their entirety).

[0302] Additional Fc domain alterations that may be incorporated into the variant Fc regions described herein include without limitation those disclosed in Ghetie et al., 1997, Nat. Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991 , J. Immunol., 147:2657-2662; Lund et al, 1992, Mol. Immunol., 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA, 92:11980- 11984; Jefferis et al, 1995, Immunol Lett., 44:111-117; Lund et al., 1995, Faseb J., 9:115- 119; Jefferis et al, 1996, Immunol Lett., 54:101-104; Lund et al, 1996, J. Immunol., 157:4963-4969; Armour et al., 1999, Eur J Immunol. 29:2613-2624; Idusogie et al, 2000, J. Immunol., 164:4178-4184; Reddy et al, 2000, J. Immunol., 164:1925-1933; Xu et al., 2000, Cell Immunol., 200:16-26; Idusogie et al, 2001 , J. Immunol., 166:2571 -2575; Shields et al., 2001 , J Biol. Chem., 276:6591 -6604; Jefferis et al, 2002, Immunol Lett., 82:57-65; Presta et al., 2002, Biochem Soc Trans., 30:487-490); U.S. Pat. Nos. 5,624,821 ; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121 ,022; 5,624,821 ; 5,648,260; 6,528,624; 6,194,551 ; 6,737,056; 6,821 ,505; 6,277,375; U.S. Patent Publication Nos. 2004 / 0002587 and PCT Publications WO 94 / 29351 ; WO 99 / 58572; WO 00 / 42072; WO 02 / 060919; WO 04 / 029207; WO 04 / 099249; WO 04 / 063351 , the contents of which are incorporated by reference herein in their entirety.

[0303] In order to treat autoantibody-mediated disorders, it would be advantageous to both remove the deleterious autoantibodies and to block the interaction of the immune complexes of these antibodies with activating Fc receptors (e.g., Fey receptors, such as CD89). Accordingly, in certain embodiments, the variant Fc region of the monovalent antigen-binding molecule exhibits increased binding to CD89 (e.g., human CD89). This is particularly advantageous in that it allows the monovalent antigen-binding molecule to additionally antagonize the immune complex-induced inflammatory response of autoantibodies being targeted for removal by FcRn inhibition. Any art recognized means of increasing affinity for CD89 (e.g., human CD89) can be employed. In certain embodiments, the monovalent antigen-binding molecule comprises a variant Fc domain or variant Fc-region comprising an N-linked glycan (e.g., at EU position 297). In this case it is possible to increase the binding affinity of the monovalent antigen-binding molecule for C89 by altering the glycan structure. Alterations of the N-linked glycan of Fc regions are well known in the art. For example, afucosylated N-linked glycans or N-glycans having a bisecting GIcNac structure have been shown to exhibit increased affinity for CD89. Accordingly, in certain embodiments, the N-linked glycan is afucosylated. Afucosylation can be achieved using any art recognized means. For example, a monovalent antigenbinding molecule can be expressed in cells lacking fucosyl transferase, such that fucose is not added to the N-linked glycan at EU position 297 of the variant Fc domain or variant Fc region (see e.g., US 8,067,232, the contents of which is incorporated by reference herein in its entirety). In certain embodiments, the N-linked glycan has a bisecting GIcNac structure. The bisecting GIcNac structure can be achieved using any art recognized means. For example, a monovalent antigen-binding molecule can be expressed in cells expressing beta1 -4-N-acetylglucosaminyltransferase III (GnTIII), such that bisecting GIcNac is added to the N-linked glycan at EU position 297 of the variant Fc domain or variant Fc region (see e.g., US 8021856, the contents of which is incorporated by reference herein in its entirety). Additionally or alternatively, alterations of the N-linked glycan structure can also be achieved by enzymatic means in vitro. To enhance the manufacturability of the monovalent antigen-binding molecules of the present invention, it is preferable that the variant Fc domains or variant Fc regions do not comprise any non-disulphide bonded cysteine residues. Accordingly, in certain embodiments the variant Fc domains or variant Fc regions do not comprise a free cysteine residue.

[0304] In certain embodiments, the variant Fc region or FcRn binding fragment thereof has altered (e.g., increased or decreased) binding affinity for an additional Fc receptor. The variant Fc region or FcRn binding fragment thereof can have altered (e.g., increased or decreased) binding affinity for one or more of Fey receptors e.g., FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). Any art recognized means of altering the affinity for an additional Fc receptor can be employed.

[0305] (v) One-armed antibodies

[0306] As noted elsewhere herein, the monovalent antigen-binding molecules of the invention encompass antibodies modified so as to be “one-armed”, wherein the single arm comprises an antigen-binding domain that binds to IgA. In some embodiments therefore, the antigenbinding molecules are one-armed IgG antibodies. In such embodiments, the one-armed IgG antibodies comprise a single Fab arm. Therefore, monovalent antigen-binding molecules of the invention encompass IgG antibodies lacking one Fab arm.

[0307] The monovalent antigen-binding molecules of the invention are asymmetrical due to the presence of a single antigen-binding domain. The presence of a single antigen-binding domain means that, in certain embodiments, only one of the Fc domains of the variant Fc region is attached to the single antigen-binding domain.

[0308] In some embodiments, the antigen-binding domain is non-covalently attached to the variant Fc region or FcRn binding fragment thereof. It is however preferred that the antigenbinding domain is covalently attached to the variant Fc region or FcRn binding fragment thereof. The antigen-binding domain may be covalently attached to either the first Fc domain or the second Fc domain.

[0309] The antigen-binding domain may be attached to the N-terminus of the first or second Fc domain. The antigen-binding domain may be attached to the C-terminus of the first or second Fc domain. Alternatively, the antigen-binding domain can be attached at a position other than the N-terminus or the C-terminus of the first or second Fc domain. In some embodiments, the C-terminus of the antigen-binding domain is attached to the N-terminus of either the first Fc domain or the second Fc domain.

[0310] In further embodiments, the antigen-binding domain is linked to the N-terminus of either the first Fc domain or the second Fc domain via a linker. In some embodiments, the linker is a non-cleavable linker. As used herein, the term “non-cleavable linker” refers to a linker that is not readily cleaved by one or more of a given enzyme (such as a protease), chemical agent, or photo-irradiation.

[0311] The linker can be a synthetic compound linker such as, for example, a chemical crosslinking agent. Non-limiting examples of suitable cross-linking agents that are available to the skilled person include N-hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2- (sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0312] In some embodiments, the linker is a peptide linker. Examples of peptide linkers are well known. Any peptide linker could be used to link an antigen-binding domain to the first or second Fc domain in the monovalent antigen-binding molecules of the invention.

[0313] Peptide linkers may be of any length. In some embodiments, the peptide linker is between about 1 and about 100 amino acids in length, between about 8 and about 40 amino acids in length, or between about 15 amino acids and about 25 amino acids in length. In some embodiments, the peptide linker is between 1 and 100 amino acids in length, between 8 and 40 amino acids in length, or between 15 and 25 amino acids in length.

[0314] In some embodiments, the peptide linker contains only glycine and / or serine residues (e.g., glycine-serine linker or GS linker). Examples of such peptide linkers include: Gly(x) Ser, where x is 0 to 6; or Ser Gly(x), where x is 0 to 6; (Gly Gly Gly Gly Ser)n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly)n, wherein n is an integer of one or more. In some embodiments, the peptide linker includes an amino acid sequence selected from the group consisting of: (GGGGS)n and (SGGGG)n, where n is 1 to 8. In some embodiments, the linker peptides are modified such that the amino acid sequence GSG (that occurs at the junction of traditional Gly / Ser linker peptide repeats) is not present. For example, in some embodiments, the peptide linker includes an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS and GGGGS(XGGGS)n, where X is any amino acid that can be inserted into the sequence and not result in a polypeptide including the sequence GSG, and n is 0 to 4.

[0315] In preferred embodiments, the antigen binding domain is a Fab (described elsewhere herein). In such embodiments, the C-terminus of the Fab heavy chain is preferably attached to the N-terminus of either the first Fc domain or the second Fc domain.

[0316] In some embodiments, one or more additional amino acids are included between the C- terminus of the Fab heavy chain and the N-terminus of the first Fc domain or the second Fc domain.

[0317] In some embodiments, the C-terminus of the Fab heavy chain is attached to the N-terminus of the first Fc domain or second Fc domain via a hinge region as defined elsewhere herein or a portion thereof.

[0318] In some embodiments, the hinge is a naturally occurring hinge region. In some embodiments, the hinge region is an IgG hinge region. In preferred embodiments, the hinge region is a human IgG hinge region. In some embodiments, the IgG hinge region is selected from an: IgG 1 , lgG2, lgG3 and lgG4 hinge region. In some embodiments, the human IgG hinge region is selected from an: lgG1 , lgG2, lgG3 and lgG4 hinge region.

[0319] (vi) Exemplary monovalent antioen-bindino molecules that bind Io A

[0320] The IgA-binding molecules of the invention can be distinguished from the prior art on the basis that they are monovalent. This is an important distinction since the current application reports, for the first time, that monovalent IgA-binding molecules achieve improved IgA clearance in vivo as compared to bivalent IgA binding molecules (i.e. improved IgA sweeping). The improvement in IgA clearance was observed in terms of the speed of IgA removal as well as the depth of response seen.

[0321] The monovalent antigen-binding molecules of the present invention may comprise the CDR, VH and / or VL domain amino acid sequences of any antigen-binding molecules (e.g. antibodies) known to exhibit binding specificity for human IgA.

[0322] Exemplary antibodies known to bind human IgA include, but are not limited to, GA1 -lgG1 , GA2-lgG1 , GA3-lgG1 and GA4-lgG1 , as disclosed in WO 2012 / 073992. The monovalent antigen-binding molecules of the invention may comprise CDR, VH and / or VL amino acid sequences derived from GA1 -IgG 1 , GA2-lgG1 , GA3-lgG1 or GA4-lgG1 .

[0323] The monovalent antigen-binding molecules having the CDR, VH and / or VL amino acid sequences recited herein may be engineered so as to exhibit pH-dependent antigen binding, as described in section (i) above. The exemplary monovalent antigen-binding molecules described herein may be engineered so as to exhibit pH-dependent antigen binding by the introduction of one or more substitutions into the variable domains. In some embodiments, the monovalent antigen-binding molecules are engineered so as to exhibit pH-dependent antigen binding by introducing one or more substitutions into one or more CDRs of the antibody. The substitutions may introduce one or more His residues into one or more sites of the variable domains, preferably the heavy chain and / or light chain CDRs so as to confer pH-dependent antigen binding. The six heavy chain and light chain CDRs combined may consist of a total of 1-10 His substitutions, optionally 1-5 His substitutions, optionally 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 His substitutions. The monovalent antigen-binding molecules may be engineered in accordance with the methods described in

[0324] WO2018 / 206748. Non-histidine substitutions may also be incorporated into variable domains, particularly the CDRs, of the pH-dependent monovalent antigen-binding molecules described herein.

[0325] Exemplary pH-dependent monovalent antigen-binding molecules in accordance with the invention are described below with reference to specific CDR, VH and / or VL sequences.

[0326] In certain embodiments, the monovalent antigen-binding molecules of the invention comprise an antigen-binding domain that comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:

[0327] HCDR3 comprising or consisting of SEQ ID NO: 3; HCDR2 comprising or consisting of SEQ ID NO: 2; HCDR1 comprising or consisting of SEQ ID NO: 1 ; LCDR3 comprising or consisting of SEQ ID NO: 6; LCDR2 comprising or consisting of SEQ ID NO: 5; and LCDR1 comprising or consisting of SEQ ID NO: 4.

[0328] In certain embodiments, the monovalent antigen-binding molecules of the invention comprise an antigen-binding domain that comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:

[0329] HCDR3 comprising or consisting of SEQ ID NO: 11 ; HCDR2 comprising or consisting of SEQ ID NO: 10; HCDR1 comprising or consisting of SEQ ID NO: 9; LCDR3 comprising or consisting of SEQ ID NO: 14; LCDR2 comprising or consisting of SEQ ID NO: 13; and LCDR1 comprising or consisting of SEQ ID NO: 12.

[0330] In certain embodiments, the monovalent antigen-binding molecules of the invention comprise an antigen-binding domain that comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:

[0331] HCDR3 comprising or consisting of SEQ ID NO: 19; HCDR2 comprising or consisting of SEQ ID NO: 18; HCDR1 comprising or consisting of SEQ ID NO: 17; LCDR3 comprising or consisting of SEQ ID NO: 22; LCDR2 comprising or consisting of SEQ ID NO: 21 ; and LCDR1 comprising or consisting of SEQ ID NO: 20.

[0332] In certain embodiments, the monovalent antigen-binding molecules of the invention comprise an antigen-binding domain that comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:

[0333] HCDR3 comprising or consisting of SEQ ID NO: 27; HCDR2 comprising or consisting of SEQ ID NO: 26; HCDR1 comprising or consisting of SEQ ID NO: 25; LCDR3 comprising or consisting of SEQ ID NO: 30; LCDR2 comprising or consisting of SEQ ID NO: 29; and LCDR1 comprising or consisting of SEQ ID NO: 28. In certain embodiments, the monovalent antigen-binding molecules of the invention comprise an antigen-binding domain that comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:

[0334] HCDR3 comprising or consisting of SEQ ID NO: 91 ; HCDR2 comprising or consisting of SEQ ID NO: 90; HCDR1 comprising or consisting of SEQ ID NO: 89; LCDR3 comprising or consisting of SEQ ID NO: 94; LCDR2 comprising or consisting of SEQ ID NO: 93; and LCDR1 comprising or consisting of SEQ ID NO: 92.

[0335] In certain embodiments, the monovalent antigen-binding molecules of the invention comprise an antigen-binding domain that comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:

[0336] HCDR3 comprising or consisting of SEQ ID NO: 99; HCDR2 comprising or consisting of SEQ ID NO: 98; HCDR1 comprising or consisting of SEQ ID NO: 97; LCDR3 comprising or consisting of SEQ ID NO: 102; LCDR2 comprising or consisting of SEQ ID NO: 101 ; and LCDR1 comprising or consisting of SEQ ID NO: 100.

[0337] In certain embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto.

[0338] In some embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0339] In certain embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto.

[0340] In some embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 15, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0341] In certain embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto.

[0342] In some embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 23, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 24.

[0343] In certain embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto.

[0344] In some embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 31 , and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 32.

[0345] In certain embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 95 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto.

[0346] In some embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 95, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 96.

[0347] In certain embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence having at least 80%, 90%, 95%, 98% 99% identity thereto.

[0348] In some embodiments, the monovalent antigen-binding molecules comprise or consist of a variable heavy chain domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 103, and a variable light chain domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 104.

[0349] The exemplary monovalent antigen-binding molecules having any of the specific CDR, VH and / or VL domains recited above may comprise any of the variant Fc regions or FcRn binding fragments thereof according to the embodiments described in sections (iii) and (iv) above.

[0350] In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a variant human IgG Fc region or FcRn binding fragment thereof, comprising a first variant human IgG Fc domain and a second variant human IgG Fc domain, wherein both the first and second Fc domains each comprise the amino acids A, A, G, Y, T, E, K, F and Y at EU positions 234, 235, 329, 252, 254, 256, 433,434 and 436, respectively. In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a variant human IgG 1 Fc region or FcRn binding fragment thereof, wherein both the first and second Fc domains each comprise the amino acids A, A, G, Y, T, E, K, F and Y at EU positions 234, 235, 329, 252, 254, 256, 433, 434 and 436, respectively.

[0351] In preferred embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a variant human IgG Fc region comprising or consisting of a first and a second variant human IgG Fc domain, wherein the first variant Fc domain comprises the amino acids A, A, G, Y, T, E, W, K, F and Y at EU positions 234, 235, 329, 252, 254, 256, 366, 433, 434 and 436, respectively; and the second Fc domain comprises the amino acids A, A, G, Y, T, E, K, F, Y, S, A and V at EU positions 234, 235, 329, 252, 254, 256, 433, 434, 436, 366, 368 and 407, respectively.

[0352] In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a variant human IgG Fc region comprising or consisting of a first and a second variant human IgG Fc domain, wherein the first variant Fc domain comprises the amino acids A, A, Y, T, E, W, K, F and Y at EU positions 234, 235, 252, 254, 256, 366, 433,434 and 436, respectively; and the second Fc domain comprises the amino acids A, A, Y, T, E, K, F, Y, S, A and V at EU positions 234, 235, 252, 254, 256, 433, 434, 436, 366, 368 and 407, respectively.

[0353] In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a variant human IgG Fc region comprising or consisting of a first and a second variant human IgG Fc domain, wherein the first variant Fc domain comprises the amino acids A, A, W, R, K, K and F at EU positions 234, 235, 366, 401 , 413, 433 and 434, respectively; and the second Fc domain comprises the amino acids A, A, R, K, K, F, S, A and V at EU positions 234, 235, 401 , 413, 433, 434, 366, 368 and 407, respectively.

[0354] In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a variant human IgG Fc region comprising or consisting of a first and a second variant human IgG Fc domain, wherein the first variant Fc domain comprises the amino acids A, A, W, R ,R, K, K and F at EU positions 234, 235, 366, 311 , 400, 413, 433 and 434, respectively; and the second Fc domain comprises the amino acids A, A, R, R, K, K, F, S, A and V at EU positions 234, 235, 311 , 400, 413, 433, 434, 366, 368 and 407, respectively. In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a first Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33 and a second Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34.

[0355] In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a first Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 35 and a second Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 36.

[0356] In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a first Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 37 and a second Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 38.

[0357] In certain embodiments, the exemplary monovalent antigen-binding molecules described herein comprise a first Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 39 and a second Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 40.

[0358] In another embodiment, the exemplary monovalent antigen-binding molecules comprise

[0359] - an antigen-binding domain that binds to IgA; and

[0360] - a variant Fc region or a FcRn binding fragment thereof, that binds to FcRn with increased affinity relative to a wild-type Fc region; wherein the variant Fc region comprises a first Fc domain and a second Fc domain; and wherein the antigen-binding domain is attached to the first Fc domain of the variant Fc region. In said embodiments, the first Fc domain comprises “knob” mutations (e.g. W at EU position 366) and the second Fc domain comprises “hole” mutations (e.g. S, A and V at EU positions 366, 368 and 407 respectively). Table 4: Heavy chain CDR sequences

[0361] Table 5: Light chain CDR sequences

[0362] Table 6: VH and VL sequences

[0363] Table 7: Fc region sequences

[0364]

[0365]

[0366] C. Polynucleotides encodinq monovalent antiqen-bindinq molecules

[0367] The invention also provides polynucleotide molecules encoding the monovalent antigenbinding molecules of the invention or fragments thereof. Also encompassed are expression vectors containing said nucleotide sequences of the invention operably linked to regulatory sequences which permit expression of the monovalent antigen-binding molecules or fragments thereof in a host cell or cell-free expression system, and a host cell or cell-free expression system containing this expression vector.

[0368] Polynucleotide molecules encoding the monovalent antigen-binding molecules of the invention include, for example, recombinant DNA molecules. The terms "nucleic acid", “polynucleotide” or a "polynucleotide molecule" as used herein interchangeably and refer to any DNA or RNA molecule, either single- or double-stranded and, if single-stranded, the molecule of its complementary sequence. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction. In some embodiments of the invention, nucleic acids or polynucleotides are "isolated". This term, when applied to a nucleic acid molecule, refers to a nucleic acid molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or non-human host organism. When applied to RNA, the term "isolated polynucleotide" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been purified / separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated polynucleotide (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0369] For recombinant production of a monovalent antigen-binding molecule according to the invention, a recombinant polynucleotide encoding it may be prepared (using standard molecular biology techniques) and inserted into a replicable vector for expression in a chosen host cell, or a cell-free expression system. Suitable host cells may be prokaryote, yeast, or higher eukaryote cells, specifically mammalian cells. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, llrlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243- 251 (1980)); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581 ; ATCC CRL 8287) or NS0 (HPA culture collections no. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM’s PERC-6 cell line. Expression vectors suitable for use in each of these host cells are also generally known in the art.

[0370] It should be noted that the term "host cell" generally refers to a cultured cell line. Whole human beings into which an expression vector encoding an antigen-binding molecule according to the invention has been introduced are explicitly excluded from the definition of a “host cell”.

[0371] D. Monovalent antiaen-bindina molecule production

[0372] In a further aspect, the invention also provides a method of producing monovalent antigenbinding molecules of the invention which comprises culturing a host cell (or cell free expression system) containing polynucleotide (i.e. an expression vector) encoding the monovalent antigen-binding molecule under conditions which permit expression of the monovalent antigen-binding molecule, and recovering the expressed monovalent antigenbinding molecule. This recombinant expression process can be used for large scale production of the monovalent antigen-binding molecules according to the invention, including molecules intended for human therapeutic use. Suitable vectors, cell lines and production processes for large scale manufacture of monovalent antigen-binding molecules (i.e. recombinant modified antibodies) suitable for in vivo therapeutic use are generally available in the art and will be well known to the skilled person.

[0373] E. Pharmaceutical compositions

[0374] The scope of the invention includes pharmaceutical compositions, containing one or a combination of monovalent antigen-binding molecules of the invention formulated with one or more pharmaceutically acceptable carriers or excipients. Such compositions may include one or a combination of (e.g., two or more different) monovalent antigen-binding molecules as described herein. Techniques for formulating monoclonal antibodies for human therapeutic use are well known in the art and are reviewed, for example, in Wang et al., Journal of Pharmaceutical Sciences, Vol.96, pp1 -26, 2007, the contents of which are incorporated herein in their entirety.

[0375] Pharmaceutically acceptable excipients that may be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0376] In certain embodiments, the pharmaceutical compositions are formulated for administration to a subject via any suitable route of administration including but not limited to intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalational, buccal e.g., sublingual), and transdermal administration. In preferred embodiments, the composition is formulated for intravenous or subcutaneous administration. F. Methods of treatment

[0377] The monovalent antigen-binding molecules and pharmaceutical compositions as described herein are intended for use in methods of treatment. The present invention thus provides monovalent antigen-binding molecules in accordance with the first aspect of the invention or pharmaceutical compositions comprising the same for use as medicaments. Further provided are methods of treating a disorder in a subject, the methods comprising administering to a patient in need thereof a therapeutically effective amount of a monovalent antigen-binding molecule in accordance with the first aspect of the invention or a pharmaceutical composition comprising the same. The invention also provides monovalent antigen-binding molecules in accordance with the first aspect of the invention or pharmaceutical compositions comprising the same for use in the treatment of a disorder in a subject in need thereof. The disorder is preferably an antibody-mediated disorder (as defined elsewhere). The subject is preferably human. All embodiments described above in relation to the monovalent antigen-binding molecules and pharmaceutical compositions of the invention are equally applicable to the methods described herein.

[0378] In certain embodiments, the disorder treated in accordance with the methods described herein is an autoantibody-mediated disorder. In certain embodiments, the disorder is an IgA-mediated disorder. In certain embodiments, the disorder is an IgA autoantibody- mediated disorder.

[0379] INCORPORATION BY REFERENCE

[0380] Various publications are cited in the foregoing description and throughout the following examples, each of which is incorporated by reference herein in its entirety.

[0381] EXAMPLES

[0382] The invention will be further understood with reference to the following non-limiting examples. Example 1 : Anti-lqA antibody development utilizing the SIMPLE antibody platform

[0383] The following example describes how anti-lgA antibodies were generated via llama immunisation.

[0384] Two llamas (Milo and Butch) were immunized with a cocktail of polyclonal immunoglobulin lgG1+lgG2+lgA (IgA from human serum, Sigma, cat. I4036). Peripheral blood lymphocytes isolated from immunized llamas were used for RNA extraction, RT-PCR and PCR-cloning of the variable domains in a single-chain variable fragment (scFv) phagemid vector. Panning phage display selections were performed for up to three rounds using human IgA Isotype Control (ThermoFisher, cat.31148), counter selecting with total human IgG (Sigma, cat. I4506) and using citrate phosphate acetate buffer (CPA) elution to enrich for pH- dependent antigen binding.

[0385] Masterplates were generated upon enrichment after at least two consecutive rounds of phage display selections both from total as well as pH-dependent elution conditions. Individual clones were grown in a 96-deep well plate and periplasmic fractions were prepared. These periplasmic extracts (P.E.) (containing Fabs), were tested in an enzyme- linked immunosorbent assay (ELISA) for binding to full- length human IgA (ThermoFisher, cat. 31148) and human IgG (Sigma, cat. I4506). Less than 3% of the selected clones cross-reacted with human IgG, demonstrating IgA specificity.

[0386] Only the Fabs that specifically bound to IgA in ELISA were further screened using surface plasmon resonance (SPR) for off-rate determination on a CM5 chip coated with full-length human IgA or human IgG immobilized at 3000 response units (RU). Binders with good affinity and specificity for human IgA at pH 7.4 were then sequenced. 244 valid sequences were obtained, containing 169 unique VH-gs-VL sequences with 56 different heavy complementarity-determining regions (HCDR3) grouped into 46 different HCDR3 families.

[0387] These unique VH-gs-VL clones were further screened using SPR to assess association and dissociation to human IgA (hulgA) and human IgG (huIgG) at pH 5.5. In addition to hulgA binding, all unique scFv variant clones were also screened for their competing properties. A CM5 chip was immobilized with recombinant human FCAR / CD89 receptor (hFcaRI) (R&D systems, cat. 3939-FA) (1000 RU), followed by sequential capture of hulgA and anti-lg A P.E. clones. Anti-lg A P.E. clones that bound to captured human IgA were considered non-competitive binders. Clones that demonstrated a faster dissociation as compared to the blank control were capable of displacing IgA from its receptor FcaRI. Next to the sequential addition, a pre-mix was made of hulgA and anti-lg A P.E. to confirm the competing properties of the different scFv variants. 99 of the total 169 analyzed anti-hulgA scFv P.E. were capable of at least partially blocking the binding of IgA to its receptor FcaRI.

[0388] A sub-set of 23 scFv P.E clones were selected based on their affinity for hulgA, the extent of pH-dependent-antigen binding and / or their competitive binding properties.

[0389] The 23 scFv P.E clones were cloned into a vector containing the sequence of the human IgG 1 with the LALA mutations (L234A, L235A) to reduce effector functions and the ABDEG™ mutations (M252Y / S254T / T256E(CH2) H433K / N434F(CH3)) to increase FcRn binding at pH 7.4 and pH 6. Antibodies were produced in HEK293 cells and purified on a protein A column.

[0390] All 23 resulting antibodies exhibited binding to human IgA. However, only 18 anti-lgA antibodies showed competition for binding of IgA to its receptor CD89.

[0391] Of these 18 anti-lgA antibodies, clone G was selected for further testing because it exhibited the following favourable properties - (i) competitive antigen-binding; (ii) pH- dependent antigen binding; and (iii) cross reactivity with rhesus IgA.

[0392] Example 2: Further testing and optimization of clone G

[0393] This example describes how the llama-derived antibody clone G was engineered in order to further improve its properties.

[0394] 2.1 Stability testino of clone G

[0395] Anti-lgA clone G from Example 1 was subjected to stability testing.

[0396] In more detail, clone G was diluted in sterile Dulbecco’s PBS (Sigma-Aldrich, cat. D8537) and incubated at 37°C for up to 4 weeks in a glass vial (Supelco, 1 .5 ml screw neck vial, cat. 854171 ). Intermittent sample collection was carried out weekly to determine the stability of the antibody. Every week, samples were subjected to a visual inspection, and after 4 weeks of stability testing, all samples were analysed via Nanodrop, Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and Biacore. A CM5 chip was coated with Motavizumab-lgA (in-house production; 4.05 mg / ml) and the association at pH 7.4 of the test samples was compared with the association of a dilution series of corresponding control samples in HBS-EP (in-house made from Cytiva, cat. BR100826) in order to assess if there was any loss of potency due to the incubation at high temperature.

[0397] After 4 weeks at 37°C, clone G had a potency loss of 12%.

[0398] In addition to potency testing, the samples were screened for the most common post- translational modifications (PTMs) using reduced peptide map analysis. PTMs included in this analysis were oxidation, deamidation, isomerization, glycosylation, and N / C terminal processing in the VH and VL. These studies identified several potential liabilities in the peptide sequences of clone G. Specifically, an isomerization site at position 98 of clone G’s VH. In addition, a clipping site between positions 52G and 53S of the HCDR2 of clone G was detected by mass spectrometry.

[0399] 2.2 Clone G optimization and engineering

[0400] The sequence of clone G was optimized by grafting the CDRs into the closest human germline sequence (Z12342|IGHV3-53*02). Screening of human IgA affinity, pH- dependency and thermostability was performed using SPR. Additionally, ELISA was used to test competition and displacement of IgA from its receptor CD89. Germlining drastically increased the pH-dependent binding to IgA by the antibody.

[0401] Further optimisation of the pH-dependent antigen binding characteristics of clone G was achieved by histidine (HIS) engineering.

[0402] A total of 17 single histidine residues were introduced into the CDRs of the VK and 26 single histidine residues were introduced into clone G’s VH. Screening for human and rhesus IgA pH-dependency was performed using SPR and ELISA. These tests revealed several spots in the VH and VK which improved pH-dependent antigen binding. Each selected histidine mutation in the VK was combined with the selected histidine mutations in the VH to generate double histidine engineered clones. These further engineered clones were then screened for human and rhesus IgA pH-dependency using SPR (see section 2.3 below) and ELISA.

[0403] Next, different histidine spots in the VH of clone G were combined into double, triple and quadruple histidine engineered variants and again screened using SPR for pH-dependent antigen binding on human and rhesus IgA. From these screening studies, a triple histidine engineered variant of clone G with a histidine residue at positions 32 (VH2), position 56 (VH9) and position 59 (VH26) showed highest pH-dependent antigen binding with both human and rhesus IgA and was therefore selected for further optimization.

[0404] The three histidine substitutions to clone G (at positions 32, 56 and 59) were then introduced into the sequence of the germlined variant of clone G to further increase pH- dependent IgA binding. Whilst there was only a minor increase in pH-dependent binding to human IgA, the three additional histidine substitutions to clone G clearly increased pH- dependent binding to rhesus IgA. Therefore both variants were selected for further characterization and comparison. They are referred to herein as “clone E” (i.e. clone G germlined variant without His substitutions at positions 32, 56 and 59) and “clone F" (i.e. clone G germlined variant with His substitutions at positions 32, 56 and 59).

[0405] Since reduced peptide map analysis identified position D98 in the VH of clone G as being prone to isomerization, this position was substituted with other amino acids. The resulting variants were screened for human, rhesus and cynomolgus monkey IgA affinity, pH- dependency and lgA:CD89 competition and displacement on Biacore and / or ELISA.

[0406] Based on these studies a variant with glutamic acid (E) at position 98 was selected for further study. The engineered variants of clone G comprising the D98E substitution are referred to herein as “clone B” (corresponding to clone E above) and “clone C” (corresponding to clone F above).

[0407] As noted above, a clipping site in HCDR2 of clone G was detected by mass spectrometry between position 52G and 53S. A follow-up stability study confirmed the presence of this potential liability in the parental clone G and the two derivative clones, clone C and clone B. In order to avoid clipping of HCDR2, positions 52G and 53S were randomly mutated in clone B and clone C. The different variants were screened for human and cynomolgus monkey IgA affinity and pH-dependent antigen binding using SPR. lgA:CD89 competition and displacement studies were performed using ELISA. The S53K variant demonstrated similar pH-dependent antigen binding and affinity to both human and cynomolgus monkey IgA relative to clone B and clone C. Moreover, a similar CD89 blocking capacity was observed for the S53K variant as compared to clone B and clone C. Given the similarities to the parental clone, the mutation S53K was selected to avoid clipping of HCDR2 in clone B and clone C. The removal of the clipping site was confirmed via an additional stability study followed by mass spectrometry analysis. The resulting two engineered variants are referred to herein as “clone D” (corresponding to clone B but with the S53K mutation) and “clone A" (corresponding to clone C but with the S53K mutation).

[0408] 2.3 oH-deoendent bindino to human and cvnomolous monkey IQA measured usino SPR A crucial property of a sweeping antibody is that it possesses pH-dependent binding to its target antigen (i.e. IgA), such that the antigen is released in the endosome due to the acidic environment. To maximize the lysosomal degradation of the antigen, re-binding of the target in the endosome should also be avoided. Therefore, affinity as well as antigenbinding at acidic pH of the anti-lg A antibodies to human and cynomolgus monkey serum IgA was analyzed using SPR.

[0409] In more detail, affinity for human IgA was determined using a CM5 chip coated with Motavizumab-lgA (UPX project 2020 222, Batch 4549; 5.04 mg / ml) by association of the test antibody at pH 7.4 and dissociation at pH 7.4 or 5.5. More in-depth characterization of the affinity and pH-dependency was done on a CM5 chip coated with anti-human Fc IgG (Jackson ImmunoResearch, cat. 109-005-098) followed by capturing the anti-lgA variants and association of 7.81 nM and 31 .25 nM IgG-depleted human IgA (Abeam, cat. Ab91025) or cynomolgus money IgA (Life diagnostics, cat. 30002-4) at pH 7.4 and dissociation at pH

[0410] 7.4 or pH 5.5. lgA:CD89 competition was analyzed by coating a 96-well half area high binding microplate (Greiner, cat. 675061) with recombinant human CD89 His-tag (Elabscience, cat. PKSH031620). After washing and blocking the plate, a dilution series of the anti-lgA antibodies in 0.1% casein-PBS, pre-incubated with a fixed concentration of human IgA (Abeam, cat. Ab91025) was added to the plate and allowed to bind for 1 hour at room temperature. Binding of IgA was detected with mouse anti-human IgA (Abeam, cat. Ab7400) and peroxidase-conjugated Donkey Anti-Mouse IgG (H+L) (Jackson Immunoresearch, cat. 715-035-150) and developed with s(SH)TMB (SDT Reagents for life, cat. sTMB) for 2-5 minutes. OD was measured at 450 nm (ref 620 nm) with a 96-well ELISA plate reader (Tecan Sunrise). lgA:CD89 displacement was analyzed with a similar set-up, but IgA was incubated 1 hour before the addition of the dilution series of anti-lg A antibodies, with a washing step in between.

[0411] Upon comparison of clones B and C, a slight improvement in pH-dependent binding of clone C to human IgA compared to clone B was observed, but this difference was more pronounced with cynomolgus monkey IgA. Whilst there was only a minor decrease in affinity with the HIS engineered variant (clone B; KD 5,02E-09 and clone C; KD 5,51 E-09), clone B reached higher RU levels for both human and cynomolgus monkey IgA.

[0412] The same SPR experimental method as described above was also used to investigate association and dissociation of variants to human and cynomolgus monkey IgA at different pHs - pH 7.4, pH 6 or pH 5. These experiments were performed in order to accommodate for differences in the pH of the endosome and lysosome. The binding reduction % at acidic pH compared to pH 7.4 was calculated based on Rmax values and are summarised in Table 8:

[0413] Table 8: Percentage (%) binding reduction of the clone E anti-lgA antibodies to human or cynomolgus monkey IgA at pH 6 and 5 compared to pH 7.4.

[0414] Reduced binding to human IgA was observed at pH 5 as compared to binding at pH 6.

[0415] Whilst only a 27% reduction in human IgA binding of clone B was observed at pH 6, the introduction of the three histidine residues in the HC resulted in 51% reduced binding at pH 6 and 100% reduced binding at pH 5. Clone B showed only a minimal reduction in binding to cynomolgus monkey IgA at pH 6 (16%) but clone C showed a 61% reduction in binding to cynomolgus monkey IgA at pH 6. 100% reduction in binding was seen for both clone B and clone C in binding experiments to cynomolgus monkey IgA at pH 5.0.

[0416] 2.4 In vivo IgA sweeping in mice

[0417] In order to explore the IgA sweeping efficacy of the anti-lgA antibodies in vivo, the anti-lg A clones were tested in in vivo experiments in a1 KI mice, which transgenically express endogenous human IgA by knocking in the human Ca Ig gene in place of the Sp region (Duchez et al. 2010).

[0418] Clone E-WT h IgG 1 and clone F-WT h IgG 1 (i.e. both still containing D98) were compared and Mota h IgG 1 WT was included as negative control. As expected, no IgA sweeping was observed with the negative control, Mota h IgG 1 WT. Initially, a steady PK profile was observed for both anti-lgA antibodies but from day 7 onwards there was faster clearance of the clone E-hlgG1 as compared to the histidine engineered clone F (Figure 1 B). The PD data (represented as % of h IgA normalized to baseline values) showed that both clones were able to clear endogenous human IgA from the circulation but that there was no real difference in the sweeping efficacy of both clones. Also, the duration of IgA sweeping was relatively prolonged, and IgA levels were still reduced by 75% 14 days post anti-lgA injection (Figure 1A).

[0419] Example 3: Variant Fc region testing in vivo

[0420] This Example summarizes the pharmacokinetic and pharmacodynamic properties of antiIgA monoclonal antibodies in cynomolgus monkeys.

[0421] The anti-lgA monoclonal antibodies tested in this Example share the same Fab (clone F as described in Example 2, section 2.2 - a germlined, His variant of original clone G) but differ in their Fc region backbones. The anti-lgA monoclonal antibodies that were tested are referred to herein as “clone F-hlgG1 HN”, “clone F-hlgG1 LALA HN” and “clone F-hlgG1 LALA ABDEG” (see Materials and Methods section 3.2 below for details of the precise substitutions in each of these Fc region backbones). All of these Fc regions comprise modifications that enhance binding to the human neonatal Fc receptor, hFcRn.

[0422] 3.1 Anti-lqA antibodies having variant Fc regions sweep circulating IgA Initial experiments explored whether the anti-lg A monoclonal antibodies were capable of sweeping circulating IgA in cynomolgus monkeys. Monkeys received a single intravenous injection (45 mg / kg) of: “clone F-hlgG1 HN”, “clone F-hlgG1 LALA HN” or “clone F-hlgG1 LALA ABDEG”; there were three monkeys per group.

[0423] Serum IgA levels in cynomolgus monkeys (0.8-4.5 mg / mL-1) were comparable to human serum IgA levels (1 -3 mg / mL-1), indicating that IgA is an abundantly present target in both species.

[0424] Serum samples were analyzed in a non-qualified PK-ELISA that was developed to measure total pharmacokinetics (PK) and the results are summarized in Figure 2. For some timepoints the concentration was below the LLOQ (lower limit of quantification) of the assay and could therefore not be determined. For most individuals, levels below the LLOQ were observed from day 21 onwards. From the total sample analysis, two PK values were potential outliers as they demonstrated a drastic drop in anti-lg A concentration that was fully recovered in the following time point.

[0425] Serum IgA levels were analysed in a non-qualified PD-MSD ELISA (Figure 3). Graphs depicting the absolute reduction in IgA levels upon IV injection of anti-lgA antibodies are summarized in Figure 4. Serum IgG levels were analyzed in a qualified PD-ELISA and results are summarized in Figure 5.

[0426] PK and PD data is reported for three anti-lgA antibodies (clone F-h IgG 1 LALA HN, clone F- hlgG1 LALA ABDEG and clone F-hlgG1 HN) after a single intravenous injection in cynomolgus monkeys. Sweeping of cynomolgus monkey IgA was observed for all anti-lgA antibodies.

[0427] The group injected with clone F-h IgG 1 HN exhibited an average IgA removal of 51 .8% (G4.1 : 41.6%, G4.2:73.1%, G4.3:40.8%). The group injected with hlgG1 LALA HN had an average IgA removal of 27.2% (G1 .1 : 42.5%, G1 .2: no sweeping, G1 .3:11 .9%) and showed the shortest effect on IgA depletion. For both of these groups, no cynomolgus monkey IgG sweeping was observed (as expected for these Fc backbones). The group injected with clone F-h IgG 1 LALA ABDEG demonstrated the best IgA sweeping effect. The total IgA reduction observed in each of the 3 male cynomolgus monkeys was 81.9% (G2.1 ), 69.3% (G2.2) and 25.7% (G2.3), respectively. This clone F-hlgG1 LALA ABDEG antibody also reduced the cynomolgus monkey IgG levels by 25%. The PK profile of the individual monkeys from this group showed some variability, which indicates that there could be a role for IgA in the clearance of the anti-lg A antibodies from the circulation.

[0428] 3.2 Materials and methods

[0429] 3.2.1 Antibodies

[0430] The anti-lgA antibodies were produced at ImmunoPrecise Antibodies (Europe) BV (Life Sciences Incubator, Yalelaan 62, 3584 CM Utrecht, The Netherlands). The three anti-lgA antibodies tested in this example share the same Fab (clone F) but have different h IgG 1 Fc backbones: hlgG1 -HN; hlgG1 -LALA-HN-delK; and hlgG1 -LALA-ABDEG-delK. delK means that the terminal lysine of the heavy chain was removed and LALA means that the L234A, L235A mutations were made in the heavy chain to reduce effector functions (Hezareh et al. 2001 ). HN and ABDEG are mutations that affect binding to the neonatal Fc receptor (FcRn); NHance® (H433K / N434F) increases FcRn binding at acidic (endosomal) pH, resulting in a prolonged half-life due to antibody recycling, whilst ABDEG™ (M252Y / S254T / T256E / H433K / N434F) enhances FcRn binding both at neutral and acidic pH.

[0431] 3.2.2 Intravenous (IV) injection of anti-loA antibodies in cvnomolous monkeys Nine male naive cynomolgus monkeys between 2,5-5 year old were hosted at PharmaLegacy Laboratories Vivarium (Pudong, Shanghai, China). The monkeys were kept in conventional rooms with 1 monkey per cage with a light cycle of 12 hours (08:00- 20:00). The animals were allowed to acclimatize for a minimum of 14 days prior to the experimental procedures. Monkeys had ad libitum access to food (Jiangsu Xietong Biotechnology Co., Ltd, China) and water for the duration of the experiment and vegetables and fruit treats were provided according to the standard protocol. Per group, three monkeys received a single intravenous slow bolus injection in the brachiocephalic vein of 45 mg / kg. Dosing of the test antibodies was performed in a staggered manner, one cynomolgus monkey of each group received the test item followed by sampling and observation. The remaining animals were only dosed the next day. Blood was collected from the cephalic, saphenous or femoral vein at different time points during the 36 day- study. Hematology, clinical chemistry and coagulation analyses were performed by Pharmalegacy Laboratories, as well as monitoring of body weight and temperature. Drug serum levels (PK) and serum IgA and IgG levels (PD) were evaluated on every bleeding timepoint. Readouts were performed using in house developed PK and PD assays; nonqualified assays were used for the PK and IgA PD, while a qualified assay was used for IgG PD.

[0432] 3.2.3 Pharmacokinetic analysis using ELISA

[0433] Pharmacokinetic analysis of clone F-hlgG1 HN, clone F-hlgG1 LALA HN and clone F- h IgG 1 LALA ABDEG was performed using an anti-human Fab that specifically recognizes HN mutations H433K / N434F. For coating of the microtiter plate, 50 pL recombinant anti- HN Fab clone 14H11 (in house produced in PBS) was added to a Maxisorp 96-well Flatbottom (ThermoScientific; cat#442404) and incubated overnight at 4°C. Plates were washed three times with 300 pL PBS-0.05% Tween20 (PBS-T) (Merck Millipore, cat#8.22184.0500) using the Biotek 405 TS Microplate Washer (Biotek). Plates were blocked for 2 hours shaking (450 rpm) at 22°C (temperature controlled incubator) with 200 pL PBS-1% casein.

[0434] A calibration curve (12 points) and three QC-samples (low QC, medium QC and high QC) of the 3 analyzed anti-lgA antibodies were made in 100% cynomolgus monkey serum (inhouse pooled; batch 20210831). For every analysis, frozen QC samples in 100% cynomolgus monkey serum were used, while the calibration curve was prepared fresh. QC samples, calibration curve and serum samples were all diluted to MRD10 (minimal required dilution) using PBS-0.1% casein -and prepared in a separate 500 pl Deepwell plate (Sigma Eppendorf®; cat# EP0030503104). Serum samples were diluted in 10% cynomolgus serum in PBS-0.1% casein. Calibration curve, QCs and samples were incubated 30 minutes at room temperature whilst shaking at 450 rpm before adding to the plate. All dilutions were done in a separate 500 pl Deepwell plate (Sigma Eppendorf®).

[0435] After washing the blocked plate three times with 300 pL PBS-T, 50 pL of the calibration curve, QC samples and samples was applied in duplicate to the plate and allowed to bind for 1 hour at 22°C whilst shaking at 450 rpm. Next, the plate was washed three times with 300 pL PBS-T and incubated for 1 hour at room temperature (450 rpm shaking) with 50 pL of biotinylated anti-HN Fab clone 13G08 (in house produced in PBS). After washing five times with 300 pL PBS-T, detection was done using a 100,000 fold dilution of Streptavidin- HRP (BD biosciences; cat#554066) in PBS-0.1% casein, shaking for 30 minutes at room temperature at 450 rpm. Lastly, the plate was washed five times with 300 pl PBS-T and 50 pL TMB solution (Merck Millipore; cat#CL07-3135989) was applied for 10 minutes, protected from direct light on an orbital shaker (450 rpm). The incubation of TMB was stopped with 50 pl 0.5M H2SO4 (Chemlab; cat#CL052615) and the absorption (450nm ref. 620nm) was measured with a Tecan Infinite Nano Microplate Reader (Tecan; cat# 30190087).

[0436] 3.2.4 Pharmacodynamic analysis for cvnomolgus monkey IgA using MSP ELISA Streptavidin pre-coated microtiter plates (MSD GOLD 96-well Streptavidin SECTOR Plate -MSD; cat#L15SA-1 ; l°Z0021733) were blocked for 1 hour shaking (450 rpm) at room temperature with 150 pL PBS-1% casein. Plates were washed three times with 300 pL PBS-0.05% Tween20 (PBS-T) (Merck Millipore, cat#8.22184.0500) using the Biotek 405 TS Microplate Washer (Biotek) and biotinylated goat anti-human serum IgA a chain specific F(ab')2 (in house tagged from Jackson; cat#109-006-01 1 ; l°154259) was captured for 1 hour, shaking (450 rpm) at room temperature.

[0437] A freshly prepared calibration curve (12 points) and two buffer QC-samples (low QC and high QC) of cynomolgus monkey IgA (life diagnostics, Inc.; cat#30002-4; l°C-D0721 A) were made in PBS-0.1 % casein . For every analysis, an additional endogenous QC, diluted to MQC levels was prepared by diluting a frozen in-house pooled aliquot of cynomolgus monkey serum 100,000 fold (in-house pooled; batch 20210831 ). Samples were prepared as a 50,000 fold dilution in PBS-0.1% casein for every time point of analysis. All dilutions were made in a separate 500 pl Deepwell plate (Sigma Eppendorf®; cat# EP0030503104).

[0438] After washing the captured plate three times with 300 pL PBS-T, 50 pL of the calibration curve, QCs and samples was applied in duplicate to the plate and allowed to bind for 1 hour at room temperature whilst shaking the plate at 450 rpm. Next, the plate was washed three times with 300 pL PBS-T and detection of the cyno IgA was done using 50 pL Sulfotagged goat anti-human serum IgA a chain specific F(ab')2 (in-house tagged from Jackson; cat#109-006-011 ; l°154259) diluted in PBS-0.1% casein and shaking for 30 minutes at room temperature at 450 rpm. Lastly, the plate was washed three times with 300 pl PBS-T and 150 pL MSD read buffer T (2x) with Surfactant (MSD; cat# R92TC-1 ; l° Y0140375) was applied for 5 minutes and measured using a MSD MESO Quickplex SQ120 reader (MSD; cat# AI0AA-0).

[0439] 3.2.5 Pharmacodynamic analysis for cvnomolgus monkey IgG using ELISA

[0440] A qualified assay was performed for determining the cynomolgus monkey IgG levels.

[0441] For coating of the microtiter plate, 100 pL recombinant mouse anti-monkey IgG (Southern Biotech; cat#4700-01 ; l°H3418-SH51 ) was added to a Maxisorp 96-well Flat-bottom (ThermoScientific; cat#442404) and incubated overnight at 4°C. Plates were washed three times with 300 pL PBS-0.05% Tween20 (PBS-T) (Merck Millipore, cat#8.22184.0500) using the Biotek 405 TS Microplate Washer (Biotek). Plates were blocked for 2 hours shaking (450 rpm) at 22°C (temperature-controlled incubator) with 250 pL PBS-1% casein. Buffers were equilibrated 30-60 min to room temperature before use.

[0442] A calibration curve (11 points) and two buffer QC-samples (low QC and high QC) of cynomolgus monkey IgG, protein A purified (MyBiosource; cat#MBS135162; l°CY-GF-816), were made in PBS-0.1% casein. For every analysis, frozen QC samples were used, while the calibration curve was prepared fresh. Additionally, an endogenous QC sample in duplicate diluted to MQC level was freshly prepared by diluting cynomolgus monkey serum (in-house pooled; batch 20210804). Serum samples were diluted in PBS-0.1% casein for every time point of analysis. All dilutions were made in a separate 2000 pl Deepwell plate (Sigma Eppendorf®; cat# EP0030504305).

[0443] After washing the blocked plate three times with 300 pL PBS-T, 100 pL of the calibration curve, QCs and samples was applied in duplicate to the plate and allowed to bind for 2 hours at 22°C whilst shaking at 450 rpm. Next, the plate was washed five times with 300 pL PBS-T and the plate was incubated for 1 hour at 22°C (450 rpm shaking) with 100 pL of the diluted detection antibody mouse anti-monkey IgG HRP (Southern Biotech; cat#4700- 05; l°H3418-YG59D) in PBS-0,1% casein. Lastly, the plate was washed five times with 300 pl PBS-T and 100 pL TMB solution (acclimatized to room temperature for 20 minutes) (Merck Millipore; cat#CL07-3135989) was applied for 15 minutes, protected from direct light on an orbital shaker (450 rpm). The incubation of TMB was stopped with 100 pl 0.5M H2SO4 (Chemlab; cat#CL052615) and the absorption (450nm ref. 620nm) was measured with a Tecan Infinite Nano Microplate Reader (Tecan; cat# 30190087). between one-armed and two-armed formats

[0444] This example demonstrates that one-armed (OA) anti- IgA antibodies with variant Fc regions having increased binding affinity to FcRn exhibit improved IgA sweeping as compared to conventional two-armed (TA) antibodies.

[0445] 4.1 OA and TA anti-loA constructs tested

[0446] To further explore anti-lgA sweeping antibodies, a series of one-armed (OA) and twoarmed (TA) antibody formats were tested in the context of anti-lgA antibodies having the clone A Fab region (a derivative of clone F having S53K and D98E substitutions). These antibodies comprised different mutations in the Fc regions as shown in Figure 6.

[0447] OA and TA ABDEG backbones

[0448] From the results obtained in the cynomolgus monkey study comparing different Fc variants that all had the same Fab (clone F), the highest level of IgA sweeping was achieved with clone F-h IgG 1 LALA ABDEG. This variant had the deepest reduction in IgA levels as well as for the longest duration (see Example 3).

[0449] To further explore the sweeping capacity of the ABDEG™ mutations (M252Y / S254T / T256E / H433K / N434F), clone A-LALA ABDEG™ was produced in a TA or an OA format using the knob-into-hole technology (Knob VH-T366W and Hole VH- T366S / L368A / Y407V) (Atwell et al. 1997) - Figure 6.

[0450] As the ABDEG™ molecule relies on FcRn for active cellular uptake and recycling of the antibody, it was hypothesised that a OA molecule might result in less steric hindrance for binding to FcRn as antibodies bind FcRn in an upside-down orientation (Pyzik et al. 2019). By reducing the steric hindrance, it was hypothesised that improved FcRn binding could be obtained and result in improved IgA sweeping. However, with a OA format the maximal size of immune complexes (IC) formed with the target IgA would be limited to dimeric and trimeric lgA:anti- IgA complexes, which could adversely affect the IgA sweeping efficacy by limiting the number of IgA molecules that can be internalized per recycling round. Additionally, more stringent mutations to avoid Fc effector functions were explored for the anti-lg A molecules since it was observed in an in vitro assay that LALA mutations (L234A / L235A) did not completely abrogate FcyR binding and affected the blocking capacity of the molecule. Therefore, a PG mutation (P329G) was added in addition to the LALA mutations in the Fc domain because this additional mutation has been reported to almost completely block residual binding to activating FcyR (Schlothauer et al. 2016).

[0451] OA and TA NHance backbones

[0452] Whilst ABDEG™ mutations exhibited sufficient IgA sweeping, IgG levels were also affected in the circulation (see Example 3). Other Fc engineering strategies designed to improve the IgA sweeping efficacy were also considered. In particular, Fc variants comprising h IgG 1 LALA HN were also explored because this variant Fc did not impact circulating IgG levels (see Example 3). Mutations to modify the surface charge or isoelectric point (pl) were also implemented in the variant Fc, thereby introducing positive patches in the C-terminus of the Fc to promote the uptake of an antigen bound antibody into the cells. As the cell membrane is negatively charged, positively charged patches on the surface of the Fc domain were hypothesised to promote cellular uptake by electrostatic attraction. The following mutations to increase the pl of the anti-lg A molecule were therefore attempted in the h IgG 1 LALA HN Fc domains:

[0453] - pl2 (D401 R / D413K),

[0454] - pl4 (Q311 R / S400R / D413K)

[0455] - Singlepl (D413K). pl2 and pl4 mutations showed the highest net charge increase (Table 9), compared to the single pl mutation that only introduces one amino acid mutation that is shared between pl2 and pl4. Additionally, pl engineering was also combined with a one-armed modified antibody format using the knob-into-hole technology (Figure 6).

[0456] Table 9: Theoretical pl of the different Fc engineered variants of clone A

[0457] 4.2 Screening of Fc variants using clone A Fab

[0458] 4.2.1 lgA:CD89 blocking capacity of clone A Fc variants

[0459] One of the modes of action of the anti-lgA antibodies described herein is their capacity to block the interaction of IgA with its receptor FcaRI (CD89). To evaluate the effect of Fc engineering on the blocking capacity, a competition and displacement ELISA and a cellbased competition assay using flow cytometry were performed.

[0460] Both the competition and displacement ELISA showed differences in the blocking capacity of the TA and OA antibody formats. Generally, OA anti-lgA variants showed reduced competing and displacing properties, which was expected due to the presence of only one Fab arm that can block binding of IgA to CD89 (EC50 values of the OA molecules should be interpreted as approximations because the bottom plateau was not always reached). Between the different TA Fc variants, no difference was observed in the competing or displacing capacities as expected (since EC50 values were similar). For the OA variants, minor shifts in the dose-response curve could be observed upon inclusion of all pl mutations. There was no clear explanation for this observation and it is possible that the pl of the molecule affects the ELISA readout (Table 10).

[0461] Table 10: EC50 values (in nM) of the hlgA:hCD89 competition and displacement properties of the different clone A-hlgG1 Fc variants. EC50 values were determined using four parameter fitting in GraphPad. EC50 values with * are approximations.

[0462] To confirm the difference in lgA:CD89 blocking capacity between OA and TA formats, an in vitro competition assay using rat basophilic leukemia (RBL) cells stably transfected with CD89 was used. In this assay, blocking of complexed IgA (RSV-F:Mota-hlgA) was evaluated - in contrast to monomeric IgA used in the ELISA setup described above. It is well known that monomeric IgA binds CD89 with low affinity (Ka of ± 106M-1), while IgA-immune complexes bind with high avidity and can crosslink the receptor (Wines et al. 1999; Wines et al. 2001). Similar to the ELISA results, reduced blocking was observed for the OA formats compared to TA formats (e.g. EC50 LALA ABDEG OA: 18.37 nM; EC50 LALA ABDEG TA: 2.71 nM). Fc variants with pl mutations reduced blocking capacity both for TA and OA antibody formats (Table 11).

[0463] Table 11 : EC50 values (in nM) of the hlgA:hCD89 competition properties of the different clone A-hlgG1 Fc variants on RBL-hCD89-GFP cells.

[0464] EC50 values were determined using four parameter fitting in GraphPad, but could not be determined for the LALA HN pl4 / pl2 OA format due to lack of curve bottom plateau.

[0465] However, upon additional analysis in RBL WT cells, aspecific binding of OA pl-engineered variants could be observed at high anti-lg A concentrations (Figure 7). It is known that introduction of positive charged patches (pl engineering) in the mAbs can lead to an increased aspecific cellular binding (and uptake), thereby masking the lgA:CD89 blocking capacity of a molecule. However, as the aspecific cell binding seemed to be more pronounced for OA formats compared to TA formats, interference of the detecting antibody with the different formats cannot be excluded.

[0466] 4.3 Characterization of FcRn-mediated cellular uptake of clone A Fc variants

[0467] In addition to pH-dependent antigen binding, active cellular uptake via Fc receptors can improve the sweeping efficacy of an antibody.

[0468] Increasing the affinity for FcRn is one way to enhance active cellular uptake. To test the hypothesis that OA antibody formats experience less steric hindrance for binding to FcRn, an FcRn occupancy assay on U937 cells was performed using flow cytometry. ARGX113 and ARGXdG (AG236) were included as positive controls - 2 Fc fragments containing ABDEG mutations and deletion of G236 in the latter variant to obstruct FcyR binding. As expected, ARGX113 had the highest FcRn occupancy (EC50 3.21 nM) followed by ARGXdG (EC50 5.56 nM), confirming the involvement of FcyR’s for better FcRn occupancy. When comparing the OA format and TA format of clone A-LALA ABDEG™, better hFcRn occupancy was observed for the OA format (EC50 19.09 nM) than for the TA format (EC50:67.19 nM) (Figure 8).

[0469] In addition, to evaluate the effect of enhanced FcRn binding and pl engineering of the antiIgA molecules on their internalisation properties, a dedicated assay in HEK-FcRn and HEK WT cells was performed using flow cytometry. In this assay, IgA was labelled with pHrodoGreen, which only fluoresces in acidic conditions as obtained in the endosome and lysosome. It was hypothesised that pl engineering could result in improved internalization due to the electrostatic attraction of the positively charged Fc to the negatively charged cell membrane (Hori et al. 2022).

[0470] In HEK-FcRn cells, more efficient IgA internalization was observed with TA formats as compared to OA formats, irrespective of the FcRn / pl mutations that were introduced in the Fc backbone. One possible explanation for this finding is that a OA format can only form dimeric or trimeric complexes with IgA, whilst a TA format can theoretically form large sized complexes, therefore internalization of a larger complex would result in a higher MFI signal due to the presence of a greater number of labelled IgA molecules. When including pl mutations into the Fc backbone, internalization was greatly improved for the pl2 and pl4 variants, but only to a limited extent for the single pl variant. However, in the control HEK- WT cells, to assess FcRn-independent internalisation, a large increase in IgA internalisation was observed for the pl2 and pl4 TA variants, indicating non-specific cell internalisation. Also for the OA formats containing pl2 or pl4 mutations, a similar internalisation signal was observed in HEK-FcRn and HEK-WT cells, again suggesting non-specific (non FcRn-dependent) cell internalisation. Only for the LALA HN Singlepl variant more internalization was seen in HEK-FcRn compared to HEK WT cells (Figure 9).

[0471] To test the probability of FcRn degradation with the anti-lgA molecules (alone and in complex with human IgA), an FcRn degradation assay using HEK-FcRn-GFP cells was performed using flow cytometry. Upon addition of anti-lgA molecules alone, no FcRn degradation could be observed for ABDEG™ containing variants, however addition of pl2 or pl4 mutations in an HN containing Fc backbone resulted in significant FcRn degradation independently of the format (OA or TA). Interestingly, no FcRn degradation was observed using the Singlepl variant, which has a milder pl effect. In contrast, complexed with human Mota-IgA all antibodies showed significant FcRn degradation and no difference could be observed between OA and TA antibody formats (Figures 10A and 10B). Only the LALA HN Singlepl variant demonstrated limited FcRn degradation in complex with its target (Figures 10A and 10B). The controls used in this assay behaved as expected; the positive control immunovant (IMVT) resulted in ±80% FcRn degradation, while the negative controls, IgA alone and untreated cells, demonstrated similar FcRn levels (Figures 10 and 10B).

[0472] 4.4 In vivo IQA sweeoino efficacy of clone A Fc variants usino Albumus™ mice

[0473] To better understand the sweeping efficacy of the different Fc engineering strategies, two in vivo studies were performed in Albumus™ mice, which express human FcRn and human albumin.

[0474] In the first study, comparison was made between the h IgG 1 LALA(PG) ABDEG TA and OA formats and the best internalizing pl-engineered variant; LALA HN pl2, in TA and OA formats. All anti-lgA molecules containing ABDEG™ mutations demonstrated a comparable PK profile, independent of the format (i.e TA or OA) (Figure 11 ). In contrast, pl-engineered variants (clone A-LALA HN pl2 TA and clone A- LALA HN pl2 OA) were detected at much lower concentrations than the expected Cmax serum concentration of a 10 mg / kg dose. This can be explained by the increased tissue distribution and increased (aspecific) cellular uptake as a consequence of the increase in pl (Figure 11 B).

[0475] Looking at the sweeping efficacy of the tested Fc variants, all clones, except clone A-hlgG1 LALA HN pl2 TA, cleared human IgA within 48 hours after the first injection but much faster IgA removal was observed for the OA formats as compared to the equivalent TA formats (Figure 11 A).

[0476] Upon comparison of ABDEG-containing and pl-engineered variants, superior IgA sweeping efficacy was observed for the ABDEG-containing variants. Upon reinjection of IgA (at Day 2), the differences between variants were even more pronounced and clearly showed that clone A-hlgG1 LALA ABDEG OA is the most effective sweeping anti-lgA Fc format (Figure 11 A).

[0477] In the second in vivo study, the effect of pl engineering was further explored, comparing the LALA HN pl2 (OA and TA) with the LALA HN Singlepl TA variant. LALA ABDEG TA and LALA HN TA were also included as Fc controls. Again, a steady PK profile was observed for LALA ABDEG TA, while LALA HN TA and LALA HN Singlepl TA were detected at lower concentrations than the theoretical Cmax serum concentration with an IP dosing of 10 mg / kg. In addition, the pl2 engineered variants were only detected at a concentration of ±1 pg / mL 2 hours post-mAb administration, but with the ti / 2 staying constant (Figure 12B).

[0478] Similar to the cynomolgus monkey study results (Example 3), improved sweeping was observed with LALA ABDEG TA compared to LALA HN TA, especially after re-injection of human Mota-IgA where almost no sweeping was observed with the latter. The effect of pl engineering on the LALA HN TA variants was minor, and improved IgA clearance only upon the second injection of human Mota-IgA. In addition, no distinction could be made between the different TA pl variants, single pl and pl2. In contrast, improved IgA clearance was only observed with a OA format of clone A-LALA HN pl2, having a comparable sweeping efficacy as clone A-LALA ABDEG from first injection onwards (Figure 12A).

[0479] To summarise the above findings, greater IgA sweeping was observed for the OA formats as compared to their equivalent TA formats. This observation applied to all Fc backbone variants tested (i.e. LALA ABDEG™ or LALA HN (with p 12 or pl4 substitutions)). Overall, the variant Fc regions comprising LALA ABDEG™ substitutions exhibited better IgA sweeping than Fc regions engineered with LALA HN substitutions.

[0480] 4.5 Materials and methods

[0481] 4.5.1 Human CD89 competition and displacement ELISA

[0482] For the competition ELISA, a 96-well half area high binding microplate (Greiner; cat#675061 ) was coated with recombinant human CD89, His-tag (Elabscience; cat#PKSH031620). After washing and blocking the plate, a dilution series of the anti-lgA antibodies in 0.1% casein in PBS (in house), pre-incubated with a fixed concentration of human serum IgA (Abeam; cat#ab91025) was added to the plate and allowed to bind for 1 hour at room temperature (shaking). Binding was detected with goat anti-human IgA, HRP conjugated (Southern Biotech; cat#1040-05) and developed with s(SH)TMB (SDT Reagents for life; cat#sTMB) for 5 minutes. OD was measured at 450 nm (ref 620 nm) with a Tecan Sunrise Microplate Reader (Tecan).

[0483] The same set-up was used for the displacement ELISA, except the IgA incubation step was performed for 1 hour (room temperature, shaking 450 rpm) before adding the dilution series of the anti-lgA antibodies, with a washing step in between.

[0484] 4.5.2 CD89 competition flow cytometry assay using RBL-CD89-GFP cells

[0485] RBL cells are an adherent rat basophilic leukemia cell line and maintained in DMEM Glutamax (Life Technologies; cat#31966047) supplemented with 10% fetal calf serum (Sigma; cat#F7524) and 1% Penicillin / Streptomycin (Sigma; cat#G7513). In this assay, RBL-WT cells or RBL cells stably transfected with human CD89-GFP were incubated with immune complexes of RSV-F (Respiratory syncytial virus fusion glycoprotein; IPA; project2021_043; batch#5131) and human Mota-IgA (IPA; project2020_222; batch#4549) . RBL-WT cells and RBL-huCD89-GFP cells were detached using Trypsin (Sigma; cat#T3924) and seeded in a 96-well V-Bottom microplate (Falcon; cat#353263). Medium was removed from the cells by a centrifugation step and the cell pellet was resuspended in a mixture of equal volumes of immune complexed human Mota-IgA (with RSV-F) with a 1 / 2 dilution series of an antibody, starting from 50 pg / mL to 97.6 ng / mL. The cells were incubated for 1 hour at 4°C, shaking at 450 rpm, followed by two washing steps with 100 pL FACS buffer (1x PBS, 0.1% BSA, 2mM EDTA; PBS - Sigma; cat#T3924, BSA - Sigma; cat#A8412, EDTA - Life Technologies; cat#15575-038). IgA on the cells was stained for 30 minutes at 4°C (shaking 450 rpm) with a mix of Goat Anti-Human IgA-PE (Southern Biotech; cat#2050-09); Fixable Viability Dye eFluor 780 (eBioscience, cat#65-0865-18) and Purified Mouse Anti-Rat CD32 (rat Fc block) (Pharmigen, cat#550270). Afterwards, cells were washed once with 150 pL and resuspended in 200 pL FACS buffer prior to readout on FACS Fortessa. Data analysis was done using FlowJo (V10.5.3).

[0486] 4.5.3 FcRn occupancy flow cytometry assay using U937 cells

[0487] U937 cells were seeded in a 96-well V-Bottom microplate (Falcon; cat#353263). A 7-step dilution range of the anti- IgA antibodies was prepared. Medium was removed from the cells by centrifugation and antibodies were added to the cells for 2 hours at 37°C, 5% CO2, shaking 450 rpm. After incubation, the plate was washed 2 times by adding 150 pL FACS buffer at pH6 followed and centrifugation. After this, viability staining was performed using the eBioscience Fixable Viability Dye eFluor 506 antibody (Thermo Scientific; cat# 65- 0866-18). Before fixation and permeabilization of the cells, 2 additional washing steps were performed with FACS buffer at pH6. For fixation and permeabilization, cells were incubated for 30 minutes in 100 pL acidic Fix / Perm buffer (pH6.0; eBioscience Fix / Perm concentrate - Thermo Scientific; cat#00-5123-43 (1 :4) in Fix / Perm diluent - Thermo Scientific; cat#00- 5223-56). After 2 washes with 150 pL acidic permeabilization buffer (pH6.0;

[0488] Permeabilization buffer - Thermo Scientific; cat#00-8333-56 (1 :10) in milliQ), FcRn staining was performed using a fluorescently labelled anti-FcRn Fab fragment recognizing IgG binding site on FcRn and Fc block (Human BD Fc Block, unlabeled; BD; cat#564220) in acidic permeabilization buffer. After staining, the plate was washedwith FACS buffer pH6.0 and read-out was performed on FACS Fortessa. Data analysis was done using FlowJo (V10.5.3).

[0489] 4.5.4 FcRn degradation flow cytometry assay using HEK-FcRn-GFP or LI937 cells The hFcRn degradation assay was done by flow cytometry using a HEK cell line expressing hFcRn-GFP. These HEK cells are an adherent human embryonic kidney cell line and maintained in growth buffer DMEM (Life Technologies; cat#31966047) supplemented with 10% fetal calf serum (Sigma; cat#F7524), 1% L-glutamine (Sigma; cat#G7513) and 1% Penicillin / Streptomycin (Sigma; cat#G7513). The cells were rinsed with PBS (Sigma; cat#D8537) and detached with trypsin for 3 minutes at 37°C (Sigma; cat#T3924). HEK-FcRn-GFP cells were seeded at in a 24-well microplate (Costar; cat#3526) and incubated overnight at 37°C, 5% CO2. Antibodies were diluted alone or with human Mota-IgA (IPA; project2020_222; batch#4549). Growth medium was removed from the cells and the cells were washed 2 times after which the antibody pre-mixes were added to the cells ON. After incubation, the supernatant was removed and cells were harvested using Trypsin (Sigma; cat#T3924). The trypsin-neutralized solution was transferred into a 96-well V-Bottom microplate (Falcon; cat#353263) and centrifuged. The supernatant was discarded and the cell pellet was stained with viability dye (eBioscience Fixable Viability Dye eFluor™780 antibody - Thermo Scientific; cat# 65-0865-14). Afterwards, cells were washed and resuspended in FACS buffer prior to readout on FACS Fortessa. Data analysis was done using FlowJo (V10.5.3).

[0490] 4.5.5 FcRn-mediated IgA internalization flow cytometry assay using HEK cells Human Mota-IgA (IPA; project2020_222; batch#4549) was labeled with pHrodo Green using the pHrodo iFL Green Microscale Protein labeling kit (Invitrogen; cat#P36015). Before seeding, HEK-FcRn cells were rinsed with PBS (Sigma; cat#D8537) and detached using cell dissociation buffer (Sigma; cat#C5789). HEK-FcRn and HEK-WT cells were seeded in a 96-well V-Bottom microplate (Falcon; cat#353263). Antibodies and pHrodo Green-Mota-hlgA were diluted in plain DMEM medium and pre-incubated at room temperature. Medium was removed from the cells by a centrifugation step and the cell pellet was resuspended in the antibody mix and incubated at 37°C. After centrifugation, cells were stained with a mix containing Fixable Viability Dye eFluor 780 (eBioscience, cat#65-0865-18)and Human BD Fc Block BD Pharmingen (BD, cat#564220). Finally, cells were washed and resuspended in FACS buffer prior to readout on FACS Fortessa. Data analysis was done using FlowJo (V10.5.3).

[0491] 4.5.6 Cvnomolgus monkey and human CD89 competition and displacement MSP

[0492] To evaluate the cynomolgus monkey cross-reactivity of the lgA:CD89 blocking capacity of the anti- IgA antibodies, an MSD ELISA was developed. An MSD GOLD 96-well Streptavidin SECTOR plate (MSD, cat#L15SA-1) was blocked with 1% BLOK Casein-PBS, washed with 1xPBS+0.05%Tween20 and captured with BIOTIN-human CD89 (in house biotinylated) or BIOTIN-cynomolgus monkey CD89 (in house biotinylated) in 0.1% Casein- PBS for 1 hour at room temperature. For the competition MSD assay, a 1 / 5 dilution series of anti-lg A antibodies was prepared with a fixed concentration of human IgA (Abeam, cat#ab91025) or cynomolgus monkey IgA (Life diagnostics; cat#3002-4) in 0.1% Casein- PBS and were pre-incubated at room temperature (shaking 450 rpm). After washing the plate, the mixture of IgA and anti-lg A antibodies was added to the plate and allowed to bind for 1 hour at room temperature (shaking 450 rpm). On the other hand, for the displacement assay, the fixed concentration of IgA was first added to the plate for 1 hour at room temperature (shaking 450 rpm) after which a washing step was performed and the 1 / 5 antiIgA dilution series was added for 30 minutes at room temperature (shaking 450 rpm).

[0493] After washing the plate, IgA was detected with a SULFO-Goat anti-human serum IgA a chain specific F(ab')2 (Sulfolabelled Fab; in house tagged - Jackson #109-006-011) and developed with Read buffer 2x (MSD, cat#R92TC-1) to be measured directly using an MSD Quickplex reader (MSD, ser#1300150206431 ).

[0494] 4.5.7 Albumus™ mice studies

[0495] 4.5.7.1 Antibodies

[0496] The antibodies (also referred to as test items) included in the study all share the same Fab ‘clone A’ but differ in Fc format and Fc engineering. All test items are two-armed antibodies except for clone A-LALA ABDEG OA and clone A-PI2 LALA HN OA, which are both one- armed antibodies illustrated with ‘OA’ in their compound name. For Fc engineering, different mutations were included and combined; LALA (L234A / L235A) and LALAPG (L234A / L235A / P329G) abolish immune effector functions, HN (H433K / N434F) and ABDEG (M252Y / S254T / T256E / H433K / N434F) increase FcRn affinity at acidic and physiological pH respectively and PI2 (D401 R / D413K) are mutations to introduce positive charged patches in the Fc and thereby increase the pl of the test item.

[0497] 4.5.7.2 IP injection of IQA and anti-loA antibodies in Albumus™ mice

[0498] 10-week old Albumus™ mice were allowed to acclimatize for at least 14 days. Mice were kept in a specific pathogen free (SPF) animal facility in individually ventilated cages with a light cycle of 12 hours (at VIB, room W218). Mice had ad libitum access to food and water. All animal experiments were performed according to institutional, national and European guidelines and were approved by the ethical committee.

[0499] For this experiment four male Albumus™ mice per group received a mix of intravenous immunoglobulin ( I VIG, 200mg / kg) intraperitoneally 3 days prior to injection of the test items (day - 3). On day 0, the mice received a single intraperitoneal (IP) injection of Mota-IgA (dose of 20 mg / kg) followed by an intraperitoneal injection of the test item (dose of 10 mg / kg) 2 hours later. A re-injection of human Mota-IgA was done on day 2.

[0500] All animals were pre-weighted before dosing and dosed according to their body weights.

[0501] For bleeding, mice were kept in a warming cabinet in order to dilate the blood vessels and increase the blood flow. Care was taken to avoid hyperthermia and dehydration. Blood samples were taken by tail snip and capillary blood collection using microvettes® with clotting activator. No more than 20 pL of blood (except for terminal bleeding) was taken per bleeding. Mice were terminated at the end of the study by inhalation euthanasia, with carbon dioxide (CO2). Test item and IgA levels were evaluated on every bleeding timepoint and readouts were performed using in house developed assays.

[0502] 4.5.7.3 Pharmacodynamic sample analysis for human IgA using ELISA

[0503] For coating of high binding half area plates (Greiner Bio-One #675061), 50 pL of AffiniPure F(ab')2Fragment Goat Anti-Human Serum IgA, achain specific antibody (Jackson ImmunoResearch #109-006-011 ) was added and incubated overnight at 4°C. Plates were washed three times with 250 pL PBS-0.05% Tween20 (PBS-T) (Merck Millipore, #8.22184) using the Biotek 405 TS Microplate Washer (Biotek) and blocked for 1 hour with 150 pL PBS-0.5% BSA (Sigma-Aldrich, #A4503).

[0504] A calibration curve (12 points) and three QC-samples (low QC, medium QC and high QC) of human Mota-IgA (UPX, 2020_222 batch 4549) were prepared. Samples were diluted using 0.1% BLOK casein (G-biosciences, #786-194). All dilutions were prepared in a 500 pL Deepwell plate (Sigma Eppendorf®; cat# EP0030503104).

[0505] After washing the blocked plate three times with 250 pL PBS-T, 50 pL of calibration curve, QC samples and samples was applied in duplicate to the plate and allowed to bind for 1 hour. Next, the plate was washed three times with 250 pL PBS-T and incubated with 50 pL F(ab')2 goat anti-human IgA secondary antibody, HRP (Invitrogen, #A24458; 1 / 4000) for 1 hour. Finally, the plate was washed three times with 250 pL PBS-T and 50 pL of TMB solution (s(HS)TMB, Stereospecific Detection Technologies #sTMB) was applied for 6 minutes, protected from direct light. The incubation of TMB was stopped with 50 pL 0.5M H2SO4 (Chemlab #052615) and the absorption (450nm ref. 620nm) was measured with a Tecan Infinite 200 PRO (Tecan, #30190087).

[0506] 4.5.7.4 Pharmacokinetic sample analysis of human IgG 1 antibodies with ABDEG™ or NHance® mutations using 14H11 / 13G08 ELISA

[0507] For coating of high binding half area plates (Greiner Bio-One #675061), 50 pL recombinant anti-HN Fab clone 14H11 (in house produced in PBS) was added and incubated overnight at 4°C. Plates were washed three times with 300 pL PBS-0.05% Tween20 (PBS-T) (Merck Millipore, cat#8.22184.0500) using the Biotek 405 TS Microplate Washer (Biotek). Plates were blocked for 1 hour shaking (450 rpm) at 22°C (temperature controlled incubator) with 200 pL PBS-1% casein (G-biosciences; #786-194). A calibration curve (12 points) and three QC-samples (low QC, medium QC and high QC) of the analyzed anti-lg A antibodies were made in 100% C57BI6 mouse serum (BIO-IVT; #MSE01SRMPNN). QC samples, calibration curve and serum samples were all diluted using PBS-0.1% casein (G- biosciences) and prepared in a separate 500 pL Deepwell plate (Sigma Eppendorf®; cat# EP0030503104). Serum samples were further diluted. Calibration curve, QCs and samples were incubated 30 minutes at room temperature whilst shaking at 450 rpm before adding to the plate. All dilutions were done in a separate 500 pL Deepwell plate (Sigma Eppendorf®; cat# EP0030503104).

[0508] After washing the blocked plate three times with 300 pL PBS-T, 50 pL of the calibration curve, QC samples and samples was applied in duplicate to the plate and allowed to bind for 1 hour at 22°C whilst shaking at 450 rpm. Next, the plate was washed three times with 300 pL PBS-T and incubated for 1 hour at room temperature (450 rpm shaking) with 50 pL of biotinylated anti-HN Fab clone 13G08 (in house produced in PBS). After washing five times with 300 pL PBS-T, detection was done using Streptavidin-HRP (BDbiosciences; cat#554066) in PBS-0.1% casein (Biorad), shaking for 30 minutes at room temperature at 450 rpm. Lastly, the plate was washed five times with 300 pL PBS-T and 50 pL TMB solution (Merck Millipore; cat#CL07-3135989) was applied for 10 minutes, protected from direct light on an orbital shaker (450 rpm). The incubation of TMB was stopped with 50 pL 0.5M H2SO4 (Chemlab; cat#CL052615) and the absorption (450nm ref. 620nm) was measured with a Tecan Infinite Nano Microplate Reader (Tecan; cat# 30190087). References

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Claims

CLAIMS1 . A monovalent antigen-binding molecule comprising:- an antigen-binding domain that binds to IgA; and- a variant Fc region or a FcRn binding fragment thereof, that binds to human FcRn with increased affinity relative to a wild-type Fc region; and wherein the variant Fc region comprises a first Fc domain and a second Fc domain.

2. The monovalent antigen-binding molecule according to claim 1 , wherein the antigen-binding molecule exhibits lower IgA binding affinity at an acidic pH than at a neutral pH.

3. The monovalent antigen-binding molecule according to claim 2, wherein the human IgA binding affinity at pH 6 is reduced by at least 25%, preferably at least 50%, as compared with the human IgA binding affinity at pH 7.4.

4. The monovalent antigen-binding molecule according to claim 2 or claim 3, wherein the human IgA binding affinity at pH 5 is reduced by at least 75%, preferably 100%, as compared with the human IgA binding affinity at pH 7.4.

5. The monovalent antigen-binding molecule according to any one of claims 1-4, wherein the antigen-binding molecule inhibits binding of IgA to an IgA receptor.

6. The monovalent antigen-binding molecule according to claim 5, wherein the IgA receptor is FcaRI (CD89) or CD71 .

7. The monovalent antigen-binding molecule according to any one of claims 1-6, wherein the antigen-binding molecule displaces IgA from an IgA receptor.

8. The monovalent antigen-binding molecule according to claim 7, wherein the antigen-binding molecule displaces IgA from a FcaRI (CD89) receptor.

9. The monovalent antigen-binding molecule according to any one of claims 1-8, wherein the antigen-binding molecule inhibits or reduces IgA immune complex formation.

10. The monovalent antigen-binding molecule according to any one of claims 1-9, wherein the antigen-binding domain is selected from: a Fab; an Fv; a scFv; and a VHH domain.11 . The monovalent antigen-binding molecule according to claim 10, wherein the antigen-binding domain is a Fab.

12. The monovalent antigen-binding molecule according to any one of claims 1-11 , wherein the antigen-binding domain is attached to the N-terminus of either the first Fc domain or the second Fc domain.

13. The monovalent antigen-binding molecule according to any one of claims 1-12, wherein the antigen-binding domain is a Fab and the C-terminus of the Fab heavy chain is attached to the N-terminus of either the first Fc domain or the second Fc domain via an IgG hinge region.

14. The monovalent antigen-binding molecule according to any one of claims 1-13, wherein the variant Fc region or FcRn binding fragment thereof binds to FcRn with increased affinity relative to a wild-type IgG Fc region.

15. The monovalent antigen-binding molecule according to any one of claims 1-14, wherein the variant Fc region or FcRn binding fragment thereof binds to human FcRn with increased affinity relative to a wild-type human IgG Fc region, preferably a wild-type human lgG1 Fc region.

16. The monovalent antigen-binding molecule according to any one of claims 1 -15, wherein the variant Fc region or FcRn binding fragment thereof binds to human FcRn with increased affinity at pH 6.0 and pH 7.4.

17. The monovalent antigen-binding molecule according to any one of claims 1 -16, wherein the binding affinity of the variant Fc region or FcRn binding fragment thereof for human FcRn at pH 6.0 is increased by at least 20x, preferably at least 30x, relative to a wild-type human lgG1 Fc region.

18. The monovalent antigen-binding molecule according to any one of claims 1 -17, wherein the binding affinity of the variant Fc region or FcRn binding fragment thereof for human FcRn at pH 6.0 is stronger than KD 15 nM.

19. The monovalent antigen-binding molecule according to any one of claims 1 -18, wherein the binding affinity of the variant Fc region or FcRn binding fragment thereof for human FcRn at pH 7.4 is stronger than KD 320 nM.

20. The monovalent antigen-binding molecule according to any one of claims 1 -19, wherein the variant Fc region or FcRn binding fragment thereof comprises at least one amino acid substitution as compared with the corresponding wild-type Fc region, wherein the at least one amino acid substitution confers increased binding affinity relative to a wildtype Fc region.21 . The monovalent antigen-binding molecule according to any one of claims 1-20, wherein the variant Fc region or FcRn binding fragment thereof is a variant human Fc region or FcRn binding fragment thereof.

22. The monovalent antigen-binding molecule according to any one of claims 1 -21 , wherein the variant Fc region or FcRn binding fragment thereof is a variant IgG Fc region or FcRn binding fragment thereof.

23. The monovalent antigen-binding molecule according to any one of claims 1-22, wherein the variant Fc region or FcRn binding fragment thereof is a variant IgG 1 Fc region or FcRn binding fragment thereof.

24. The monovalent antigen-binding molecule according to any one of claims 1-23, wherein the variant Fc region or FcRn binding fragment thereof comprises the amino acids:(i) Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively; or(ii) Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively.

25. The monovalent antigen-binding molecule according to any one of claims 1-24, wherein the first Fc domain or second Fc domain comprises the amino acids:(i) Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively; or(ii) Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively.

26. The monovalent antigen-binding molecule according to any one of claims 1-25, wherein the first Fc domain and the second Fc domain comprise the amino acids:(i) Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively; or(ii) Y, T, E, K, F and Y at EU positions 252, 254, 256, 433, 434 and 436, respectively.

27. The monovalent antigen-binding molecule according to any one of claims 20-26, wherein the variant Fc region or the FcRn binding fragment thereof comprise at least one additional amino acid substitution as compared with the corresponding wild-type Fc region; and wherein the at least one additional substitution reduces or eliminates Fc effector function.

28. The monovalent antigen-binding molecule according to any one of claims 20-27, wherein the first Fc domain and / or second Fc domain comprise the amino acids:(i) A and A at EU positions 234, 235; and optionally(ii) G at EU position 329.

29. The monovalent antigen-binding molecule according to any one of claims 20-28, wherein the first Fc domain and the second Fc domain comprise the amino acids A, A, G, Y, T, E, K, F and Y at EU positions 234, 235, 329, 252, 254, 256, 433, 434 and 436, respectively.

30. The monovalent antigen-binding molecule according to any one of claims 20-29, wherein the variant Fc region or the FcRn binding fragment thereof comprise at least one additional amino acid substitution as compared with the corresponding wild-type Fc region; and wherein the at least one substitution promotes dimerisation between the first Fc domain and the second Fc domain.31 . The monovalent antigen-binding molecule according to claim 30, wherein the first Fc domain and the second Fc domain comprise knob-into-holes amino acid substitutions.

32. The monovalent antigen-binding molecule according to any one of claims 20-31 , wherein the first Fc domain comprises the amino acid W at EU position 366; and the second Fc domain comprises the amino acids S, A and V at EU positions 366, 368 and 407, respectively.

33. The monovalent antigen-binding molecule according to any one of claims 20-32, wherein the first Fc domain comprises the amino acids A, A, G, Y, T, E, W, K, F and Y at EU positions 234, 235, 329, 252, 254, 256, 366, 433, 434 and 436, respectively; and the second Fc domain comprises the amino acids A, A, G, Y, T, E, K, F, Y, S, A and V at EU positions 234, 235, 329, 252, 254, 256, 433, 434, 436, 366, 368 and 407, respectively.

34. The monovalent antigen-binding molecule according to claim 33, wherein the antigen-binding domain is a Fab attached to the first Fc domain.

35. The monovalent antigen-binding molecule according to any one of claims 1-34, wherein the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 and / or the second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34.

36. The monovalent antigen-binding molecule according to any one of claims 1-35, wherein the first Fc domain and the second Fc domain do not comprise an N-linked glycan at EU position 297.

37. The monovalent antigen-binding molecule according to any one of claims 1-35, wherein the first Fc domain and second Fc domain comprise an afucosylated N-linked glycan at EU position 297.

38. The monovalent antigen-binding molecule according to any one of claims 1-35, wherein the first Fc domain and second Fc domain comprise an N-linked glycan having a bisecting GIcNac at EU position 297 of the Fc domains.

39. The monovalent antigen-binding molecule according to any one of claims 1-38, wherein the monovalent antigen-binding molecule is a humanised or germlined variant of a non-human antibody.

40. The monovalent antigen-binding molecule according to claim 39, wherein the non- human antibody is camelid-derived.41 . The monovalent antigen-binding molecule according to any one of claims 1-40, wherein the antigen-binding molecule is a modified IgG antibody having only one Fab arm.

42. The monovalent antigen-binding molecule according to claim 41 , wherein the modified IgG antibody is a modified lgG1 antibody.

43. The monovalent antigen-binding molecule according to any one of claims 1-42, wherein the monovalent antigen-binding molecule binds to membrane-bound IgA or free IgA.

44. An isolated polynucleotide or polynucleotides, which encode the monovalent antigen-binding molecule of any one of claims 1 -43.

45. An expression vector comprising the polynucleotide or polynucleotides of claim 44 operably linked to regulatory sequences which permit expression of the monovalent antigen-binding molecule.

46. A host cell or cell-free expression system containing the expression vector of claim 45.

47. A method of producing a recombinant monovalent antigen-binding molecule which comprises culturing the host cell or cell free expression system of claim 46 under conditions which permit expression of the monovalent antigen-binding molecule and recovering the expressed monovalent antigen-binding molecule.

48. A pharmaceutical composition comprising a monovalent antigen-binding molecule according to any one of claims 1 -43 and at least one pharmaceutically acceptable carrier or excipient.

49. A monovalent antigen-binding molecule according to any one of claims 1 -43 or a pharmaceutical composition according to claim 48 for use as a medicament.

50. A method of treating a disorder in a subject, wherein the method comprises administering to a patient in need thereof a therapeutically effective amount of a monovalent antigen-binding molecule according to any one of claims 1 -43 or a pharmaceutical composition according to claim 48.51 . The method of claim 50, wherein the disorder is an IgA-mediated disorder.

52. The method of claim 50 or claim 51 , wherein the disorder is an IgA autoantibody- mediated disorder.

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