Antibody

Abstract

The invention relates to antibodies, and specifically to anti-amyloid-beta (Aβ) antibodies. The invention extends to compositions comprising the antibodies, including pharmaceutical compositions and kits. The invention also extends to methods of making and using the antibodies, for example in therapy and diagnosis of Aβ-mediated 5 conditions, such as Alzheimer's disease.

Description

[0001] Antibody

[0002] The present invention relates to antibodies, and specifically to anti-amyloid-beta (Ag) antibodies. The invention extends to compositions comprising the antibodies, including pharmaceutical compositions and kits. The invention also extends to methods of making and using the antibodies, for example in therapy and diagnosis of Ap-mediated conditions, such as Alzheimer's disease.

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterised by the accumulation of misfolded and aggregated proteins in the brain. Many AD drug discovery programs focus on extracellular deposits of amyloid-beta (Ag) as a therapeutic target, and passive immunotherapy using monoclonal antibodies against aggregated or fibrillar Ap has been one of the most developed and successful methods to treat AD. The recent FDA approval of three full effector hlgGl Ap-targeting antibodies (Aducanumab, Lecanemab and Donanumab) for patients with mild cognitive impairment or early Alzheimer's disease has ended decades of failure and heralds the start of a potential new era of drugs to clear abnormally folded proteins.

[0004] In July / August 2024, the UK Medicines and Healthcare product Regulatory Agency approved Lecanemab and Donanemab for use in people with early Alzheimer's Disease, but the National Institute for Health and Care Excellence (NICE) determined that these therapeutic antibodies were not cost-effective due to cost of additional monitoring for adverse effects. The European Medicines Agency (EMA) also refused the marketing approval for Lecanemab based on its assessment of the same data, stating that the benefits of the drug did not outweigh its potential harms. Indeed, despite initially promising results, widespread approval and success rate of these amyloid clearing antibodies has been hampered by vascular and inflammatory adverse effects, with up to 30% of patients affected.

[0005] Removal of aggregated amyloid from the brain relies on interaction with Fey receptors expressed on microglia. Engaging Fey receptors on microglia promotes phagocytosis of antibody-bound amyloid, but this is accompanied by inflammatory adverse effects, as mentioned above. An effective way to reduce the inflammatory effects is to remove glycans or amino acid substitutions in the Fc domain of the antibody (e.g., N297A in the CH2 domain), but this significantly reduces the capacity to clear amyloid.

[0006] Accordingly, it has not been possible to produce an antibody which is immune-specific for amyloid-beta, and is able to clear amyloid plaques, but without the negative vascular and inflammatory side effects.There is, therefore, a need to produce improved antibodies which bind to amyloidbeta, and which do not result in adverse side effects.

[0007] The inventors conducted a series of Fc-engineering experiments on antibodies to improve the efficacy of amyloid immunotherapy, and have synthesised an improved therapeutic anti-amyloid-beta (AP) antibody that effectively removes amyloid-beta aggregates and plaques from AD-affected mouse brains, but with reduced adverse effects. The amyloid specific antibodies selectively bind to targets Fc gamma R1 (FcyRI), and have been shown to effectively bind to, and remove, amyloid-beta aggregates through phagocytosis, while exhibiting reduced vascular and inflammatory adverse effects. Accordingly, these FcyRI-selective antibodies are expected to be an improvement on known anti-amyloid-beta (AP) antibodies which are not FcyRI-selective.

[0008] Thus, in a first aspect of the invention, there is provided an anti-amyloid beta antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an Fc region, which selectively targets Fc gamma RI (FcyRI).

[0009] As described in the examples, to test if Fc-engineering could be used to influence the capacity of antibodies to clear amyloid, the inventors generated a panel of humanized IgGl anti-Ap antibodies, based on the parent (wild-type) antibody, "h3D6 / Bapineuzumab", and including an aglycosylated form of the parent ("agly hlgGl 3D6"), and also FcyRI-selective Fc variants ("agly-Fc5 hlgGl 3D6"). The ability of these Fc variants to clear Ap plaques and induce inflammation was assessed in vivo using an experimental model of AD and in vitro using the THP-1 human macrophage cell line. The results revealed that the FcyRI-selective (Fc5) variants of Bapineuzumab (Fc5-Bapi) is as efficient in clearing antibody-bound amyloid as the wild-type variant. However, advantageously, the inventors also showed that human IgGl-bapi-Fc5 has surprisingly reduced inflammatory effects compared to the parent clone, hlgGl-Bapi. These results provide the first pro of- of- co nee pt data that Fc engineering can lead to optimized anti-amyloid immunotherapy by reducing adverse effects.

[0010] It will be appreciated that there are two main final forms of amyloid beta (AP), namely AP40 and AP42. It will be appreciated that AP40 comprises 40 amino acids, and that AP42 comprises 42 amino acids.In one embodiment, the amino acid sequence of human amyloid-beta (AP) may be provided herein as SEQ ID No: 1, as follows:

[0011] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAI IGLMVGGVVIA

[0012] [SEQ ID No: 1]

[0013] It will be appreciated that the amino acid sequence of human amyloid-beta (AP) as provided in SEQ ID No: 1 represents AP42, and comprises 42 residues.

[0014] In one embodiment, the anti-amyloid beta antibody may bind to the N-terminus of AP42.

[0015] In another embodiment, the amino acid sequence of human amyloid-beta (AP) may be provided herein as SEQ ID No: 23, as follows:

[0016] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAI IGLMVGGVV

[0017] [SEQ ID No: 23]

[0018] It will be appreciated that the amino acid sequence of human amyloid-beta (AP) as provided in SEQ ID No: 23 represents AP40, and comprises 40 residues.

[0019] Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 1, or a variant or fragment thereof, and / or SEQ ID No: 23, or a variant or fragment thereof.

[0020] In one embodiment, the antibody or antigen-binding fragment thereof may bind to an epitope comprising or consisting of amino acid residues 1-4 of SEQ ID No: 1 and / or SEQ ID No: 23, which may be provided herein as SEQ ID No: 2, as follows:

[0021] DAEF

[0022] [SEQ ID No: 2]

[0023] Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof. The epitope as provided in SEQ ID No: 2 represents the epitope to which the antibody or antigenbinding fragment thereof binds when the antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Bapineuzumab.In another embodiment, the antibody or antigen-binding fragment thereof may bind to an epitope comprising or consisting of amino acid residues 3-7 of SEQ ID No: 1 and / or SEQ ID No: 23, which may be provided herein as SEQ ID No: 24, as follows:

[0024] EFRH

[0025] [SEQ ID No: 24]

[0026] Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 24, or a variant or fragment thereof. The epitope as provided in SEQ ID No: 24 represents the epitope to which the antibody or antigenbinding fragment thereof binds when the antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Aducanumab.

[0027] In yet another embodiment, the antibody or antigen-binding fragment thereof may bind to an epitope comprising or consisting of amino acid residues 1-17 of SEQ ID No: 1 and / or SEQ ID No: 23, which may be provided herein as SEQ ID No: 25, as follows:

[0028] DAEFRHDSGYEVHHQKL

[0029] [SEQ ID No: 25]

[0030] Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 25, or a variant or fragment thereof. The epitope as provided in SEQ ID No: 25 represents the epitope to which the antibody or antigenbinding fragment thereof binds when the antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Lecanemab.

[0031] In yet another embodiment, the antibody or antigen-binding fragment thereof may bind to an epitope comprising or consisting of amino acid residues 4-7 of SEQ ID No: 1 and / or SEQ ID No: 23, which may be provided herein as SEQ ID No: 26, as follows:

[0032] FRHD

[0033] [SEQ ID No: 26]

[0034] Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 26, or a variant or fragment thereof. The epitopeas provided in SEQ ID No: 26 represents the epitope to which the antibody or antigenbinding fragment thereof binds when the antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Donanemab.

[0035] The epitope may be linear or conformational. The term "linear epitope" can mean an epitope comprising or consisting of amino acid residues that form a sequence together in the primary sequence of the protein antigen, i.e., sequential or continuous epitope. The linear epitope may be present on folded beta amyloid.

[0036] The term "conformational epitope" can mean an epitope consisting of amino acid residues, at least some of which are separated from others in the primary sequence of the protein antigen, but which together assemble in the 3D structure and are recognised by an antibody, i.e., discontinuous or non-sequential epitope.

[0037] The invention extends to both whole antibodies (i.e., immunoglobulins) with immunospecificity for beta amyloid, as well as to antigen-binding fragments or regions of the corresponding full-length antibody, provided that the fragment selectively targets Fc gamma R1 (FcyRI). As such, any reference to an antibody fragment herein will include only fragments which selectively target Fc gamma R1 (FcyRI)

[0038] The antibody or antigen-binding fragment thereof may be monovalent, divalent or polyvalent. Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulphide bridge with a light chain (L). Divalent antibodies are tetramer (H2L2) comprising two dimers associated by at least one disulphide bridge. Polyvalent antibodies may also be produced, for example by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences in the remainder of the chain. The variable region includes several hypervariable regions, or Complementarity Determining Regions (CDRs), that form the antigen-binding site of the antibody molecule and determine its specificity for the antigen, i.e., beta amyloid, or variant or fragment thereof (e.g., an epitope). On either side of the CDRs of the heavy and light chains is a framework region, a relatively conserved sequence of amino acids that anchors and orients the CDRs. Antibody fragments may include a bi-specific antibody (BsAb).The heavy chain constant region typically comprises three domains, CHI, CH2, and CHS. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0039] Each heavy chain and light chain generally comprise three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 -CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[0040] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 6, e, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. In one embodiment, the antibody or antigen-binding fragment thereof is of the IgG antibody class. In a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment thereof is of the IgGl antibody class, most typically human IgGl. IgGl typically improves the stability of the antibody or antigen-binding fragment thereof.

[0041] In some embodiments, the antibody or antigen binding fragment thereof is thermally stable. In some embodiments, the antibody or antigen binding fragment thereof has a Tml of greater than 65°C, 66°C, 67°C, 68°C, or 69°C. In some embodiments, the antibody or antigen binding fragment thereof has a Tml of greater than 70°C, 71°C, 72°C, or 73°C.

[0042] The light chain from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain.

[0043] The constant region consists of one of five heavy chain sequences (p, y,

[0044]

[0045] a, or e) and one of two light chain sequences (K or A). The heavy chain constant region sequences determine the isotype of the antibody and the effector functions of the molecule.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof is isolated or purified.In one embodiment, the antibody or antigen-binding fragment thereof comprises a polyclonal antibody, or an antigen-binding fragment thereof. The antibody or antigenbinding fragment thereof may be generated in a rabbit, mouse, or rat.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof is obtained by immunising a host animal with a beta-amyloid protein, or a variant or fragment thereof, and then collecting the antibody or antigen-binding fragment thereof. The host animal may be a rabbit.

[0048] In another embodiment, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof. The antibody or fragment thereof of may be mammalian. In one embodiment, the antibody of the invention is a human antibody. As used herein, the term "human antibody" can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity for beta amyloid, or a variant or fragment thereof. An amino acid sequence, which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence identity when compared to a reference sequence. Such identity is definitively known or recognisable as representing the amino acid sequence of the particular human antibody. Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids. Such a human antibody or fragment thereof maintains its function of selectively binding to beta amyloid, or a variant or fragment thereof.

[0049] The term "human monoclonal antibody" can include a monoclonal antibody with substantially or entirely human CDR amino acid sequences produced, for example by recombinant methods, such as production by a phage library, by lymphocytes or by hybridoma cells.

[0050] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool ofhybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (by so-called "affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0051] The term "humanised antibody" can mean an antibody from a non-human species (e.g., mouse or rabbit) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.

[0052] The antibody may be a recombinant antibody. The term "recombinant human antibody" can include a human antibody produced using recombinant DNA technology.

[0053] The term "antigen-binding fragment" can mean a region of the antibody having specific binding affinity for its target antigen, i.e., beta amyloid or a variant or fragment thereof. In one embodiment, the fragment is an epitope. The epitope may be linear or conformational. The antigen-binding region may be a hypervariable CDR or a functional portion thereof. The term "functional portion" of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen. The functional portion of a CDR may comprise a ligand which specifically binds to beta amyloid, or a fragment thereof. It will be appreciated, however, that in all embodiments, the antigen-binding fragment selectively targets Fc gamma RI (FcyRI).

[0054] The term "CDR" can mean a hypervariable region in the heavy and light variable chains. There may be one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e., the three-dimensional combining site with which the antigen binds or specifically reacts. It has, however, been postulated that there may be four CDRs in the heavy chains of some antibodies.

[0055] The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.The amino acid sequence boundaries of a CDR can be determined by using any of a number of known numbering schemes, including those described by Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme).

[0056] The term "functional fragment" of an antibody can mean a portion of the antibody which retains a functional activity. A functional activity can be, for example antigen binding activity or specificity. A functional activity can also be, for example, an effector function provided by an antibody constant region. In this regard, it is important that the functional fragment retains selectivity for targeting Fc gamma R1 (FcyRI). The term "functional fragment" is also intended to include, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art. Human monoclonal antibody functional fragments include, for example individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single chain Fv (scFv); and Fc fragments. Alternatively, as discussed hereinafter, and as exemplified, the Fc fragment of the antibody may be modified by introducing amino acid substitutions into the Fc region, which result in it selectively targeting Fc gamma RI (FcyRI).

[0057] The term "VL fragment" can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs. A VL fragment can further include light chain constant region sequences.

[0058] The term "VH fragment" can mean a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.

[0059] The term "Fd fragment" can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e., VH and CH-1. The "Fd fragment" does not include the light chain, or the second and third constant regions of the heavy chain.

[0060] The term "Fv fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy andlight chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about 110 amino acids of both the heavy and light chains.

[0061] The term "Fab fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains. Thus, a Fab fragment additionally includes, for example, amino acid residues from about 110 to about 220 of the heavy and light chains.

[0062] The term "Fab' fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.

[0063] The term "F(ab')2 fragment" can mean a bivalent antigen-binding fragment of a human monoclonal antibody. An F(ab')2 fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

[0064] The term "single chain Fv (scFv)" can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.

[0065] The term "bispecific antibody (BsAb)" can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.

[0066] One skilled in the art knows that the exact boundaries of a fragment of an antibody are not important, so long as the fragment maintains a functional activity. Using well-known recombinant methods, one skilled in the art can engineer a polynucleotide sequence to express a functional fragment with any endpoints desired for a particular application. A functional fragment of the antibody may comprise or consist of a fragment with substantially the same heavy and light chain variable regions as the human antibody.In one embodiment, the antibody or antigen-binding fragment thereof, with respect to the first aspect of the invention, is immunospecific for an epitope within beta amyloid. The antigen-binding fragment thereof may comprise or consist of any of the fragments selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F (ab')2 and Fc fragment.

[0067] The antigen-binding fragment thereof may be a single domain antibody (sdAb), otherwise referred to as a nanobody, which the skilled person would understand is an antibody fragment consisting of a single monomeric variable antibody domain.

[0068] The antigen-binding fragment thereof may comprise or consist of any one of the antigen binding region sequences of the VL, any one of the antigen binding region sequences of the VH, or a combination of VL and VH antigen binding regions of a human antibody. The appropriate number and combination of VH and VL antigen binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the antigen-binding fragment. Functional fragments or antigen-binding fragments of antibodies may be readily produced and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods and chemical synthesis. Proteolytic methods for the isolation of functional fragments comprise using human antibodies as a starting material. Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin. The appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required. For example, papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc fragment. Pepsin cleavage, for example, results in a bivalent F (ab') fragment. An F(ab')2 fragment of the invention may be further reduced using, for example, DTT or 2-mercaptoethanol to produce two monovalent Fab' fragments.

[0069] Functional or antigen-binding fragments of antibodies produced by proteolysis may be purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments may be removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.

[0070] The antibody or antigen-binding fragment thereof may be produced by recombinant methodology. In one embodiment, one initially isolates a polynucleotide encodingdesired regions of the antibody heavy and light chains. Such regions may include, for example, all or part of the variable region of the heavy and light chains. In one embodiment, such regions can particularly include the antigen binding regions of the heavy and light chains, optionally the antigen binding sites, and optionally the CDRs.

[0071] The polynucleotide encoding the antibody or antigen-binding fragment thereof according to the invention may be produced using methods known to those skilled in the art. The polynucleotide encoding the antibody or antigen-binding fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art. Alternatively, smaller fragments may be synthesized and joined to form a larger functional fragment using recombinant methods known in the art.

[0072] As used herein, the term "immunospecificity" can mean the binding region of the antibody or antigen-binding fragment thereof is capable of immunoreacting with beta amyloid, or a variant or fragment thereof, by specifically binding therewith. The antibody or antigen-binding fragment thereof can selectively interact with an antigen (beta amyloid) with an affinity constant of approximately 10'4to 10'13M’1, optionally 10'6to 10'9M’1, or optionally, 10'10to 10'12M’1.

[0073] The term "immunoreact" can mean the binding region is capable of eliciting an immune response upon binding with beta amyloid, or an epitope thereof.

[0074] The term "epitope" can mean any region of an antigen with the ability to elicit, and combine with, a binding region of the antibody or antigen-binding fragment thereof. The epitope may be linear. This can mean that the antibody interacts with a plurality of continuous amino acids of the antigen, and so the epitope can consist of these defined amino acids. Alternatively, the epitope may be conformational, i.e., non-linear or discontinuous. This can mean that the antibody interacts with multiple, distinct segments from the primary amino acid sequence of the antigen.

[0075] Thus, the antibody or antigen-binding fragment thereof may comprise a heavy chain. The heavy chain may be selected from the group consisting of IgA; IgD; IgE; IgG and IgM. In one embodiment, the heavy chain is an IgG. In a typical embodiment, the heavy chain may be an IgGl. However, the heavy chain may be an IgG2. The heavy chain may be an IgG3. The heavy chain may be an IgG4.

[0076] As described in Example 1, the inventors produced a panel of antibodies comprising amyloid-beta-binding CDRs derived from currently available monoclonals.In a first embodiment, therefore, the anti-amyloid beta antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Bapineuzumab (Bapi). It will be appreciated that Bapineuzumab is an anti-Ap antibody. It will also be appreciated that Bapineuzumab is a humanised version of the murine antibody 3D6 expressed by the hybridoma deposited under ATCC under No. PTA-5130. Bapineuzumab is described in US8613920B2.

[0077] In a second embodiment, the anti-amyloid beta antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Aducanumab. It will be appreciated that Aducanumab is an anti-Ap antibody. It will also be appreciated that Aducanumab is a fully human antibody comprising a glycosylated human IgGl heavy chain and a human kappa light chain. Aducanumab is described in US20240043513A1.

[0078] In a third embodiment, the anti-amyloid beta antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Lecanemab. It will be appreciated that Lecanemab is an anti-Ap antibody. It will also be appreciated that Lecanemab is a humanised IgGl monoclonal version of mAbl58, which is a murine monoclonal antibody raised to target protofibrils, as disclosed in WO 2007 / 108756.

[0079] In a fourth embodiment, the anti-amyloid beta antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody, Donanumab. It will be appreciated that Donanumab is an anti-N3pGlu AP antibody. It will also be appreciated that Donanumab is a human engineered anti-N3pGlu AP antibody. Donanumab is described in US8679498B2.

[0080] The VH and VL chains of three of the above amyloid beta-binding antibodies, namely Bapineuzumab, Aducanumab, and Lecanemab, were combined with the constant regions of either mouse mIgG2a or human hlgGl. It will be appreciated, however, that the inventors envisage combining the VH and VL chains of any amyloid betabinding antibody, including Donanumab, with the constant regions of either mouse mTgG2a or human hlgGl. It will be appreciated that the below exemplars provide a robust proof of concept for combining the VH and VL chains of amyloid beta-binding antibodies in general with the constant regions of either mouse mTgG2a or human hlgGl. Table 1 provides the VH and VL sequences for exemplars Bapineuzumab, Aducanumab, and Lecanemab, and Table 2 provides the constant region sequences forthe human IgGl CH and mouse IgG2a chains. Each of these antibodies are defined below in detail.

[0081] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as h3D6 / Bapineuzumab. The parent (wild-type) antibody called "h3D6 / Bapineuzumab" may be produced by subcloning the DNA encoding the variable regions of both heavy and light chains from Table 1 into expression vectors which contained mouse or human IgG constant regions shown in Table 2 via Hindlll / Spel (heavy chain) and Hindlll / BsiWI (light chain).

[0082] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof:

[0083] NYGMS

[0084] [SEQ ID No: 3]

[0085] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 3 represents residues 30-35 of SEQ ID No: 6.

[0086] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof.

[0087] SIRSGGGRTYYSDNVKG

[0088] [SEQ ID No: 4]

[0089] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 4 represents residues 50-66 of SEQ ID No: 6.

[0090] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof.

[0091] YDHYSGSSDY

[0092] [SEQ ID No: 5]

[0093] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 5 represents residues 99-108 of SEQ ID No: 6.In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 3, a CDR-H2 domain comprising or consisting of SEQ ID No: 4 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 5.

[0094] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 6, or a variant or fragment thereof.

[0095] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0096] [SEQ ID No: 6]

[0097] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 7, or a variant or fragment thereof.

[0098] KSSQSLLDSDGKTYLN

[0099] [SEQ ID No: 7]

[0100] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 7 represents residues 24-39 of SEQ ID No: 10.

[0101] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 8, or a variant or fragment thereof.

[0102] LVSKLDS

[0103] [SEQ ID No: 8]

[0104] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 8 represents residues 55-61 of SEQ ID No: 10.

[0105] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 9, or a variant or fragment thereof.WQGTHFPRT

[0106] [SEQ ID No: 9]

[0107] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 9 represents residues 94-102 of SEQ ID No: 10.

[0108] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 7, a CDR-L2 domain comprising or consisting of SEQ ID No: 8, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 9.

[0109] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 10, or a variant or fragment thereof.

[0110] DWMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKI.DSGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0111] [SEQ ID No: 10]

[0112] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0113] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 3, a CDR-H2 domain comprising or consisting of SEQ ID No: 4, a CDR-H3 domain comprising or consisting of SEQ ID No: 5, a CDR-L1 domain comprising or consisting of SEQ ID No: 7, a CDR-L2 domain comprising or consisting of SEQ ID No: 8, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 9.

[0114] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 6, and a light chain variable region comprising or consisting of SEQ ID No: 10.

[0115] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (CH) region (which may be human IgGl CH region), comprising or consisting of a sequence as substantially set out in SEQ ID No: 11, or a variant or fragment thereof.QVQLVQSGGG WQPGRSLRL SCAASGFTFS RYTIHWVRQA PGKGLEWVAV MSYNGNNKHY ADSVNGRFTI SRNDSKNTLY LNMNSLRPED TAVYYCARIR DTAMFFAHWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCWVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0116] [SEQ ID No: 11]

[0117] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (CH) region (which may be mouse IgG2a CH region), comprising or consisting of a sequence as substantially set out in SEQ ID No: 12, or a variant or fragment thereof.

[0118] AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV TSSTWPSQSI TCNVAHPASS TKVDKKIEPR GPTIKPCPPC KCPAPNLLGG PSVFIFPPKI KDVLMISLSP IVTCVWDVS EDDPDVQISW FVNNVEVHTA QTQTHREDYN STLRVVSALP IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE MTKKQVTLTC MVTDFMPEDI YVEWTNNGKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW VERNSYSCSV VHEGLHNHHT TKSFSRTPGK

[0119] [SEQ ID No: 12]

[0120] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (CL) region (which may be human IgGl CL region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 13, or a variant or fragment thereof.

[0121] RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

[0122] [SEQ ID No: 13]

[0123] In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (CL) region (which may be mouse IgG2a CL region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 14, or a variant or fragment thereof.

[0124] RTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYER HNSYTCEATHKTSTSPIVKSFNRNEC

[0125] [SEQ ID No: 14]

[0126] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11 or 12, and a light chain constant region comprising or consisting of SEQ ID No: 13 or 14.Typically, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11, and a light chain constant region comprising or consisting of SEQ ID No: 13.

[0127] It will be appreciated that instead of h3D6 / Bapineuzumab, the antibody or antigenbinding fragment thereof may comprise beta amyloid-binding CDRs derived from Aducanumab. In this embodiment, the antibody or antigen-binding fragment thereof of the invention may again be produced by subcloning the DNA encoding the variable regions of both heavy and light chains from Table 1 into expression vectors which contained mouse or human IgG constant regions shown in Table 2 via Hindlll / Spel (heavy chain) and Hindlll / BsiWI (light chain).

[0128] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 15, or a variant or fragment thereof:

[0129] SYGMH

[0130] [SEQ ID No: 15]

[0131] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 15 represents residues 31-35 of SEQ ID No: 18.

[0132] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 16, or a variant or fragment thereof.

[0133] VIWFDGTKKYYTDSVKG

[0134] [SEQ ID No: 16]

[0135] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 16 represents residues 50-66 of SEQ ID No: 18.

[0136] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 17, or a variant or fragment thereof.

[0137] DRGIGARRGPYYMDV

[0138] [SEQ ID No: 17]It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 17 represents residues 99-113 of SEQ ID No: 18.

[0139] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 15, a CDR-H2 domain comprising or consisting of SEQ ID No: 16 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 17.

[0140] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 18, or a variant or fragment thereof.

[0141] QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYL QMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0142] [SEQ ID No: 18]

[0143] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 19, or a variant or fragment thereof.

[0144] RASQSISSYLN

[0145] [SEQ ID No: 19]

[0146] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 19 represents residues 24-34 of SEQ ID No: 22.

[0147] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 20, or a variant or fragment thereof.

[0148] AASSLQS

[0149] [SEQ ID No: 20]

[0150] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 20 represents residues 50-56 of SEQ ID No: 22.In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 21, or a variant or fragment thereof.

[0151] QQSYSTPLT

[0152] [SEQ ID No: 21]

[0153] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 21 represents residues 89-97 of SEQ ID No: 22.

[0154] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 19, a CDR-L2 domain comprising or consisting of SEQ ID No: 20, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 21.

[0155] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 22, or a variant or fragment thereof.

[0156] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0157] [SEQ ID No: 22]

[0158] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0159] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 15, a CDR-H2 domain comprising or consisting of SEQ ID No: 16, a CDR-H3 domain comprising or consisting of SEQ ID No: 17, a CDR-L1 domain comprising or consisting of SEQ ID No: 19, a CDR-L2 domain comprising or consisting of SEQ ID No: 20, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 21.

[0160] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 18, and / or a light chain variable region comprising or consisting of SEQ ID No: 22.In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (CH) region (which may be human IgGl CH region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 11, or a variant or fragment thereof.

[0161] QVQLVQSGGG WQPGRSLRL SCAASGFTFS RYTIHWVRQA PGKGLEWVAV MSYNGNNKHY ADSVNGRFTI SRNDSKNTLY LNMNSLRPED TAVYYCARIR DTAMFFAHWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCWVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0162] [SEQ ID No: 11]

[0163] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (CH) region (which may be mouse IgG2a CH region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 12, or a variant or fragment thereof.

[0164] AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV TSSTWPSQSI TCNVAHPASS TKVDKKIEPR GPTIKPCPPC KCPAPNLLGG PSVFIFPPKI KDVLMISLSP IVTCVWDVS EDDPDVQISW FVNNVEVHTA QTQTHREDYN STLRVVSALP IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE MTKKQVTLTC MVTDFMPEDI YVEWTNNGKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW VERNSYSCSV VHEGLHNHHT TKSFSRTPGK

[0165] [SEQ ID No: 12]

[0166] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (CL) region (which may be human IgGl CL region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 13, or a variant or fragment thereof.

[0167] RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

[0168] [SEQ ID No: 13]

[0169] In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (CL) region (which may be mouse IgG2a CL region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 14, or a variant or fragment thereof.

[0170] RTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYER HNSYTCEATHKTSTSPIVKSFNRNEC

[0171] [SEQ ID No: 14]In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11 or 12, and a light chain constant region comprising or consisting of SEQ ID No: 13 or 14.

[0172] Typically, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11, and a light chain constant region comprising or consisting of SEQ ID No: 13.

[0173] It will be appreciated that instead of h3D6 / Bapineuzumab or Aducanumab, the antibody or antigen-binding fragment thereof may comprise beta amyloid-binding CDRs derived from Lecanemab. In this embodiment, the antibody or antigen-binding fragment thereof of the invention may again be produced by subcloning the DNA encoding the variable regions of both heavy and light chains described below into expression vectors which contained mouse or human IgG constant regions shown in Table 2 via Hindlll / Spel (heavy chain) and Hindlll / BsiWI (light chain).

[0174] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 27 , or a variant or fragment thereof:

[0175] SFGMH

[0176] [SEQ ID No: 27]

[0177] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 27 represents residues 31-35 of SEQ ID No: 30.

[0178] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 28, or a variant or fragment thereof.

[0179] YISSGSSTI YYGDTVKG

[0180] [SEQ ID No: 28]

[0181] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 28 represents residues 50-66 of SEQ ID No: 30.

[0182] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 29, or a variant or fragment thereof.EGGYYYGRS YYTMDY

[0183] [SEQ ID No: 29]

[0184] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 17 represents residues 99-113 of SEQ ID No: 30.

[0185] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 27 , a CDR-H2 domain comprising or consisting of SEQ ID No: 28 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 29.

[0186] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 30, or a variant or fragment thereof.

[0187] EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNAKNSLFL QMSSLRAEDTAVYYCAREGGYYYGRSYYTMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0188] [SEQ ID No: 30]

[0189] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 31, or a variant or fragment thereof.

[0190] RSSQS IVHSNGNTYLE

[0191] [SEQ ID No: 31]

[0192] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 31 represents residues 24-39 of SEQ ID No: 34.

[0193] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 32, or a variant or fragment thereof.

[0194] KVSNRFS

[0195] [SEQ ID No: 32]It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 32 represents residues 55-61 of SEQ ID No: 34.

[0196] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 33, or a variant or fragment thereof.

[0197] FQGSHVPPT

[0198] [SEQ ID No: 33]

[0199] It will be appreciated that the amino acid sequence as substantially set out in SEQ ID No: 33 represents residues 94-102 of SEQ ID No: 34.

[0200] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 31, a CDR-L2 domain comprising or consisting of SEQ ID No: 32, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 33.

[0201] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 34, or a variant or fragment thereof.

[0202] DWMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRIS RVEAEDVGI YYCFQGSHVPPTFGPGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0203] [SEQ ID No: 34]

[0204] In one embodiment, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0205] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 27 , a CDR-H2 domain comprising or consisting of SEQ ID No: 28, a CDR-H3 domain comprising or consisting of SEQ ID No: 29, a CDR-L1 domain comprising or consisting of SEQ ID No: 31, a CDR-L2 domain comprising or consisting of SEQ ID No: 32, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 33.In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 30, and a light chain variable region comprising or consisting of SEQ ID No: 34.

[0206] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (CH) region (which may be human IgGl CH region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 11, or a variant or fragment thereof.

[0207] QVQLVQSGGG WQPGRSLRL SCAASGFTFS RYTIHWVRQA PGKGLEWVAV MSYNGNNKHY ADSVNGRFTI SRNDSKNTLY LNMNSLRPED TAVYYCARIR DTAMFFAHWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCWVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0208] [SEQ ID No: 11]

[0209] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (CH) region (which may be mouse IgG2a CH region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 12, or a variant or fragment thereof.

[0210] AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV TSSTWPSQSI TCNVAHPASS TKVDKKIEPR GPTIKPCPPC KCPAPNLLGG PSVFIFPPKI KDVLMISLSP IVTCVWDVS EDDPDVQISW FVNNVEVHTA QTQTHREDYN STLRVVSALP IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE MTKKQVTLTC MVTDFMPEDI YVEWTNNGKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW VERNSYSCSV VHEGLHNHHT TKSFSRTPGK

[0211] [SEQ ID No: 12]

[0212] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (CL) region (which may be human IgGl CL region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 13, or a variant or fragment thereof.

[0213] RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

[0214] [SEQ ID No: 13]

[0215] In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (CL) region (which may be mouse IgG2a CL region), comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 14, or a variant or fragment thereof.RTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYER HNSYTCEATHKTSTSPIVKSFNRNEC

[0216] [SEQ ID No: 14]

[0217] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11 or 12, and a light chain constant region comprising or consisting of SEQ ID No: 13 or 14.

[0218] Typically, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11, and a light chain constant region comprising or consisting of SEQ ID No: 13.

[0219] It will be appreciated that the beta-amyloid-binding CDRs may be instead derived from Donanumab, and which can be combined with either of the human or mouse constant regions, typically the human regions, described above.

[0220] In a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment thereof is aglycosylated. As described in the examples, an aglycosylated embodiment of the parent antibody, 3D6 / Bapineuzumab, is referred to as "agly hlgGl 3D6".

[0221] The skilled person will appreciate how to produce an aglycosylated antibody. For example, certain amino acid residues, which are normally glycosylated, may be modified so that they are replaced with an amino acid which may not be conjugated with a glycan to become glycosylated.

[0222] In an embodiment, one such amino acid residue which may be modified may be N297 with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11. Typically, the N297 amino acid residue may be modified to alanine, with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11.

[0223] It will be appreciated that the numbering of mutations in the constant region starts at A-118 (heavy chain) and R-108 (light chain), as different antibodies have different lengths of VH and VL.

[0224] Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises N297A.Known antibodies which bind to beta amyloid comprise an Fc region which either binds to Clq (and so thereby activate the complement system) and / or bind to Fc gamma receptors other than Fc gamma RI (and so therefore activate microglial cells or macrophages), which means that they suffer the problem of adverse vascular and inflammatory effects. This potential of prior art antibodies to bind to other Fc gamma Receptors results in the adverse side effects.

[0225] In contrast, however, the antibody or antigen-binding fragment of the invention selectively targets Fc gamma RI (FcyRI) in preference to Fc gamma RII (FcyRII) / Fc gamma RIIA (FcyRIIA) and Fc gamma RIII (FcyRIII). Starting with the aglycosylated antibody, agly hlgGl 3D6, the inventors then produced a variant antibody called "agly-Fc5 hlgGl 3D6", which had a modified Fc region.

[0226] It will be appreciated that Fc gamma RIIA (FcyRIIA) is the activatory FcyRII receptor in human.

[0227] As described in the Examples, the inventors have surprisingly demonstrated that the antibodies and antigen-binding fragments referred to herein, are able to significantly target beta amyloid, while also selectively targeting, i.e., binding, Fc gamma RI (FcyRI).

[0228] In an embodiment, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 0.1 nM and and 500 nM, between 0.5 nM and 450 nM, or between 1 nM and 400 nM. Typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 1.5 nM and 300 nM, between 2 nM and 200 nM, or between 2.5 nM and 100 nM. More typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 3 nM and 50 nM, between 3.5 nM and 25nM, or between 4 nM and 15 nM. Yet more typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 4.5 nM and 10 nM, between 5 nM and 9 nM, between 5.1 nM and 8 nM, between 5.2 nM and 7 nM, or between 5.3 nM and 6 nM. Most typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 5.6 nM and 5.7 nM.In another embodiment, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 1 nM and 5 nM, between 2 nM and 4 nM, or between 2.5 nM and 3.5 nM. Typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 3.1 nM and 3.2 nM.

[0229] In another embodiment, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 10 nM and 50 nM, between 20 nM and 40 nM, or between 25 nM and 35 nM. Typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) may be between 29 nM and 33 nM.

[0230] In an embodiment, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RII (FcyRII) may be greater than 10 nM, 50 nM, or 100 nM. Typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RII (FcyRII) may be greater than 250 nM, 500 nM, or 1000 nM. More typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RII (FcyRII) may be greater than 5 pM, 10 pM, or 50 pM.

[0231] In an embodiment, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RIII (FcyRIII) may be greater than 10 nM, 50 nM, or 100 nM. Typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RIII (FcyRIII) may be greater than 250 nM, 500 nM, or 1000 nM. More typically, the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RIII (FcyRITI) may be greater than 5 pM, 10 pM, or 50 pM.

[0232] In a typical embodiment, the antibody or antigen-binding fragment thereof comprises a modified E382 residue and / or a modified M428 residue, with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11.Typically, the E382 amino acid residue may be modified to valine. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises E382V.

[0233] Typically, the M428 amino acid residue may be modified to isoleucine. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises M428I.

[0234] Thus, in one embodiment, the antibody or antigen-binding fragment thereof comprises a modified N297 residue, a modified E382 residue, and a modified M428 residue.

[0235] In one embodiment, the antibody or antigen-binding fragment thereof comprises N297A, E382V, and M428I.

[0236] The inventors have also surprisingly discovered that further Fc mutations result in yet higher FcyRI binding affinity. For example, the inventors generated FcyRI-selective Fc variants containing the amino acid substitutions N297A, Q295R, L328W, P331A, I332Y, E382V, and M428L, known as Fc701. Indeed, the inventors have surprisingly discovered that an aglycosylated Fc variant ("Fc701") with the mutations Q295R, L328W, P331A, I332Y, E382V, and M428L results in 120-fold improved binding capacity to FcyRI when compared to wild type aglycosylated IgG, and excellent FcyRI selectivity and no binding to FcyRIIb. Furthermore, the inventors have discovered that said Fc mutations result in higher pH-dependent FcRn binding compared to wild type aglycosylated IgGl. In addition, the inventors have surprisingly discovered that these amino acid substitutions improve selectivity for FcyRI and extend half-life in vivo.

[0237] In one embodiment, therefore, the antibody or antigen-binding fragment thereof comprises a modified Q295 residue, a modified L328 residue, a modified P331 residue, a modified 1332 residue, a modified E382 residue, and / or a modified M428 residue. In a typical embodiment, the antibody or antigen-binding fragment thereof comprises a modified Q295 residue, a L328 residue, a P331 residue, a 1332 residue, a E382 residue, and a M428 residue, with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11.

[0238] It will be appreciated that the above mentioned six Fc mutations may be made in addition to the aglycosylation described above. Accordingly, in one embodiment, the anti-amyloid beta antibody or antigen-binding fragment is aglycosylated. In anotherembodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises N297A.

[0239] Typically, the Q295 amino acid residue may be modified to arginine. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises Q295R.

[0240] Typically, the L328 amino acid residue may be modified to tryptophan. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises L328W.

[0241] Typically, the P331 amino acid residue may be modified to alanine. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises P331A.

[0242] Typically, the 1332 amino acid residue may be modified to tyrosine. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises I332Y.

[0243] Typically, the E382 amino acid residue may be modified to valine. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises E382V.

[0244] Typically, the M428 amino acid residue may be modified to leucine. Therefore, in a typical embodiment, the anti-amyloid beta antibody or antigen-binding fragment comprises M428L.

[0245] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof, consisting of a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: the heavy chain variable region CDR1 amino acid sequences are selected from the group consisting of SEQ ID NOs: 3 and 12, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the heavy chain variable region CDR2 amino acid sequences are selected from the group consisting of SEQ ID NOs: 4 and 13, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the heavy chain variable region CDR3 amino acid sequences are selected from the group consisting of SEQ ID NOs: 5 and 14, or variant sequences thereof comprising 1,2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable regions CDR1 amino acid sequences are selected from the group consisting of SEQ ID NOs: 7 and 16, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable regions CDR2 amino acid sequences are selected from the group consisting of SEQ ID NOs: 8 and 17, or variant sequences thereof comprising 1 , 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable regions of CDR3 amino acid sequences are selected from the group consisting of SEQ ID NOs: 9 and 18, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof.

[0246] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95, 97 or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 15; the light chain variable region comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95, 97 or 99%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 19. Alternatively the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises no more than 5 amino acid, or no more than 4 amino acid, or no more than 3 amino acid, or no more than 2 or no more than 1 amino acid change compared to the amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 15; the light chain variable region comprises no more than 5 amino acid, or no more than 4 amino acid, or no more than 3 amino acid, or no more than 2 or no more than 1 amino acid change compared to the amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 19. In this context, the term "change" refers to insertions, deletions and / or substitutions.

[0247] In one embodiment, the antibody or antigen-binding fragment according to the first aspect, may comprise a mutated Fc fragment. For example, the antibody or antigenbinding fragment may be mutated by introducing amino acid substitutions into the Fc region, which silence or reduce the effector function of the antibody, and / or enhance Fc function. Accordingly, in one embodiment, the Fc fragment comprises one or more amino acid substitution that silences or reduces the effector function of the antibody or antigen-binding fragment thereof.In one embodiment, the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: L234A, L235A, and P329G, according to EU numbering. In one embodiment, the Fc fragment comprises the amino acid substitutions L234A, L235A, and P329G. In one embodiment, the Fc fragment comprises the amino acid substitutions L234A and L235A.

[0248] In another embodiment, the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: N325S and L328F, according to EU numbering. In one embodiment, the Fc fragment comprises the amino acid substitutions N325S and L328F.

[0249] In another embodiment, the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: M252Y, S254T, and T256E, according to EU numbering. In one embodiment, the Fc fragment comprises the amino acid substitutions M252Y, S254T, and T256E. Advantageously, these amino acid substitutions improve antibody affinity for FcRn and extend half-life in vivo.

[0250] Advantageously, the anti-beta-amyloid nature of the antibody or antigen-binding fragment according to the first aspect of the invention means that it has significant utility as a therapeutic agent in its own right, and may be used in the treatment, amelioration or prevention of a condition involving beta-amyloid.

[0251] Accordingly, in a second aspect of the invention, there is provided an antibody or antigen-binding fragment thereof according to the first aspect, for use in therapy.

[0252] In a third aspect of the invention, there is provided an antibody or antigen-binding fragment thereof according to the first aspect, for use in treating, preventing or ameliorating a beta-amyloid-mediated neurodegenerative disorder.

[0253] According to a fourth aspect of the invention, there is provided a method of treating, preventing or ameliorating a neurodegenerative disorder in a subject, the method comprising administering, or having administered, to a patient in need of such treatment, a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first aspect.

[0254] Examples of neurodegenerative disorder, which may be treated, may be selected from a group consisting of: Alzheimer's disease; Parkinson's disease; Huntington's disease;Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); Frontotemporal Dementia, and Dementia with Lewy Bodies.

[0255] In one embodiment, the neurodegenerative disorder, which is treated, is Alzheimer's disease, Parkinson's disease, or Motor Neurone disease.

[0256] In another embodiment, the neurodegenerative disorder, which is treated, is Alzheimer's disease.

[0257] It will be appreciated that antibodies or antigen-binding fragments thereof according to the invention (referred to herein as "agents") may be used in a monotherapy (e.g., the use of an antibody alone), for treating, ameliorating or preventing the neurodegenerative disorder. Alternatively, agents according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing a neurodegenerative disorder, such as anti-inflammatory drugs or anti-o-synuclein agents, such as anti o-synuclein antibody.

[0258] The agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.

[0259] Medicaments comprising agents of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g., intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.

[0260] Agents and medicaments according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site, typically the brain. Suchdevices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g., at least daily injection).

[0261] In one embodiment, agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

[0262] It will be appreciated that the amount of the antibody (i.e., agent) that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent, and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

[0263] Generally, a daily dose of between O.Olpg / kg of body weight and lOOmg / kg of body weight of agent according to the invention may be used for treating, ameliorating, or preventing the neurodegenerative disorder, depending upon which agent. In some embodiments, the daily dose of agent is between 1 p g / kg of body weight and lOOmg / kg of body weight, optionally between lOpg / kg and lOmg / kg body weight, and optionally between approximately 100 p g / kg and lOmg / kg body weight.

[0264] The agent may be administered before, during or after onset of the neurodegenerative disorder. Daily doses may be given as a single administration (e.g., a single daily injection). Alternatively, the agent may require administration twice or more times during a day. As an example, agents may be administered as two (or more depending upon the severity of the condition being treated) daily doses of between 0.07pg and 700 mg (i.e., assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses. Known procedures, such as thoseconventionally employed by the pharmaceutical industry (e.g., in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).

[0265] In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect, and optionally a pharmaceutically acceptable vehicle.

[0266] The pharmaceutical composition may be anti-beta amyloid, i.e., a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of a beta amyloid-mediated condition.

[0267] The invention also provides in a sixth aspect, a process for making the pharmaceutical composition according to the fifth aspect, the process comprising combining a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined in the first aspect, with a pharmaceutically acceptable vehicle.

[0268] The antibody or antigen-binding fragment thereof may be as defined with respect to the first aspect.

[0269] A "subject" may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g., a horse), pets, or may be used in other veterinary applications. In some embodiments, the subject is a human being.

[0270] A "therapeutically effective amount" of the antibody or antigen-binding fragment thereof is any amount which, when administered to a subject, is the amount of agent that is needed to treat the neurodegenerative disorder, or produce the desired effect.

[0271] For example, the therapeutically effective amount of antibody or antigen-binding fragment thereof used may be from about 0.1 ng / kg to about 100 mg / kg, and optionally from about 1 ng / kg to about 10 mg / kg. In one embodiment, the amount of antibody or antigen-binding fragment thereof is an amount from about 10 ng / kg to about 10 mg / kg, and optionally from about 50 ng / kg to about 5 mg / kg.A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0272] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tabletdisintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets may contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0273] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0274] The agents and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0275] In a seventh aspect of the invention, there is provided a polynucleotide sequence encoding the antibody, or antigen-binding fragment thereof as defined in the first aspect.

[0276] In an eighth aspect of the invention, there is provided an expression cassette comprising a polynucleotide sequence according to the seventh aspect.

[0277] The polynucleotide sequence encoding the antibody, or antigen-binding fragment of the invention may be harboured in a recombinant vector, for example a recombinant vector for delivery into a host cell of interest to enable production of the antibody, or antigen-binding fragment thereof.

[0278] Accordingly, in a ninth aspect of the invention, there is provided a recombinant vector comprising the expression cassette according to the eighth aspect.

[0279] The vector encoding the antibody, or antigen-binding fragment may for example be a plasmid, cosmid or phage and / or be a viral vector. Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with thenucleotide sequences. The nucleotide sequences may be a DNA sequence, and it is this DNA sequence which encodes the antibody, or antigen-binding fragment.

[0280] Recombinant vectors encoding the antibody, or antigen-binding fragment may also include other functional elements. For example, they may further comprise a variety of other functional elements including a suitable promoter for initiating transgene expression upon introduction of the vector in a host cell. For instance, the vector may be capable of autonomously replicating in the nucleus of the host cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g., by homologous recombination) are envisaged. Suitable promoters may include the SV40 promoter, CMV, EFla, PGK, viral long terminal repeats, as well as inducible promoters, such as the Tetracycline inducible system, as examples. The cassette or vector may also comprise a terminator, such as the Beta globin, SV40 polyadenylation sequences or synthetic polyadenylation sequences. The recombinant vector may also comprise a promoter or regulator or enhancer to control expression of the nucleic acid as required.

[0281] The vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e., to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA. For example, ampicillin, neomycin, puromycin or chloramphenicol resistance is envisaged. Alternatively, the selectable marker gene may be in a different vector to be used simultaneously with the vector containing the transgene. The cassette or vector may also comprise DNA involved with regulating expression of the nucleotide sequence, or for targeting the expressed polypeptide to a certain part of the host cell.

[0282] Purified vector may be inserted directly into a host cell by suitable means, e.g., direct endocytotic uptake. The vector may be introduced directly into a host cell (e.g., a eukaryotic or prokaryotic cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, calcium phosphate, cationic lipid-based lipofection, polymer or dendrimer-based methods or ballistic bombardment.

[0283] Alternatively, vectors of the invention may be introduced directly into a host cell using a particle gun.Alternatively, the delivery system may provide the polynucleotide to the host cell without it being incorporated in a vector. For instance, the nucleic acid molecule may be incorporated within a liposome or virus particle. Alternatively, a "naked" polynucleotide may be inserted into a host cell by a suitable means e.g., direct endocytotic uptake.

[0284] In a tenth aspect of the invention, there is provided a host cell comprising the polynucleotide sequence according to the seventh aspect, the expression cassette according to the eighth aspect, or the vector according to the ninth aspect.

[0285] The host cell may be a eukaryotic or prokaryotic host cell. In one embodiment, the host cell is a eukaryotic host cell. In one embodiment, the host cell is a mammalian host cell such as NSO murine myeloma cells, PER.C6® human cells, Human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells. In one embodiment, the host cell is a CHO cell.

[0286] In an eleventh aspect, there is provided a method of preparing the antibody, or antigen-binding fragment according to the first aspect, the method comprising: a) introducing, into a host cell, the vector of the ninth aspect; and

[0287] b) culturing the host cell under conditions to result in the production of the antibody, or antigen-binding fragment according to the first aspect.

[0288] The host cell of step a) may be a eukaryotic or prokaryotic host cell. In one embodiment, the host cell is a eukaryotic host cell. In one embodiment, the host cell is a mammalian host cell such as NSO murine myeloma cells, PER.C6® human cells, Human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells. In one embodiment, the host cell is a CHO cell.

[0289] The method may further comprise (c) harvesting, centrifuging and / or filtering the cell culture media to obtain a cell culture supernatant comprising the antibody or antigen binding fragment thereof.

[0290] The method may further comprise (d) separating and purifying the antibody or antigen binding fragment thereof from the cell culture supernatant. In one embodiment, purification is performed by at least one chromatographic step.

[0291] Suitable chromatographic steps include affinity chromatography and / or ion exchange chromatography. In some embodiments, affinity chromatography is protein Achromatography. Ion exchange chromatography may be anionic exchange chromatography and / or cationic exchange chromatography.

[0292] In some embodiments, step (d) comprises separating and purifying the antibody or antigen binding fragment thereof from the cell culture supernatant by:

[0293] i) protein A chromatography;

[0294] ii) anionic exchange chromatography; and / or

[0295] iii) cationic exchange chromatography.

[0296] The method may further comprise (e) filtering the purified antibody or antigen binding fragment thereof resulting from step (d). In some embodiments, step (e) comprises virus filtration. Thus, in some embodiments the purified antibody or antigen binding fragment thereof resulting from step (d) is filtered using a virus filtration membrane. Suitable membranes would be known to those skilled in the art.

[0297] As discussed herein, beta amyloid plays a role in many neurodegenerative conditions. Thus, given that the antibodies of the invention are able to bind to beta amyloid, the antibodies or antigen-binding fragments thereof may be used as a robust diagnostic tool by detecting the presence, and determining the concentration of beta amyloid.

[0298] Thus, in a twelfth aspect, there is provided the antibody or antibody binding fragment of according to the first aspect, for use in diagnosis or prognosis.

[0299] In one embodiment, the diagnosis or prognosis may comprise imaging.

[0300] According to a thirteenth aspect of the invention, there is provided the antibody or antibody binding fragment of according to the first aspect, for use in diagnosing or prognosing a neurodegenerative disorder.

[0301] According to the fourteenth aspect, there is provided a method of diagnosing or prognosing a neurodegenerative disorder in a subject, the method comprising detecting beta amyloid in a biological sample obtained from the subject with the antibody or antibody binding fragment of according to the first aspect.

[0302] Examples of neurodegenerative disorder, which may be diagnosed or prognosed, may be selected from a group consisting of: Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, andtype 3; Amyotrophic Lateral Sclerosis (ALS); Frontotemporal Dementia, and Dementia with Lewy Bodies.

[0303] In one embodiment, the neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, or Motor Neurone disease. In another embodiment, the neurodegenerative disorder is Alzheimer's disease.

[0304] The method may be an in vitro or ex vivo method. In some embodiments, the method is an in vitro method.

[0305] The use or method may comprise determining the level of expression of beta amyloid in a subject, optionally wherein (i) an increase in the concentration of beta amyloid in the biological sample when compared to a reference concentration from a healthy control population is indicative of a neurodegenerative disorder or a poor prognosis; and / or (ii) a decrease in the concentration of beta amyloid in the biological sample when compared to a reference concentration from a healthy control population is indicative of a improvement in a neurodegenerative disorder or a good prognosis.

[0306] According to the fifteenth aspect of the invention, there is provided a kit for diagnosing a subject suffering from a neurodegenerative disorder, or for providing a prognosis of the subject's condition, the kit comprising an antibody or antigen-binding fragment thereof according to the first aspect for detecting beta amyloid in a sample from a test subject.

[0307] The kit may further comprise instructions for use and / or a receptacle for obtaining a biological sample from a subject.

[0308] Prognosis may relate to determining the therapeutic outcome in a subject that has been diagnosed with the neurodegenerative disorder. Prognosis may relate to predicting the rate of progression or improvement and / or the duration of the neurodegenerative disorder in a subject, the probability of survival, and / or the efficacy of various treatment regimes. Thus, a poor prognosis may be indicative of progression of the neurodegenerative disorder, low probability of survival and reduced efficacy of a treatment regime. A favourable prognosis may be indicative of resolution of the neurodegenerative disorder, high probability of survival and increased efficacy of a treatment regime.In some embodiments, the sample comprises a biological sample. The sample may be any material that is obtainable from a subject from which protein is obtainable.

[0309] The biological sample may be tissue or a biological fluid. The biological sample may be any material that is obtainable from the subject from which endothelial, smooth muscle and / or interstitial cells are obtainable. Furthermore, the sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, breast milk, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, lymph, granuloma, cancer biopsy or combinations thereof.

[0310] The sample may comprise blood, urine, tissue etc. In one embodiment, the biological sample comprises a blood sample. The blood may be venous or arterial blood. Blood samples may be assayed immediately. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before the method is conducted. Alternatively, the blood sample may be stored at room temperature, for example between 18 to 22 degrees Celsius, before the method is conducted. The blood sample may comprise comprises blood serum. The blood sample may comprise blood plasma. The detection may be carried out on whole blood or peripheral blood.

[0311] The blood may be further processed before the use of the first aspect is performed. For instance, an anticoagulant, such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Thus, the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting.

[0312] It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially the amino acid / nucleotide / peptide sequence", "variant" and "fragment", can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of any one of the sequences referred to herein, for example 40% identity with any of the sequences identified herein.Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, optionally greater than 70%, optionally greater than 75%, and optionally greater than 80% sequence identity to any of the sequences referred to are also envisaged. In some embodiments, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, optionally at least 90% identity, optionally at least 92% identity, optionally at least 95% identity, optionally at least 97% identity, optionally at least 98% identity and, optionally at least 99% identity with any of the sequences referred to herein.

[0313] The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g., BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g., functional form and constants.

[0314] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

[0315] Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson etal., 1997, Nucleic Acids Research, 24, 4876-4882) is one way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be awarethat it may be necessary to vary these and other parameters for optimal sequence alignment.

[0316] In some embodiments, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. In some embodiments, overhangs are included in the calculation. Hence, one method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N / T)*100.

[0317] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in, for example, in those described herein that are amino acid sequences.

[0318] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

[0319] All of the features described herein (including any accompanying claims, abstracts, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive.

[0320] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figure, in which:-

[0321] Figure 1 shows immunohistochemistry data illustrating binding of amyloid antibodies to human amyloid (A[342) in brain tissue from APP / PS1 transgenic mice, for: (i) a wildtype antibody human IgGl "h3D6" (i.e., Bapineuzumab); (ii) an aglycosylated form (N297A) of the wildtype human IgGl: "agly hlgGl 3D6"; (iii) an FcyRI-selective Fc variant of the aglycosylated human IgGl ("Fc5 variant"; E382V / M428I): "agly-Fc5 hlgGl 3D6"; and a "secondary antibody control". The data show that hlgGl 3D6, a-gly-Fc5 hlgGl 3D6, and a-gly hlgGl 3D6 show similar binding patterns in APP / PS1 mouse tissue.

[0322] Figure 2 is CisBio affinity data for: (i) the wild-type antibody human IgGl "hlgGl" 3D6; (ii) the aglycosylated form of the wild-type human IgGl: "agly hlgGl" 3D6; and (iii) the FcyRI-selective Fc variant of the aglycosylated human IgGl: "agly- hlgGl Fc5" 3D6. The data show that the Fc5 mutation results in retained binding to RcgRI. The data show that hlgGl aglycosylated Fc5 3D6 has a reduced affinity to FcyRI when compared to the wild-type hlgGl 3D6, but increased affinity to FcyRI when compared to the aglycosylated form of the wild-type hlgGl. hlgGl 3D6 shows the highest affinity: Kd (M) hlgGl 3D6 = 10 -8.3, Kd (M) agly-Fc5 hlgGl 3D6 = 10-7.3 and Kd (M) a-gly hlgGl 3D6 = 10 -6.3.

[0323] Figure 3 is immunohistochemistry data showing IgG labelled structures in brain tissue from APP / PS1 transgenic mice treated with A) Bapineuzumab hlgGl, B) a-glycosylated Fc5 Bapineuzumab hlgGl, C) a-glycosylated Bapineuzumab hlgGl, D) Bapineuzumab mIgG2a. Tissue was collected and analysed 2 weeks after stereotaxic administration ofthe antibodies. IgG labelled structures are shown in brown, with representative images from the ipsilateral hippocampus and cortex.

[0324] Figure 4 shows retained IgG and neuroinflammation in brain tissue from APP / PS1 mice treated with anti-amyloid antibodies. A) Immunofluorescence showing retained human IgG (as a measure of amyloid load) in brain tissue from APP / PS1 transgenic mice (IgG, green) 2 weeks after stereotaxic injection. Data shows strongest IgG immunoreactivity for a-gly hlgG 3D6, suggesting binding of the antibody to its target but limited removal. No difference in IgG immunoreactivity for hlgGl 3D6 and a-gly-Fc5 hlgGl 3D6 is observed, suggesting comparable efficacy to clear amyloid from brain tissue following stereotaxic injection. The lowest immunoreactivity was observed following injection of Mouse IgG2a 3D. Immunofluorescence from the contralateral hemisphere was used as control. B) Immunofluorescence histochemistry for CD64 (red, row 2), CD68 (red, row 3), and GFAP (red, row 4). CD64 expression shows minimal change following administration of a-gly 3D6 or a-gly-Fc5 3D6, whereas increased expression of CD64 is observed following administration of amyloid-targeting antibodies with full effector function (hlgGl and mIgG2a). A similar pattern is observed for CD68 expression (red, row 3), suggesting increased microglial activation only occurs following administration of therapeutic antibodies with full effector function. No change in expression of GFAP. C-E) Quantification of immunofluorescence for C) IgG, D) CD64, and E) CD68. N = 5 animals per treatment group. Data were analysed by one-way ANOVA and Tukey post hoc test and presented as mean + / - SD. Statistical analyses were performed comparing the mean of each treatment group with the mean of mIgG2a. *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001.

[0325] Figure 5 shows antibody-mediated phagocytosis by THP-1 cells. Representative images of cellular uptake of fluorescent microspheres, covalently linked to amyloidbeta 1-42 peptide. Data show that antibody opsonised microspheres significantly enhance uptake of fluorescence spheres. Images were taken at four different time points (15 min, 1 h, and 24h) with an x20 objective. Scale bar = 150 um.

[0326] Figure 6 shows antibody-mediated inflammation by THP-1 cells. Amyloid-targeting antibodies were coated on a cell culture plate followed by addition of THP-1 cells. Cells were collected 24h later for qPCR analysis of pro-inflammatory cytokines. Data show that a-gly-Fc5 3D6 results in reduced levels of IL-6 and IL-1 b mRNA expression, when compared to hlgGl 3D6 with full effector function. This experiment suggests reduced inflammatory effects despite effective engagement with amyloid. Genes ofproinflammatory cytokines TNF-a, IL-6 and IL- lb are expressed as fold change over a control sample with no antibody and after normalizing to GAPDH. Data were analysed by two-way ANOVA and Tukey post hoc test and presented as mean + / - SD (n=4).

[0327] Figure 7 shows fluorescent immunohistochemistry stains of amyloid and hlgG in a hlgGl bapineuzumab / 3D6 treated APP / PS1 transgenic mouse brain. The brain tissue section was stained with Amytracker-520 (green), anti-human IgG Rhodamine Red (red), and dapi (blue). The tissue sample contains a hlgGl bapineuzumab-treated (ipsilateral) hemisphere. The images were captured at 40x magnification. Scale bar = 100pm. IgG and amyloid colocalise, suggesting remaining IgG is bound to amyloid and can be used as a measure of remaining amyloid deposits.

[0328] Figure 8 is a diagram outlining binding capacity to FcyR following antibody engineering. FcyRI has the capacity to bind very strongly to mlgG2a, strongly to hlgGl, less strongly to hlgGl-agly-Fc5, and minimally to hlgGl-agly. FcyRII has the capacity to bind weakly to mlgG2a, weakly to hlgGl, not at all to hlgGl-agly-Fc5, and not at all to hlgGl-agly. FcyRIII has the capacity to bind weakly to mlgG2a, weakly to hlgGl, not at all to hlgGl-agly-Fc5, and not at all to hlgGl-agly. FcyRIV has the capacity to bind very strongly to mlgG2a, less strongly to hlgGl, not at all to hlgGl-agly-Fc5, and not at all to hlgGl-agly.

[0329] Figure 9 shows immunofluorescence showing binding of amyloid antibodies to human amyloid (AP42) in brain tissue from APP / PS1 transgenic mice. Data show that hlgGl 3D6 and 3D6-701 bind to structures resembling amyloid plaques. Data also show binding of Lecanemab hlgGl, a-gly-hlgGl Lecanemab (NA), Lecanemab-Fc5, and Lecanemab-701 to structures resembling amyloid plaques. The Fc variants of Lecanemab appear to show similar binding patterns in APP / PS1 mouse tissue, suggesting that Fc engineering did not influence specificity to amyloid. 20x magnification.

[0330] Figure 10 shows binding of anti-AP antibody Fc variants to AP plaques in APP / PS1 mouse brain sections. Representative immunohistochemical images of AP staining in the cortical region of APP / PS1 mouse brain sections acquired on lOx and 40x objectives. Boxed regions indicate areas shown at higher magnification in the corresponding inset images. Sections were stained using BAPI hlgGl, LECA hlgGl, LECA agly-hlgGl, LECA agly-Fc5 hlgGl, or LECA agly-Fc701 hlgGl primary antibodies. A secondary-antibody-only control is also included.Figure 11 shows anti-AP antibody Fc variants differentially promote THP-1 phagocytosis of Ag-conjugated fluorescent microspheres. (A) Representative images showing phagocytosis of Ag-conjugated fluorescent microspheres by THP-1 cells following incubation with microspheres opsonised with either isotypic control, BAPI hlgGl, LECA hlgGl, LECA agly-hlgGl, LECA agly-Fc5 hlgGl, or LECA agly-Fc701 hlgGl antibodies. Images were acquired at 1 h, 4 h, and 24 h following incubation using a 20x objective. Scale bar: 150 pm. (B) Representative higher power images showing phagocytosis of Ag-conjugated fluorescent microspheres by THP-1 cells following incubation with microspheres opsonised with either isotypic control, BAPI hlgGl, LECA hlgGl, LECA agly-hlgGl, LECA agly-Fc5 hlgGl, or LECA agly-Fc701 hlgGl antibodies. Images were acquired at 24 h (C) Representative z-stack images confirming internalisation of Ap-conjugated fluorescent microspheres within THP-1 cells. Scale bar: 40 pm. (D) Bar charts showing the mean percentage of phagocytic THP-1 cells following incubation with Ap-conjugated fluorescent microspheres opsonised with anti-amyloid Fc variants after 1 h, 4 h, and 24 h. Data are presented as mean ± 1 standard deviation (n = 3 independent experiments). Comparisons represent one-way ANOVA with Dunnett's post-hoc test where "ns" = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** =p < 0.0001. (E) Diagram of experimental design.

[0331] Figure 12 shows anti-AP antibody Fc variants induce distinct inflammatory transcriptional responses in THP-1 cells. Bar charts showing qPCR analysis of (A) IL-6 and (B) IL-lfi expression in THP-1 cells treated with either BAPI hlgGl, LECA hlgGl, LECA agly-hlgGl, LECA agly-Fc5 hlgGl, or LECA agly-Fc701 hlgGl antibodies. Gene expression is shown as fold change relative to the average of untreated controls, calculated using the 2-AACt method and normalised to GAPDH expression. Bars represent mean fold change ± 1 standard deviation, with overlaid data points indicating individual measurements. THP-1 cDNA was generated from two technical replicates per condition across two independent experiments (n = 4 total measurements). Comparisons represent one-way ANOVA with Dunnett's post-hoc test where "ns" = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001,

[0332] **** =p < 0.0001.

[0333] Figure 13 shows SPR sensorgrams showing binding of 3D6 antibody variants (Figure 13; left panel) and Lecanemab antibody variants (Figure 13; right panel) to human FcyRI. The HBS-EP+ buffer control is shown as baseline. For 3D6, the wild-type Fc (hlgGl) exhibits strong binding, while the a-gly (NA) variant displays significantly abrogated binding, which is rescued with the Fc5 and Fc701 mutations, with the 3D6Fc701 showing a slower off-rate than the 3D6 Fc5 variant. For the lecanemab variants equivalent data is shown.

[0334] Figure 14 shows SPR sensorgrams showing binding of 3D6 (Figure 14; left panel) and Lecanemab (Figure 14; right panel) antibody variants to human FcyRIIa. For both antibody series (3D6 and Lecanemab) wild-type IgG (hl) display clear binding to FcyRIIa whilst the Fc-engineered variants (a-gly (NA), Fc5, Fc701) exhibit no binding. HBS-EP+ buffer serves as a negative control.

[0335] Figure 15 shows SPR sensorgrams showing binding of 3D6 (Figure 15; left panel) and Lecanemab (Figure 15; right panel) antibody variants to human FcyRIIb. For both antibody series (3D6 and Lecanemab) wild-type IgG (hl) display clear binding to FcyRIIb whilst the Fc-engineered variants (a-gly (NA), Fc5, Fc701) exhibit no binding. HBS-EP+ buffer serves as a negative control.

[0336] Figure 16 shows SPR sensorgrams showing binding of 3D6 (Figure 16; left panel) and Lecanemab (Figure 16; right panel) antibody variants to human FcyRIIIa. For both antibody series (3D6 and Lecanemab) wild-type IgG (hl) display clear binding to FcyRIIIa whilst the Fc-engineered variants (a-gly (NA), Fc5, Fc701) exhibit no binding. HBS-EP+ buffer serves as a negative control.

[0337] Figure 17 shows SPR sensorgrams showing binding of different concentrations of 3D6 antibody variants (Figure 17; top panel) and different concentrations of Lecanemab antibody variants (Figure 17; bottom panel) to human FcyRI. The HBS-EP+ buffer control is shown as baseline. For 3D6, the wild-type Fc (hlgGl) exhibits strong binding. The 3D6 Fc5 and Fc701 variants bind to human FcyRI, with the 3D6 Fc701 showing a slower off-rate than the 3D6 Fc5 variant. For the lecanemab variants equivalent data is shown.

[0338] Figure 18 shows the kinetic parameters of 3D6 / Bapi and Lecanemab Fc variants for human FcyRI binding.

[0339] Examples

[0340] To test if Fc-engineering could be used to influence the capacity of antibodies to clear amyloid, the inventors generated a panel of humanized IgGl anti-AP antibodies, based on the parent (wild-type) antibody, "h3D6 / Bapineuzumab", and including an aglycosylated form (N297A) of the parent ("agly hlgGl 3D6"), and also FcyRI-selective Fc variants ("agly-Fc5 hlgGl 3D6"), containing the amino acid substitutionsE382V and M428I. The ability of these Fc variants to clear AP plaques and induce inflammation was assessed in vivo using an experimental mouse model of AD and in vitro using the THP-1 human macrophage cell line. The results surprisingly revealed that the novel Fc5 variant of Bapineuzumab, with selective binding to FcyRI (Fc5-Bapi), is as efficient in clearing antibody-bound amyloid as the wild-type aglycosylated variant. Moreover, the inventors also discovered that this same engineered antibody, h!gGl-bapi-Fc5, has reduced inflammatory effects compared to hlgGl-Bapi.

[0341] The inventors also generated a panel of IgGl anti-Ag antibodies, based on the parent (wild-type) antibody, "Lecanemab", and including an aglycosylated form of the parent, and also FcyRI-selective Fc variants containing the amino acid substitutions N297A, Q295R, L328W, P331A, I332Y, E382V, and M428L, known as Fc701. The ability of these variants to bind human amyloid in brain tissue using an experimental mouse model of AD was assessed, as well as FcyRI selectivity. The results surprisingly revealed that Fc701 mutations result in 120-fold improved binding capacity to FcyRI when compared to wild type aglycosylated IgG, and excellent FcyRI selectivity and no binding to FcyRIIb.

[0342] These unexpected results provide the first proof-of-concept data that Fc engineering can lead to optimised anti-amyloid immunotherapy by reducing adverse effects.

[0343] Materials and Methods

[0344] Antibodies

[0345] Recombinant versions of the Ap-targeting antibody (FDA UNII: NC11WKO35D) with constant regions; mIgG2a and hlgGl, aglycosylated hlgG (N297A) or aglyosylated hIgGl-Fc5 (N297A, E382V, M428I - EU numbering). The recombinant antibodies were produced by subcloning DNA of variable regions (comprising the beta amyloid-binding CDRs) of both heavy and light chains (Table 1) (The International Immunogenetics Information Systems®), synthesised by GeneArt, into expression vectors (pEE6.4 and pEE12.4 from Lonza) containing various mouse or human IgG constant regions (Table 2) via Hindlll / Spel (heavy chain) and Hindlll / BsiWI (light chain).

[0346] Table 1 - Sequences of the variable regions of the recombinant antibodies used Amino acid sequences of variable heavy chain (VH) and light chain (VL) comprising the CDRs for Bapineuzumab, Aducanumab and Lecanemab.

[0347]

[0348]

[0349] Table 2 - Sequences of the constant regions of the recombinant antibodies used Amino acid sequences of constant heavy chain (CH) and light chain (CL) regions for human IgGl and mouse IgG2a (uniprot.org).

[0350]

[0351]

[0352] To generate aglycosylated hlgGl (N297A), Asparagine (N) at position 297 was changed to Alanine (A) N297.

[0353] To generate aglycosylated h!gGl-Fc5 mutant (N297A, E382V, M428I), Asparagine (N) at position 297 was changed to Alanine (A), Glutamic acid at position 382 was changed to Valine (E382V), and Glutamic acid at position 428 Methionine at position 428 was changed to Isoleucine (M428I).

[0354] Using Invitrogen ExpiCHO expression system for transient transfection of the plasmid, the antibodies were expressed in ExpiCHO cells and purified from the cell culture supernatant using protein A or protein G affinity purification columns (such as a Protein A MabSelect SuRe column). Finally, the concentrations of the antibodies were measured and calculated using a Nanodrop and a molar extinction coefficient of 1.45. All antibodies were checked to contain < 1% aggregate determined by HPLC and tested to contain < 5EU endotoxin per 1 mg antibody assessed by the Endosafe-PTS portable test system (Charles River Laboratories, L'Arbresle, France).

[0355] Abeta antibody binding assay

[0356] Binding characteristics of the recombinant antibodies was assessed by immunohistochemistry using brain tissue from APP / PS1 transgenic mice, which express human amyloid precursor protein and progressive amyloid deposition in the brain. Brains were sectioned coronally into 10 micron sections using a cryostat and mounted onto plus-charged glass slides. Sections containing hippocampus and cortex were processed for antibody binding to amyloid. Sections were dried for 30 minutes at 37°C and fixed in cold 100% ethanol for 10 minutes. After incubation with blocking buffer containing 2% Bovine Serum Albumin and 10% goat serum (Invitrogen), the recombinant antibodies (hlgGl, aglycosylated hlgGl and aglycosylated) were added at 5 ug / ml for overnight incubation. Sections were washed with PBS and incubated with a secondary biotinylated goat-anti-human IgG to detect binding of the recombinant antibodies to the amyloid in the tissue. Following incubation of the secondary antibody, sections were washed, incubated with HRP-conjugatedAvidin / Biotin complex for 30 minutes, and followed by DAB / H2O2 chromogen solution. Sections were then counterstained with hematoxylin, dehydrated, and mounted using xylene. After drying, sections were analysed using a light microscope.

[0357] FcyRI binding assay

[0358] Binding of the recombinant antibodies to FcyRI was carried out using a commercially available Cisbio assay, using manufacturer's instructions.

[0359] Animals and stereotaxic surgery

[0360] APP / PS1 transgenic mice were anesthetised and secured onto a stereotaxic frame. A mid-sagittal incision was made exposing the skull, and one burr hole was made for a unilateral injection into the hippocampus at coordinates (-2.0, +1.7). A Hamilton syringe (33 gauge) was inserted -1.4 mm into the brain and 2 ul of antibody (1 mg / ml) was slowly injected using a 33 gauge Hamilton syringe 33 gauge. The wound was closed and sutured with 5.0 Mersilk and mice allowed to recover in a 28°C chamber. Mice were kept in 12 h light-dark cycles with free access to food and water. Mice were randomised into 4 groups (mIgG2a 3D6, hlgG 3D6, aglycosylated hlgG 3D6, and aglycosylated Fc5 variant of hlgGl 3D6,) (n = 4 / 5 per treatment group). Sutures were used to close the wound and mice were left to recover in a thermoregulated environment. In the following 7 days, tissue healing and animal recovery was monitored daily (twice on the first 2 days p.i.), by use of the Wolfensohn scoring system. Antibodies were injected at Lundbeck, Denmark. PPL: 19b: 2014-15-0201-00339 CIO.

[0361] Tissue processing

[0362] 14 days after the stereotaxic injections of the antibodies, mice were terminally anaesthetised and transcardially perfused with heparinised saline / 4% PFA. The brain was removed and mounted in optimal cutting temperature medium (OCT, Sakura Finetek, Thatcham, UK) and tissue was kept at -80°C until processing for immunohistochemistry.

[0363] Immunofluorescence and immunohistochemistry

[0364] Brains were sectioned coronally into 10 micron sections using a cryostat and mounted onto plus-charged glass slides. Sections were dried for 30 minutes at 37°C and fixed in cold 100% ethanol for 10 minutes. After incubation with blocking buffer containing 2% Bovine Serum Albumin and 10% goat serum (Invitrogen), 30 minutes prior primary antibodies were added and incubated at 4°C overnight. Immunofluorescence was performed using rat-anti-mouse (AT152-9, 1:500, Biorad) or rat-anti-mouseCD68 (FA11, 1:500, Biorad) followed by goat-anti-rat IgG A568 (1:500 Invitrogen) to identify microglia / macrophages. FITC donkey anti-human (Jackson Laboratory, AB_2340513 (1:500, Sigma) or FITC goat anti-mouse (1:500, Invitrogen) was used to detect remaining hlgGl recombinant anti-amyloid antibodies following stereotaxic injection. Rabbit anti-GFAP (1:2000, Dako) was used to detect astrocytes. Following antibody incubations, sections were washed, incubated with DAPI for nuclear counter staining (Sigma), and mounted in Mowiol.

[0365] Immunohistochemistry was performed using rat-anti-mouse CD64 or CD68 mAbs followed by rabbit-anti-rat biotinylated (1:250, Vector) and hematoxylin for nuclear counter staining (Sigma).

[0366] Human THP-1 cell culture and effector function assay

[0367] The human monocyte cell line THP-1 was cultured in RPMI 1640 medium (Gibco) supplemented with 10 % heat inactivated foetal calf serum (FCS, Sigma), 100 uM penicillin, 2 mM L-glutamine, and 1 mM sodium pyruvate. Cells were incubated at 37°C with 5 % CO2. Cells were passaged twice per week with a 1:3 dilution to keep cultures at cell densities below 0.5 x 106 cells / ml. To monitor AP uptake by THP-1 cells, 0.5 mg of human AP peptide (1-42, Abeam) was conjugated to fluorescent carboxylate-modified microspheres (1.0 pm, yellow-green). Conjugation was performed by using a two-step l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) coupling chemistry that activates the surface of carboxyl groups on the microsphere particles and couples the microspheres to primary amine groups on the AP peptide via amine bond formation. Following amyloid coupling, fluorescent microspheres were blocked with 1 ml of 1 x PBS plus 1% bovine serum albumin (BSA) and stored at 4°C until required. To monitor antibody-mediated uptake, conjugated amyloid microspheres were added to hlgG 3D6, aglycosylated 3D6, or aglycosylated Fc5-3D6 in a 96-well plate at 37°C to allow binding of the antibodies to the Ap-conjugated microspheres. After 2 h, 100 ul of THP-1 cells (1.4 x 105 cells / ml) was added. THP-1 cells treated with AP conjugated amyloid microspheres in the absence of therapeutic antibodies were included as control. Cells were imaged with EVOS fluorescent microscope at different time points: 15 min, 1 h, and 24 h post-incubation at 37°C.

[0368] To measure the inflammatory response of the amyloid antibodies, 500 pl of 5 ug / ml hlgGl 3D6, aglycosylated hlgGl 3D6, or aglycosylated Fc5 hlgGl 3D6 were incubated in a 12-well plate for 2 h. Then, 1 ml suspension of THP-1 cells (1 x 106cells / ml) was added to each well and left incubating overnight at 5% CO2 and 37°C. For the control,cells without incubation with plate-bound antibody were included. After 24 h, cells were centrifuged for 6 min at 12000 rpm and RNA extracted using TRIzol method according manufacturer's instructions (Ambion). In order to perform RT-qPCR, first- strand complementary DNA (cDNA) was synthesised from the RNA samples by reverse transcription using TaqMan Reverse Transcription Reagents (Invitrogen). RT-qPCR was performed by use of the SYBR Green technology using primers, as shown in Table 3, specific for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tumour necrosis factor-alpha (TNF-o), and interleukin 6 (IL-6).

[0369] Table 3. Primer sequences

[0370]

[0371] Statistical analysis

[0372] Data was analysed using a Graph-pad prism software. Initially, data sets were tested for Gaussian distribution using the D'Agostino-Pearson omnibus test. If data sets were not normally distributed, the data were transformed using the function Y=log(Y+l), and normality tests were repeated for transformed data sets. Depending on the number of independent variables, experiments were initially tested using one or two- way ANOVA and then the Tukey post hoc test, which is corrected for multiple comparisons. Statistical analyses were performed comparing the mean of each treatment group with the mean of the antibody with the highest binding affinity to FcyRI - mIgG2a or hlgG. All graphs display mean and standard deviation.

[0373] Immunofluorescence

[0374] Immunofluorescence was performed to compare binding of anti-amyloid antibody variants to amyloid-0 in APP / PS1 mouse brain sections. Coronal brain sections (10 pm) from a 10-month-old APP / PS1 mouse were air-dried, fixed in 100% ethanol for 10 min at 4 °C, washed in PBS, and blocked in PBS supplemented with 10% goat serum (Merck, G9023) and 2% bovine serum albumin (Sigma-Aldrich, A7906) for 30min at room temperature. Anti-amyloid antibody variants (2 pg / mL) were applied to the tissue overnight at 4 °C in a humidified chamber.

[0375] The following day, sections were washed and incubated with FITC-conjugated donkey anti-human IgG (1:250; Jackson ImmunoResearch, 709-095-149)) for 45 min at room temperature. Sections were washed and incubated with DAPI (1:2000; Sigma, F6057)) for 5 min then washed again with PBS. Sections were mounted with 10 pL Mowiol Mountant (Sigma, 81381)and cover slipped before storing at 4 °C in darkness. Images of selected brain regions (cortex and hippocampus) were acquired on a Leica DM5000B microscope with a DFC300 FX camera using lOx and 40x objectives, while keeping constant exposure settings for each objective. FITC (green) and DAPI (blue) channels were merged.

[0376] Phagocytosis of opsonised Aft-conjugated fluorescent microspheres

[0377] Antibodies used (5 pg / mL): Isotype control hlgGl, BAPI hlgGl, LECA hlgG, LECA agly-hlgGl, LECA agly-Fc5 hlgGl, LECA agly-Fc701 hlgGl

[0378] THP-1 human monocytic cells were cultured in RPMI-1640 medium, supplemented with Pen Penicillin-streptomycin,(100 pM), L-glutamine (2 mM) Sodium pyruvate (1 mM) and 10% Foetal bovine serum. at 37 °C and 5% CO2. Fluorescent carboxylate-modified microspheres (1.0 pm, yellow-green 505 / 515; Invitrogen, F8823) (1% w / v) were conjugated to human AP1-42 peptide (Abeam, abl20301) and suspended in 1 x PBS + 1% bovine serum albumin.

[0379] Antibodies (5 ug / ml) and human AP1-42 peptide conjugated microspheres (diluted 1:10 in culture medium), were mixed (25 pL each), and incubated at 37°C for 2 h for opsonisation. 10,000 THP-1 cells in 80 pL were then transferred to a 96-well plate. Following opsonisation, 20 pL of microsphere / antibody solution was added to 10,000 THP-1 cells making up a total volume of 100 ul.

[0380] Cells were incubated at 37 °C and 5% CO2 and imaged at 1, 4, and 24 h using a 20x objective and GFP channel on an EVOS M5000 microscope using identical exposure settings. Z-stack imaging was performed to validate microsphere internalisation. Images were analysed in ImageJ (vl.54 g) to calculate the proportion of microspherecontaining cells and data were plotted in GraphPad Prism (vlO.6.1).

[0381] Statistical analysis was performed in GraphPad Prism. One-way analysis of variance (ANOVA) with Dunnett's post-hoc test was used with LECA hlgGl set as reference formultiple comparisons (a = 0.05). Data are presented as mean ± SD and statistical thresholds are indicated on graphs.

[0382] Inflammatory gene expression

[0383] Antibodies used (5 pg / mL): BAPI hlgGl, LECA hlgGl, LECA agly-hlgGl, LECA agly-Fc5 hlgGl, LECA agly-Fc701 hlgGl.

[0384] Antibodies were diluted 1:100 in RPMI 1640 and 500 pL transferred to a 12-well plate for to allow immobilization to the plate. After 2h at room temperature, antibody solutions were removed, THP-1 cells (1 x 106cells / well) added to the well and incubated overnight at 37 °C and 5% CO2.

[0385] Total RNA was extracted from THP-1 cells using the RNeasy Plus Micro Kit (Qiagen, 74034) according to the manufacturer's instructions. Briefly, cells were pelleted, lysed in RLT buffer supplemented with p-mercaptoethanol, and homogenised by vortexing. Ethanol was added to the lysate before loading onto RNeasy spin columns. Genomic DNA was removed by on-column DNase I digestion and RNA was eluted in 30 pL RNase-free water. RNA concentration and purity were measured on a NanoDrop ND-1000 and samples were stored at -80 °C.

[0386] THP-1 RNA was reverse transcribed using TaqMan™ reverse transcription reagents (Invitrogen, N8080234) according to the manufacturer's instructions. No-reverse transcriptase controls were prepared parallelly. Reactions were run on a Biometra UNO-Thermoblock thermocycler (Analytik Jena) at 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. cDNA was subsequently diluted 1:5 with RNase-free water and stored at -20 °C.

[0387] RT-qPCR reactions (20 pL total volume) contained cDNA (5 pL), iTaq™ Universal SYBR® Green Supermix (10 pL, Biorad), and 300 nM of both forward and reverse primers in RNase-free water. Amplification was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) with critical threshold (Ct) set to 0.1, under the following conditions:

[0388] 1. 95 °C for 10 min

[0389] 2. 95 °C for 15 s

[0390] 3. 60 °C for 1 min

[0391] 4. Steps 2-3 repeated 50 times

[0392] 5. 72 °C for 10 min

[0393] 6. 95 °C for 10 min7. 55-90 °C in 0.2 °C increments

[0394] 8. 4 °C (hold)

[0395] Amplicon specificity was subsequently confirmed by melting curve analysis. Relative IL-6 and IL-lfi expression were calculated using the 2“AACt method(43), normalising each sample to GAPDH and to the average of control conditions. Fold change values were plotted in GraphPad Prism.

[0396] Statistical analysis was performed in GraphPad Prism. One-way analysis of variance (ANOVA) with Dunnett's post-hoc test was used with LECA hlgGl set as reference for multiple comparisons (a = 0.05). Data are presented as mean ± SD and statistical thresholds are indicated on graphs.

[0397] Surface plasmon resonance

[0398] Binding of the 3D6 and Lecanemab antibodies, including wild-type (WT) and Fc variants, to recombinant proteins containing the extracellular domain of human Fc gamma receptors (FcyR) obtained from R&D Systems was analysed using a Biacore T100 system upgraded to T200 sensitivity (Cytiva). An anti-His antibody was immobilised on a Biacore CM5 sensor chip via a His Capture Kit (Cytiva). Recombinant human FcyRs (FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa) were captured via His Tag at a concentration of 2.5 or 5pg / ml, using a flow rate of 10 pl / min for 2 min. Antibodies were then injected at lOOnM in HBS-EP+ running buffer (HEPES-buffered saline containing 3mM EDTA and 0.05% Surfactant P20). Each interaction was monitored with a 3-minute association phase followed by a 3-minute dissociation phase at a flow rate of 30 pl / min. Sensor surfaces were regenerated between samples using 10 mM Glycine (pH 1.5) for 1 minute at 30 pl / min. Binding responses were reference-subtracted using a control flow cell.

[0399] Example 1 - Generation of antibodies which bind to amyloid beta and which is selective for FcvRI

[0400] The inventors first produced a panel of antibodies based on the VH and VL chains of Bapineuzumab (Bapi) for binding to amyloid beta combined with the constant regions of either mouse m!gG2a or human hlgGl. It is also envisaged that other amyloid beta-specific monoclonal antibodies could be used for the CDRs (VH and VL), such as Aducanumab and Donanumab. Table 1 provides the VH and VL sequences for the exemplars Bapineuzumab and Lecanemab, and also for Aducanumab, and Table 2 provides the constant region sequences for the human IgGl CH and mouse IgG2 chains.1) "3D6" wild-type - The parent (wild-type) antibody called "h3D6 / Bapineuzumab" was produced by subcloning the DNA encoding the variable regions of both heavy and light chains from Table 1 into expression vectors which contained mouse or human IgG constant regions shown in Table 2 via Hindlll / Spel (heavy chain) and Hindlll / BsiWI (light chain).

[0401] 2) "agly hlgGl 3D6" variant - An aglycosylated variant of the parent antibody, 3D6, called "agly hlgGl 3D6" was produced by changing the Asparagine (N) at position 297 to Alanine (A) N297, i.e., N297A.

[0402] 3) "agly-Fc5 hlgGl 3D6" variant - The Fc region of the aglycosylated variant was then modified to produce an aglycosylated h!gGl-Fc5 (N297A, E382V, M428I) mutant by making the following three modifications in the CH3 chain, i.e., Asparagine (N) at position 297 was changed to Alanine (A), Glutamic acid at position 382 was changed to Valine (E382V), and Glutamic acid at position 428 Methionine at position 428 was changed to Isoleucine (M428I).

[0403] In addition to the above aglycosylated hIgGl-Fc5 (N297A, E382V, M428I) mutant based on Bapineuzumab and Lecanemab, the inventors also envisage making the same FcyRI-selective variant based on Aducanumab and Donanumab.

[0404] 4) "hIgGl-Fc701" variant - The Fc region of the aglycosylated variant was modified to produce an aglycosylated hIgGl-Fc701 (N297A, Q295R, L328W, P331A, I332Y, E382V, M428I).

[0405] Example 2 - Immunohistochemistry showing binding of amyloid antibodies to human amyloid (AB42) in brain tissue from APP / PS1 transgenic mice

[0406] Referring now to Figure 1, it is shown that hlgGl 3D6, aglycosylated-Fc5 hlgGl 3D6, and aglycosylated hlgGl 3D6 antibodies show similar binding patterns to beta amyloid in APP / PS1 mouse tissue. These results indicate that the Fc-engineering of the recombinant 3D6 (Bapineuzumab) has no deleterious effect on the specificity of the antibodies binding to beta amyloid antigen or epitope.

[0407] Example 3 - Relative binding strength of the recombinant antibodies to FcvRI The Cisbio assay was used to determine the relative binding strength of the recombinant antibodies to FcyRI. Referring to Figure 2, it is shown that aglycosylated hlgGl and hlgGl aglycosylated-Fc5 hlgGl 3D6 have a reduced affinity to FcyRI whencompared to hlgGl 3D6. hlgGl 3D6 shows the highest affinity: Kd (M) hlgGl 3D6 = 10 -8.3, Kd (M) agly-Fc5 hlgGl 3D6 = 10-7.3 and Kd (M) a-gly hlgGl 3D6 = 10 -6.3. These results indicate that removing glycans from the N297 position in the CH2 region of hlgGl significantly reduces the binding of the antibody to FcyRI, but that introducing the three Fc5 mutations (N297A, E382V, M428I) in the CH3 region restores binding to FcyRI.

[0408] Future studies will confirm lack of binding to FcyRII and FcyRIII.

[0409] Example 4 - Immunohistochemistry to indicate amyloid removal.

[0410] Referring to Figure 3, immunohistochemistry was used to detect remaining IgG immunoreactivity in brain tissue (hippocampus and cortex) from APP / PS1 transgenic mice 2 weeks after stereotaxic injection. The results are used as an indicator of amyloid removal. Data presented in Figure 3 show that the presence of IgG immunoreactivity of human IgG for mice receiving aglycosylated hlgGl 3D6, suggesting binding of this antibody to amyloid, but limited removal. Compared to aglycosylated hlgGl 3D6, the IgG immunoreactivity for the wild-type hlgGl 3D6 parent antibody and the aglycosylated-Fc5 hlgGl 3D6 variant is reduced, suggesting comparable binding and removal of amyloid following stereotaxic injection of the antibodies. The lowest immunoreactivity was observed following injection of mouse IgG2a 3D6, and the contralateral hemisphere was used as a control.

[0411] Example 5 - Immunofluorescence to confirm presence of amyloid antibodies to human amyloid

[0412] Referring to Figure 4, immunofluorescence was used to confirm the remaining presence of amyloid antibodies specifically binding to human amyloid in brain tissue from APP / PS1 transgenic mice (IgG, green), measured 2 weeks after stereotaxic injection. The data show strongest IgG immunoreactivity for aglycosylated hlgG 3D6, suggesting binding of the antibody to its target, but limited removal. No difference is seen in IgG immunoreactivity for the hlgGl 3D6 parent and aglycosylated-Fc5 hlgGl 3D6 antibodies is observed, suggesting comparable efficacy to clear amyloid from brain tissue following stereotaxic injection. The lowest immunoreactivity was observed following injection of mouse IgG2a 3D6, suggesting most effective removal of amyloid occurs with the IgG version and contralateral hemisphere was used as control.

[0413] Immunofluorescence for CD64 expression (red, row 2) shows minimal change following administration of the aglycosylated hlgGl 3D6 or the aglycosyalated hlgGl Fc5 3D6 variant, whereas treatment of amyloid-targeting antibodies with full effectorfunction (hlgGl and m!gG2a) results in increased expression of CD64. A similar pattern is observed for CD68 expression (red, row 3), suggesting increased microglial activation only occurs following administration of therapeutic antibodies with full effector function. No change was seen in the expression of GFAP. Quantification of immunofluorescence (N = 5 animals per treatment group) shows that amyloid-binding antibodies according to the invention with selective binding to FcyRI (aglycosylated-Fc5 hlgG 3D6) can remove amyloid as efficiently as antibodies with full effector functions (hlgGl, m!gG2a 3D6). The data also show that selective binding to FcyRI (by the aglycosylated-Fc5 hlgG 3D6 variant) results in reduced microglial activation as measured by expression levels of FcyRI (CD64) and CD68. The lowest levels of remaining IgG were observed in the brain sections of mice treated with mIgG2a, as this isotype binds to mouse microglial FcyRs with the highest affinity, thereby promoting phagocytosis of AB.

[0414] Example 6 - Phagocytosis of AB-coated beads

[0415] Antibody-mediated phagocytosis by THP-1 cells was used to investigate the uptake of amyloid-coated beads as a measure of phagocytosis. Referring to Figure 5, there are shown representative images of cellular uptake of fluorescent microspheres covalently linked to amyloid-beta 1-42 peptide. Data show that antibody opsonised microspheres significantly enhance uptake of fluorescence spheres. Images were taken at three different time points (15 min, 1 h, and 24 h) with an x20 objective lens. The data show comparable uptake of amyloid-conjugated beads following incubation with hlgGl 3D6 parent and aglycosylated-Fc5 hlgGl 3D6 variant measured at 24 h. The assay also shows the uptake of amyloid-conjugated beads bound to aglycosylated hlgGl 3D6, but to a lesser extent as compared to the non-engineered parent or the Fc5-mutant. These data indicate that phagocytosis capacity is not affected by introducing the Fc5 mutation.

[0416] Example 7 - In vitro inflammation assay

[0417] To investigate whether the Fc5 mutant anti-AB antibody with reduced affinity to FcyRI has reduced inflammatory effects, an SYBR Green RT-qPCR assay was performed. Amyloid-targeting antibodies were coated on a cell culture plate (overnight) followed by addition of THP-1 cells. Cells were collected 24 h later for qPCR analysis of pro-inflammatory cytokines. Referring to Figure 6, there are shown the data of genes of proinflammatory cytokines TNF-a, IL-6, and IL- lb expressed as a fold change over a control sample with no antibody and after normalising to GAPDH. Data were analysed by two-way ANOVA and Tukey post hoc test and presented as mean + / - SD (n=4). These data indicate that the aglycosylated-Fc5 3D6 variant results in reduced levels ofIL-6 and IL-1 b mRNA expression, when compared to the hlgGl 3D6 parent with full effector function. This experiment therefore suggests reduced inflammatory effects are experienced by the aglycosylated-Fc5 3D6 variant despite effective engagement with amyloid.

[0418] Example 8 - Binding properties of Lecanemab Fc variants

[0419] Sections from an APP / PS1 mouse brain enriched for cortical and hippocampal regions were used to compare the Ag-binding properties of Lecanemab Fc variants.

[0420] Representative immunofluorescence images are shown in Figure 9 and 10. All Lecanemab variants bound to AP, resembling plaques. BAPI hlgGl was used as positive control to compare binding properties. No non-specific binding patterns were detected in the 2° antibody control.

[0421] Conclusions: Fc engineering of Lecanemab hlgGl does not influence binding to amyloid.

[0422] Example 9 - Phagocytosis of amyloid-coniugated microspheres

[0423] A phagocytosis assay was carried out to assess phagocytic activity of anti-amyloid antibody variants. Representative images as shown in Figure 11A and 11B show the relative differences in amyloid-conjugated fluorescent microsphere uptake. Z-stack imaging was performed to verify the intracellular localisation of fluorescent microspheres within THP-1 cells, supporting phagocytic uptake. Representative z-stack images are shown in Figure 11C. Graphs quantifying the mean percentage of phagocytic cells across treatments are provided in Figure 11D.

[0424] At 1 h post-treatment, a similar proportion of cells positive for microsphere uptake was seen across anti-amyloid antibody variants ranging from 9.79% (LECA hlgGl), to 7.81% (LECA Fc5) to 10.09% (BAPI hlgGl). The isotype control was significantly lower at 4.26% of cells positive for microsphere uptake (p < 0.05).

[0425] At 4 h post-treatment, BAPI / 3D6 hlgGl induced the highest levels of phagocytic activity (24.63%), followed by LECA Fc701 (22.92%), LECA hlgGl (20.41%), LECA Fc5 (18.45%), LECA a-gly (13.31%), and the isotype control (10.72%). Compared with LECA hlgGl, phagocytosis was significantly lower for the isotype control (p < 0.01) and LECA a-gly (p < 0.05).

[0426] At 24 h post-treatment (Figure2A and 2B), BAPI / 3D6 hlgGl induced the highest phagocytic activity (54.91%), followed by LECA Fc701 (49.85%), LECA hlgGl(46.31%), LECA Fc5 (40.75%), LECA a-gly (24.46%), and the isotype control (15.69%). Relative to LECA hlgGl, the isotype control (p < 0.0001) and LECA a-gly (p < 0.001) showed significantly lower phagocytic activity.

[0427] Conclusions: hlgGl Lecanemab effectively takes up amyloid-conjugated spheres, comparable to hlgGl bapineuzumab. The Lecanemab Fc5 and Fc701 variants retain the ability to phagocytose amyloid-conjugated spheres.

[0428] Example 10 - Pro-inflammatory gene expression

[0429] THP-1 cells were treated with anti-amyloid antibody variants to compare their effects on inflammatory gene expression, IL-6 and IL-lfi mRNA levels. Results are shown in Figures 12A-B.

[0430] IL-6 mRNA expression was increased in THP-1 cells following exposure to immobilised BAPI hlgGl and LECA hlgGl. LECA hlgGl induced the highest levels of IL-6 mRNA expression (2.95-fold relative to untreated cell controls), followed by BAPI hlgGl (2.74-fold). Exposure of THP-1 cells to LECA Fc5 (1.35-fold), LECA Fc701 (1.14-fold), and LECA a-gly (1.02-fold) induced lower levels of IL-6 mRNA with levels close to untreated cell control. Compared with LECA hlgGl, IL-6 expression was significantly lower for LECA Fc5 (p < 0.01), LECA a-gly (P<0.001), and LECA Fc701 (p < 0.001). IL-lfi induction was increased in THP-1 cells following exposure to immobilised BAPI hlgGl and LECA hlgGl. Levels of IL-lfi mRNA increased in THP-1 cells following exposure to LECA hlgGl (14.73-fold) and BAPI hlgGl (12.87-fold), LECA Fc701 (9.83-fold), LECA Fc5 (8.02-fold), and LECA a-gly (4.05-fold). Relative to LECA hlgGl, IL-10 expression was significantly lower for LECA Fc701 (p < 0.05), LECA Fc5 (p < 0.01), and LECA a-gly (p < 0.0001).

[0431] Conclusions: hlgGl Lecanemab induces cytokine production, comparable to hlgGl bapineuzumab. The Lecanemab Fc5 and Fc701 variants show reduced IL-6 and IL- ip production by THP-1 effector cells.

[0432] Example 11 - FcvR-binding properties of antibody Fc variants

[0433] Surface Plasmon Resonance (SPR) was performed to compare the binding properties of amyloid antibodies to the human Fey receptors FcyRI, FcyRIIa, FcyRIIb, and FcyRIIIa Sensorgrams are shown in Figure 13-17. Measurements at a single antibody concentration (100 nM), is shown reflecting receptor engagement rather than binding affinity.All antibodies bound FcyRI, with LECA hlgGl displaying the strongest peak binding response (1202.31 RU), followed by BAPI hlgGl (1065.66 RU), LECA Fc5 (842.17 RU), LECA Fc701 (807.12 RU), and LECA a-gly (283.47 RU). Gradual disengagement from FcyRI upon dissociation was observed for all antibodies except LECA a-gly, which showed a steep loss of signal, reflecting weak binding to FcyRI. The Fc701 shows a slower offrate than the Fc5 variant. BAPI hlgGl and LECA hlgGl antibodies displayed binding to FcyRIIa, FcyRIIb, and FcyRIIIa, while none of the aglycosylated antibodies gave measurable responses. BAPI hlgGl and LECA hlgGl antibodies engaged most strongly with FcyRIIa and FcyRIIIa, while FcyRIIb produced the faintest signal.

[0434] Figure 18 shows the kinetic parameters of 3D6 / Bapi and Lecanemab Fc variants for human FcyRI binding. These data confirm a slower off-rate of the 3D6 Fc701 variant, when compared to the Fc5 variant, as shown in Figure 13.

[0435] Conclusions: binding to FcyRI: hlgGl variants show strongest binding to FcyRI, followed by Fc701 and Fc5 variants, with Fc701 showing a slower offrate than Fc5 variants. The a-glycosylated (NA) variants show weak binding to FcyRI.

[0436] Discussion

[0437] The inventors' data shows that introducing the Fc5 or Fc701 mutations in aglycosylated hlgGl does not affect the specificity of the antibody, and confirms that the exemplified mutant hlgGl has capacity to bind to FcyRI. In vivo experiments using APP / PS1 transgenic mice show that Fc5 hlgGl 3D6 (Bapineuzumab) clears amyloid to the same extend as the non-engineered hlgGl version of 3D6. These experiments also show that this clearance of amyloid occurs in the absence of microglial activation. These observations were confirmed and extended using an in vitro phagocytosis assay using human THP-1 monocytes, showing effective uptake of antibody bound amyloid beads in the absence of cytokine production. In addition, experiments using APP / PS1 transgenic mouse brain tissue show that all Fc variants of 3D6 and Lecanemab, including the Fc5 and-Fc701 variants bind to structures resembling amyloid plaques. The Fc701 mutations improve selectivity for FcyRI and extend half-life in vivo.

[0438] Collectively, these data show that the inventors' novel amyloid-specific antibodies with selective binding to FcyRI retain the ability for it to phagocytose amyloid, but with reduced inflammation. Accordingly, the inventors have shown that the introduction of these Fc5 or Fc701 mutations results in antibodies with properties that can enhance immunotherapeutic effects by reducing adverse effects. Furthermore, the inventors believe that their novel approach can be applied not only to Bapineuzumab andLecanemab, but also to other hlgGl anti-amyloid antibodies, such as Aducanumab and Donanumab.

[0439] Conclusions

[0440] The innovative approach disclosed herein to generate an amyloid-targeting mAb that effectively clears amyloid, without inducing adverse effects, has great commercial application and a clear impact on people living with Alzheimer's Disease.

Claims

Claims1. An anti-amyloid beta antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an Fc region, which selectively targets Fc gamma RI (FcyRI).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to an epitope comprising or consisting of SEQ ID No: 2, SEQ ID No: 24, SEQ ID No: 25, and / or SEQ ID No: 26.

3. The antibody or antigen-binding fragment thereof according to either claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of IgA; IgD; IgE; IgG and IgM, optionally wherein the heavy chain is an IgGl.

4. The anti-amyloid beta antibody or antigen-binding fragment thereof according to any preceding claim, wherein the anti-amyloid beta antibody or antigenbinding fragment thereof is aglycosylated.

5. The anti-amyloid beta antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a modified amino acid residue, wherein the modified amino acid residue is N297 with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11.

6. The anti-amyloid beta antibody or antigen-binding fragment thereof according to claim 5, wherein the N297 amino acid residue is modified to alanine (N297A), with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11.

7. The anti-amyloid beta antibody or antigen-binding fragment thereof according to any preceding claim, wherein the selectivity with which the Fc region of the anti-amyloid beta antibody or antigen-binding fragment thereof targets Fc gamma RI (FcyRI) is between 3 nM and 50 nM, or between 4 nM and 15 nM.

8. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises amodified E382 residue and / or a modified M428 residue, with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the E382 amino acid residue is modified to valine (E382V) and / or the M428 amino acid residue is modified to isoleucine (M428I).

10. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a modified Q295 residue, a modified L328 residue, a modified P331 residue, a modified 1332 residue, a modified E382 residue, and / or a modified M428 residue, with reference to the heavy chain constant (CH) region provided in SEQ ID No: 11.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the Q295 amino acid residue is modified to arginine (Q295R), the L328 amino acid residue is modified to tryptophan (L328W), the P331 amino acid residue is modified to alanine (P331A), the 1332 amino acid residue is modified to tyrosine (I332Y), the E382 amino acid residue is modified to valine (E382V), and / or the M428 amino acid residue is modified to leucine (M428L).

12. The anti-amyloid beta antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises amyloid-beta-binding CDRs from the monoclonal antibody Bapineuzumab, Aducanumab, Lecanemab, and / or Donanumab.

13. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 3, a CDR-H2 domain comprising or consisting of SEQ ID No: 4, a CDR-H3 domain comprising or consisting of SEQ ID No: 5, a CDR-L1 domain comprising or consisting of SEQ ID No: 7, a CDR-L2 domain comprising or consisting of SEQ ID No: 8, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 9.

14. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 6, and / or a light chain variable region comprising or consisting of SEQ ID No: 10.

15. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11, and a light chain constant region comprising or consisting of SEQ ID No: 13.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 15, a CDR-H2 domain comprising or consisting of SEQ ID No: 16, a CDR-H3 domain comprising or consisting of SEQ ID No: 17, a CDR-L1 domain comprising or consisting of SEQ ID No: 19, a CDR-L2 domain comprising or consisting of SEQ ID No: 20, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 21.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-12 or 16, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 18, and / or a light chain variable region comprising or consisting of SEQ ID No: 22.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, 16, or 17, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11, and a light chain constant region comprising or consisting of SEQ ID No: 13.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 27 , a CDR-H2 domain comprising or consisting of SEQ ID No: 28, a CDR-H3 domain comprising or consisting of SEQ ID No: 29, a CDR-L1 domain comprising or consisting of SEQ ID No: 31, a CDR-L2 domain comprising or consisting of SEQ ID No: 32, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 33.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1-12 or 19, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 30, and a light chain variable region comprising or consisting of SEQ ID No: 34.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, 19, or 20, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 11, and a light chain constant region comprising or consisting of SEQ ID No: 13.

22. The antibody or antigen-binding fragment thereof according to any preceding claim, for use in therapy.

23. The antibody or antigen-binding fragment thereof according to any preceding claim, for use in treating, preventing or ameliorating a beta-amyloid-mediated neurodegenerative disorder.

24. The antibody or antigen-binding fragment thereof, for use according to claim 23, wherein the neurodegenerative disorder is selected from a group consisting of: Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); Frontotemporal Dementia, and Dementia with Lewy Bodies.

25. The antibody or antigen-binding fragment thereof, for use according to claim 23 or 24, wherein the neurodegenerative disorder is Alzheimer's disease.

26. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-21, and optionally a pharmaceutically acceptable vehicle.

27. A process for making the pharmaceutical composition according to claim 26, the process comprising combining a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined in any one of claims 1- 21, with a pharmaceutically acceptable vehicle.

28. A polynucleotide sequence encoding the antibody, or antigen-binding fragment thereof according to any one of claims 1-21.

29. An expression cassette comprising a polynucleotide sequence according to claim 28.

30. A recombinant vector comprising the expression cassette according to claim 29.

31. A host cell comprising the polynucleotide sequence according to claim 28, the expression cassette according to claim 29, or the vector according to claim 30.

32. A method of preparing the antibody, or antigen-binding fragment according to any one of claims 1-21, the method comprising:a) introducing, into a host cell, the vector of claim 30; andb) culturing the host cell under conditions to result in the production of the antibody, or antigen-binding fragment according to any one of claims 1-21.

33. The antibody or antibody binding fragment of according to any one of claims 1- 21, for use in diagnosis or prognosis.

34. The antibody or antibody binding fragment of according to any one of claim 1- 21, for use in diagnosing or prognosing a neurodegenerative disorder.

35. A method of diagnosing or prognosing a neurodegenerative disorder in a subject, the method comprising detecting beta amyloid in a biological sample obtained from the subject with the antibody or antibody binding fragment of according to any one of claims 1-21.

36. The method according to claim 35, wherein the neurodegenerative disorder is selected from a group consisting of: Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); Frontotemporal Dementia, and Dementia with Lewy Bodies.

37. The method according to either claim 35 or 36, wherein the neurodegenerative disorder is Alzheimer's disease.

38. A kit for diagnosing a subject suffering from a neurodegenerative disorder, or for providing a prognosis of the subject's condition, the kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1- 21 for detecting beta amyloid in a sample from a test subject.

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