IL322931A - Humanised antibody against amyloid beta 42 - Google Patents

Description

WO 2024/194298 PCT/EP2024/057283 HUMANISED ANTIBODY AGAINST AMYLOID BETA 42 Field of the inventionThe present invention relates to a new humanised antibody, and its use in the treatment of an amyloid disease such as Alzheimer’s Disease and related disorders.

BackgroundAlzheimer’s Disease (AD) is a progressive neurological disorder characterised by the loss of synaptic function eventually leading to neuronal death. One of the main histopathological features of AD is the extracellular deposition of insoluble amyloid aggregates of the amyloid- (AP) peptide in plaques, essentially insoluble fibrillar assemblies, in the brain, and the role of these plaques in the etiology of AD has been the subject of intense study. Various antibodies have been developed which target the plaques. These antibodies are typically either selective for aggregated forms of Ap or specific for a pyroglutamated form of Ap. However, so far, the clinical benefit of such antibodies has been very limited.Recent work has shown that plaques are not in fact the most toxic forms of Ap, and that the neurotoxicity of Ap stems mostly from soluble oligomeric aggregates of the amyloid-p-1-42 peptide (Ap42). Remarkably, of all Ap present in AD patients’ brains, only a very small fraction of soluble aggregated oligomeric Ap42 carries most of the neurotoxic potential (Hong etaL, Acta Neuropathologica 136, 19-40, 2018). In other words, the natural toxic form of Ap is of very low abundance, and this has hampered the development of beneficial therapies, as therapeutics which target or cross-react with inert, non-toxic, forms of Ap may never reach the true toxic species.A monoclonal antibody has been developed by the present applicant Alzinova AB (WO 2012/120035; Sandberg etaL, Alz Res Therapy 14, 196, 2022). This antibody, named ALZ-201 (mAb20), is specific for a subset of soluble oligomeric Ap42 but does not bind to other forms of Ap (e.g. to insoluble aggregates or plaques, or to monomeric or non-aggregated AP). ALZ-201 was generated using as the immunogen a stabilised form of the Ap42 peptide, Ap42- CC, which constrains the conformation of the peptide, inhibiting its fibrillization Ap42CC accumulates as toxic prefibrillar oligomers with p structure and is being developed by Alzinova as a vaccine candidate, named ALZ-101 (WO WO 2024/194298 PCT/EP2024/057283 2009/128772), which is presently in clinical trials (NCT05328115). The resulting ALZ-201 antibody thus recognises a conformational epitope on the stabilised soluble oligomeric antigen (i.e. on the vaccine candidate). The conformational specificity of the antibody has been confirmed, and it has been shown to neutralise the neurotoxic effect of post-mortem brain extracts from patients with AD in mouse primary neuronal cultures; when the AD brain extracts were immunodepleted using ALZ-201, the loss of nuclei, neurites, synapses, and branching was reduced as compared to non-depleted extracts, comparable to the reduction in neurotoxicity achieved by immunodepletion using a pan-specific anti-Ap antibody (4G8) which binds to all forms of Ap (Sandberg etal., Alz Res Therapy 14, 196, 2022). Further non-clinical efficacy assays have been performed in learning tests in zebrafish embryos after injecting human extracts into the brain (Sandberg etal., poster #43003 presented at the Alzheimer’s Association International Conference, USA, 2020, July 26-30). These data again demonstrate that the toxic effects of AD brain extracts may be prevented by immunodepletion with ALZ-201.Accordingly, despite targeting only a small fraction of patient-derived Ap, studies have shown that ALZ-201 has a high neutralising effect on neurotoxicity of patient-derived Ap. In light of such promising pre-clinical studies, monoclonal antibody (mAb) ALZ-201 is being developed for clinical use. To this end, a humanised antibody is required.

Summary of the inventionIn a first aspect of the invention, provided herein is an antibody comprising an antigen-binding domain capable of binding specifically to Ap42 prefibrillar oligomers with p structure, said antigen binding domain comprising:(i) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO.1; or(ii) a light chain variable region (VL) comprising the sequence of SEQ ID NO.2;or a combination thereof.In particular, the antibody, or more particularly the binding domain thereof, does not bind, or exhibits substantially no or negligible binding, to monomers or unstructured oligomers of Ap (e.g. of Ap42), nor to fibrillar forms of Ap (e.g. of Ap42).

WO 2024/194298 PCT/EP2024/057283 The antibody may be presented in different formats. It may be monovalent or bivalent. Further, it may be in a single chain format, or it may comprise two or more separate chains, e.g. 4 chains. It may comprise both the VH and VL regions, for example a polypeptide, or chain, comprising a VH region, and a polypeptide, or chain, comprising a VL region. However, binding proteins in the form of, or comprising, single domain antibodies are also known, and are included herein, which comprise only VH or only VL.The antibody may be an intact or full-length antibody or a fragment thereof, notably a fragment that retains the antigen-binding domain of the antibody. Accordingly, the antibody may contain one or more, e.g. two, antigen-binding domains.In an embodiment, the heavy chain variable region (VH) consists of the sequence of SEQ ID NO.1. In an embodiment, the light chain variable region (VL) consists of the sequence of SEQ ID NO.2.The antibody may be provided in an immunoglobulin (ig) format, and more particularly an Ig format comprising 2 heavy and 2 light chains, or as a fragment thereof. However, as described in more detail below the antibody may be in the form of any construct comprising the antigen-binding domain. This may include for example fusion proteins comprising the antibody and another protein, or bi-specific constructs.Accordingly, in one embodiment, the antibody is in the form of a full-length Ig antibody, or an antigen-binding fragment thereof (that is, a fragment which comprises the antigen binding domain). In a specific embodiment, the antibody is an IgG antibody, or a fragment thereof.Thus, the antibody may comprise a first chain (a heavy chain) with the VH region and a heavy chain constant region, and a second chain (a light chain) with the VL region and a light chain constant region. In a particular embodiment, the light chain constant region is a kappa (k) constant region. In another particular embodiment, the heavy chain constant region is an IgG constant region, more particularly an IgG 1 constant region. The antibody may comprise one or more of each of said first and second chains, e.g. 2 heavy chains, and 2 light chains.In a specific embodiment, the antibody is an lgG1 Kappa antibody, or a fragment thereof.In another aspect, provided herein is a conjugate comprising the antibody as defined herein linked to at least one diagnostic agent.

WO 2024/194298 PCT/EP2024/057283 In a further aspect, provided herein is an antibody as defined herein for use in therapy.In another aspect, provided herein is an antibody or a conjugate thereof for use in diagnosis practiced on a human or animal body (i.e. in vivo diagnosis).In a further aspect, provided herein is a pharmaceutical composition comprising an antibody as defined herein, in admixture with at least one pharmaceutically acceptable carrier or excipient.In a still further aspect, provided herein is an antibody or a pharmaceutical composition as defined herein, for use in the treatment of an amyloid disease.In a still further aspect, provided herein is a conjugate as defined herein for use in in vivo diagnosis of an amyloid disease.A related aspect provides use of an antibody as defined herein in the manufacture of a medicament for use in the therapy of an amyloid disease.A further related aspect provides a method of treatment of an amyloid disease, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, or pharmaceutical composition, as defined herein.The various medical uses and methods set out above are particularly for the treatment or prevention of an amyloid disease.In certain embodiments of the various uses and methods above the amyloid disease may be any one of Alzheimer’s Disease (AD), Down’s syndrome, or Inclusion Body Myositis (IBM).The subject may be any human or animal subject, particularly a human.Another aspect provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody as defined herein, or a VH and/or VL region thereof.It will be understood in this regard that the antibody may be composed of, or may comprise, one or more protein chains, or sub-units, e.g. 2 or more, and in such a form the individual chains or subunits, e.g. the individual molecules (i.e. polypeptides) comprising the VH and/or VL regions may be encoded by separate nucleic acid molecules.Accordingly, this aspect can be seen to provide one or more nucleic acid molecules comprising nucleotide sequences that encode the antibody as defined herein.Also provided herein is a vector comprising the nucleic acid molecule, or the one or more nucleic acid molecules, as defined herein. Analogously, this aspect WO 2024/194298 PCT/EP2024/057283 can also be seen to provide one or more expression vectors comprising the one or more nucleic acid molecules as defined herein.In an embodiment the vector(s) is(are) an expression vector(s).The vector may be a viral vector. Accordingly, also provided is a virus comprising one or more nucleic acid molecules as defined herein.Yet another aspect provides a host cell comprising a vector (e.g. an expression vector) or a nucleic acid molecule as defined herein (or more particularly the one or more vectors or nucleic acid molecules as defined herein), or a host cell expressing an antibody as defined herein.The host cell can be prokaryotic or eukaryotic. In an embodiment the host cell is a mammalian host cell.A yet further aspect provides a method for producing an antibody as defined herein, said method comprising culturing a host cell as defined herein under conditions suitable for expression of the antibody.The method may further comprise obtaining (e.g. collecting or isolating) the antibody from the host cell or from the culture (e.g. from the growth medium or supernatant after culture).The method may further comprise the step of introducing into the host cell one or more nucleic acid molecules or vectors as defined herein.

Detailed description of the inventionThe murine antibody ALZ-201 was humanised by grafting the complementarity-determining regions (CDRs), as defined by the Kabat nomenclature, from each of the light chain variable region (VL) and heavy chain variable region (VH) into a human germline VL and VH respectively that was selected to be as close as possible, in terms of sequence homology, to the corresponding murine VL and VH.The term “VH” (or VH domain) or "heavy chain variable region" refers to the variable region of a heavy chain of an antibody molecule. The VH comprises three heavy chain CDRs (VHCDRs) termed VHCDR1, VHCDR2 and VHCDR3 from the amino terminus to carboxy terminus.The term “VL” (or VL domain) or "light chain variable region" refers to the variable region of a light chain of an antibody molecule. The VL comprises three light chain CDRs (VLCDRs) termed VLCDR1, VLCDR2 and VLCDR3 from the amino terminus to the carboxy terminus.

WO 2024/194298 PCT/EP2024/057283 The CDRs are regions of hypervariability within variable regions.The heavy chain and light chain variable regions each also have four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions are more conserved and separate the CDRs.A number of factors need to be considered in designing a functional humanised antibody, and humanization is not a straightforward process.As noted above, and described in more detail in Example 1 below, in preparation for humanisation a molecular model of antibody ALZ-201 was constructed, itself not a trivial task requiring extensive human input, and this was used to guide the humanisation design. Guided by the molecular model, the first step in the humanisation design was performed in silica, again involving various design considerations and options, including the selection of particular human germline sequences, and of residues in the human framework regions to be “back- mutated ”, that is to be substituted with the parental murine amino acid. Further, during the design process, residues in the CDRs were selected for “germ-lining” (i.e. substitution with their human germline counterparts). This led to the design of different humanised heavy chain variable region (VH) sequences based on different human germline VH sequences, and 8 different humanised light chain variable region (VL) sequences, based on 4 different human germline VL sequences. Thus, various different possible humanised VH/VL combinations were derived, representing different options for humanised variants of ALZ-201. In the next step, various selection criteria were used to narrow the list of 48 combinations to 18, from which 16 combinations were selected for experimental testing, alongside a chimeric variant of ALZ-201 herein denoted either chALZ-201 or mAb15. The candidate combinations were subject to iterative ranking based on degree of, or more particularly, % humanness (% identity with human amino acid residues, or put another way, % sequence identity of the humanised VH and VL sequences with the human V gene sequences; the % sequence identity to human homologous antibodies ranged from 81.0% to 88.7%), and on the results of experimental tests on expression yield, affinity, and thermal stability. More particularly, 6 candidates were selected for further testing of affinity (EC50 as determined by ELISA) and temperature stability, and the selections and testing were again narrowed down to 5 for testing of affinity by SPR (Kd (nM) as determined by Biacore), then to two top candidates for testing of expression yield, WO 2024/194298 PCT/EP2024/057283 leading ultimately to the selection of the lead candidate antibody, identified as Abin the Examples below.The humanised antibody which has been obtained is defined by particular VH and VL sequences. It is well known in the art that antibodies may be provided in various formats and configurations, including various artificial constructs, beyond the classical format of immunoglobulins and their fragments. As well as antibodies and constructs comprising both a VH and VL region, so-called single-domain antibodies may be prepared, which comprise only VH, or only VL. Thus, provided herein is generally an antibody which is defined by the humanised VH and/or VL sequences of the humanised ALZ-201 derivative that has been obtained. The antibody comprises an, or at least one, antigen binding domain (or alternatively put, an antigen-binding unit). Since the antigen binding domain is obtained, or derived from an antibody, the antibody may be referred to, or regarded as an antibody- based binding protein, or as a binding protein comprising an antibody-derived binding domain.The antigen-binding domain of the antibody comprises at least one VH region or at least one VL region. In an embodiment the antigen binding domain comprises at least one VH region and at least one VL region.The binding protein may comprise one or more antigen-binding domains. Each antigen-binding domain may comprise a (or at least one) VH region, a (or at least one) VL region, or both a VH and a VL domain (or a least one VH region and at least one VL region). In a typical antibody format, the binding protein comprises two antigen-binding domains (in other words it is bivalent), and more particularly each said antigen-binding domain comprises a VH and a VL region. However, as discussed further below both monovalent antibodies or antibody fragments are known, which comprise a single antigen-binding domain. In such an embodiment the single antigen-binding domain may comprise both a VH and a VL region. Further, and noted above, the binding protein may comprise an antigen-binding domain comprising solely a VH region or solely a VL region, e.g. as a single-domain antibody, for example a VHH antibody, or a camelid antibody.As used herein, the terms "a" and "an" are used in the sense that they include "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated.

WO 2024/194298 PCT/EP2024/057283 In addition, where the terms comprise , comprises , has or having , or other equivalent terms are used herein, then in some more specific embodiments, these terms include the term “consists of or “consists essentially of, or other equivalent terms.As noted above, the VH region comprises an amino acid sequence as set forth in SEQ ID NO.1.

VH (SEQ ID NO. 1)QVTLKESGPTLVKPTQTLTLTCTFSGFSLSTFGSGVSWIRQPPGKALEWLAHIYW DDDKHYNPSLKSRLTITKDTSKNQVVLTITNMDPVDTATYFCARRESHYYGSGYYF DYWGQGTLVTVSS The VL region comprises an amino acid sequence as set forth in SEQ ID NO. 2.

VL (SEQ ID NO. 2)DIQLTQSPSSLSASVGDRVTITCRASSSISYMHWYQQKPGKAPKPWIYATSNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWRSDPLTFGGGTKVEIK As also noted above each of these variable region sequences comprises complementarity determining regions (CDRs, which are underlined in the sequences above), and four framework regions (FR).The Kabat numbering scheme is a widely adopted standard for numbering and assigning CDR residues in an antibody based on sequence alignment data (Johnson G. and Wu T.T., Nucleic Acids Res. 28, 214-18, 2000). It assigns the VHCDR1 to residues 31-35B (position 35 is a defined insertion point, and the terminology 35B is understood in the art to convey this), the VHCDR2 to residues 50-65, and the VHCDR3 to residues 95-102. Conversely, it assigns the VLCDRto residues 24-34, the VLCDR1 to residues 50-56, and the VLCDR3 to residues 89-97.The Chothia CDR definitions were later introduced based on available antibody structures (Chothia, C. and Lesk, A.M., J. Mol. Biol. 196, 910, 1987). The AbM method (K.R. Abhinandan K.R. and Andrew C.R. Martin A.C.R., Molecular Immunology 45, 3832-3839, 2008) utilises a modified version of the Chothia scheme and assigns the VHCDR1 to residues 26-35B, VHCDR2 to residues 50-58, WO 2024/194298 PCT/EP2024/057283 and VHCDR3 to residues 95-102. Conversely, VLCDR1 is assigned to residues 24- 34, VLCDR2 to residues 50-56, and VLCDR3 to residues 89-97. These CDRs are underlined in SEQ ID NO 1 and SEQ ID NO 2 above.The AbM scheme was used to define the CDR sequences of the parental antibody (WO2012120035A1). These CDR sequences correspond to the CDR sequences of the parental antibody ALZ-201, except VLCDR1 which for the chosen humanised antibody is comprised of residues RASSSISYMH (SEQ ID NO. 17) whereas for the non-humanised parental antibody ALZ-201 it is comprised of residues RASSSVSYMH (SEQ ID NO. 18). Hence, the amino acid position 29 of the humanised derivative is an isoleucine instead of a valine.An alternative method for assigning CDRs is referred to as the IMTG scheme (Lefranc M-P et al., Developmental & Comparative Immunology 27, 55-77, 2003). Utilising this scheme would assign the VHCDR1 to residues 26-35B, VHCDR2 to residues 51-56, and VHCDR3 to residues 93-102. Conversely, VLCDR1 would be assigned to residues 27-32, VLCDR2 to residues 50-51, and VLCDR3 to residues 89-97.The antibody, as noted above, may take various forms, including antibody fragments. All such forms are included. As noted above the antibody may comprise one or more antigen-binding domains, or one or more VH and/or one or more VL regions. The VH and/or VL regions may be comprised in a single chain (polypeptide) or on separate chains (polypeptides). The antibody may accordingly comprise one or more polypeptide chains, e.g. 2 or 4 polypeptides. An individual polypeptide may comprise one or more VH and/or more one or more VL regions, e.g. both a VH and a VL region etc.Accordingly, an antigen-binding domain may comprise one or more polypeptides (chains), each comprising one or more VH and/or one or more VL regions.Further, a polypeptide (chain) comprising a variable region sequence may comprise all or part of a constant region sequence, for example one, two or all 3 of CH1, CH2 and CHS of a heavy chain constant region in the case of VH, and all or part of the light chain constant region (CL) in the case of VL. Since the VH and VL herein have been humanised, in particular the constant region sequence is a human, or humanised, sequence, especially a human sequence.Thus, the term “antibody ” includes all known forms of antibody, including whole, or full-length, antibodies, or any antigen-binding fragments thereof, or single WO 2024/194298 PCT/EP2024/057283 chains or single-chain derivatives thereof, as well as synthetic or artificial antibody constructs which comprise at least one VH or at least one VL region as defined herein, and multimers thereof, e.g. dimeric, trimeric or higher order multimeric antibodies. It will be understood that the term includes recombinant and engineered antibodies.More broadly, the term “antibody ” can be seen to include any binding protein (which may be referred to as an immunological binding protein) which comprises an antigen-binding domain, specifically an antigen-binding domain derived from an antibody. Accordingly, the term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region obtained or derived from an antibody. In a preferred embodiment the antibody or fragment thereof comprises at least one VH region and a least one VL region.In one embodiment, the antibody is an immunoglobulin antibody, and more specifically an antibody comprising at least 2 heavy chains and at least 2 light chains, or a fragment thereof.In any format herein, wherein the antibody comprises constant regions, the heavy chain comprises the VH herein and all or part of a heavy chain constant region and the light chain comprises the VL herein and all or part of a light chain constant region. When the antibody comprises a full complement of constant regions from the heavy and light chains it is referred to as a whole antibody, or full- length antibody. Such full-length/whole antibodies represent one preferred embodiment.Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM and any of these are included, although IgA and IgG are preferred, particularly IgG. Several of these are further divided into subclasses or isotypes, such as lgG1, lgG2, lgG3, lgG4, and the like, for example camelid antibodies are IgG antibodies which often have lgG2 or lgG3 constant domains. All sub-classes are included herein. The heavy-chain constant domains that correspond to the difference classes of immunoglobulins are termed a, 5, 8, y and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Suitable heavy chain constant regions are known and available in the art.The light chains of mammalian antibodies are assigned to one of two clearly distinct types: kappa (k) and lambda (X), and either of these may be used.

WO 2024/194298 PCT/EP2024/057283 Again, suitable light chain constant region sequences are known and available in the art.In an embodiment the heavy chain constant region is or comprises all or part of the human IgG 1 constant region having the amino acid sequence set forth in SEQ ID NO.3 or an amino acid sequence having at least 90% sequence identity thereto.

SEQ ID NO. 3, human lqG1 constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KIn another embodiment, the light chain constant region is or comprises all or a part of the human kappa constant region having the amino acid sequence set forth in SEQ ID NO. 4 or an amino acid sequence having at least 90% sequence identity thereto.

SEQ ID NO. 4 Human kappa constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In more particular embodiments, the amino acid sequence has at least 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 3 or 4 respectively.Thus, some variation in the constant region sequences in the binding proteins/antibodies herein is allowed for.Sequences which have at least 90% sequence identity to a stated reference sequence may be referred to as substantially identical or substantially similar sequences. Such sequences may include single or multiple base or amino acid alterations (additions, substitutions, insertions or deletions) to the sequences. Amino acid substitutions may be conservative substitutions. Such sequence modifications may be made for various reasons, including for example, for ease of production, stability, pharmacokinetics of the antibody etc.

WO 2024/194298 PCT/EP2024/057283 A conservative amino acid substitution is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). In other examples, families of amino acid residues can be grouped based on hydrophobic side groups or hydrophilic side groups.Sequence identity may be assessed by any convenient method. However, for determining the degree of identity between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used together with BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1, so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.Generally, computer programs will be employed for such calculations. Programs that compare and align pairs of sequences, like ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST (Altschul etal., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, WO 2024/194298 PCT/EP2024/057283 BLASTN, or GOG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998). The LALIGN pairwise sequence alignment program is available from EMBL-EBI.Sequences according to the present invention having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity may be determined using any of the available programs, for example the LALIGN program, with default parameters.In one embodiment the antibody is an IgG antibody, and more particularly an lgG1 antibody, ora fragment thereof.In one particular embodiment the antibody is an IgGkappa antibody, or a fragment thereof.In another embodiment the antigen binding domain comprises:(i) a heavy chain sequence comprising the VH sequence of SEQ IDNO. 1 linked to the heavy chain constant region sequence of SEQ ID NO. 3 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 3; and/or(ii) a light chain sequence comprising the VL sequence of SEQ ID NO. linked to the light chain constant region sequence of SEQ ID NO. or an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 4.Antibody fragments that retain the ability of their full-length parent antibody to bind to antigen or which, put another way, comprise an antigen-binding domain, include: Fab', Fab, F(ab')2, Fd, Fv, single domain antibodies (DABs), e.g. consisting of a VH domain (also known as VHH antibodies), and nanobodies.Other antibody formats known in the art and encompassed herein include single chain formats including scFv (single chain Fv), dsFv, ds-scFv, linear antibodies, TandAbs, minibody, diabody, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lambda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-lg (dual variable domain antibody, bispecific format); scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting") and such like.

WO 2024/194298 PCT/EP2024/057283 Single domain antibodies include camelid antibodies, vNAR (shark) antibodies, VH antibodies or VL antibodies.The techniques for preparing the various antibody formats, fragments and constructs are well known in the art.Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be produced by recombinant techniques, cell-free expression systems, or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art.As noted above, in certain embodiments, the antibody, or antibody fragment, comprises all or a portion of a heavy chain constant region. Furthermore, the antibody or antibody fragment can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region, or a portion thereof. All or part of such constant regions may be produced naturally or may be wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art, and exemplified above.In other embodiments no constant regions, e.g. no heavy chain or light chain constant regions, are present, e.g. a variable domain or heavy chain variable domain (VH) is the only part of an antibody that is present.The antibodies herein, and the nucleic acids that encode them, are artificial (e.g. engineered or synthetic) constructs that do not occur in nature, and do not correspond to molecules that occur naturally. In other words, the antibodies and nucleic acids are non-native. This is implicit from their composition.In an embodiment, the antibody may be provided in the form or a conjugate comprising the binding protein linked or attached (i.e. conjugated) to another entity. This may be another protein or polypeptide component, which may be conjugated to the binding protein via a peptide bond or peptide linker, in which case the conjugate may be regarded as a fusion protein comprising the binding partner linked (or fusion to) a fusion partner. Alternatively, the other entity may be non- proteinaceous in nature, for example it may be a small molecule or other chemical or physical entity (e.g. polymer, label etc.) and may be linked to the binding protein WO 2024/194298 PCT/EP2024/057283 chemically be procedures and linking groups well known in the art. As will be described in more detail below, such a fusion partner or conjugated entity may be a therapeutic or diagnostic agent.A person skilled in the art will appreciate that the antibodies and antibody fragments, may be prepared in any of several ways well known and described in the art, but are conveniently prepared using recombinant methods.To that end, nucleic acid molecules (e.g. one or more nucleic acid molecules) comprising nucleotide sequences that encode the antibodies as defined herein or parts or fragments thereof represent other aspects herein.In this regard, based on the VH and VL amino acid sequences of SEQ ID NO.s 1 and 2, appropriate encoding nucleotide sequences can be devised. These may be codon-optimised for expression in a desired host cell according to techniques and principles well known in the art. In an embodiment the nucleotide sequences are codon-optimised for expression in mammalian cells.An exemplary nucleotide sequence encoding the VH sequence of SEQ ID NO. 1 is set forth in SEQ ID NO. 5 below and an exemplary nucleotide sequence encoding the VL sequence of SEQ ID NO. 2 is set forth in SEQ ID NO. 6 below. Here, the first nine nucleotides, GCCGCCACC, constitute the protein translation initiation site. Both SEQ ID NO.5 and 6 have been optimised for expression in CHO cells and include signal peptides for protein secretion (underlined nucleotides).

SEQ ID NO.5GCCGCCACCATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCTGCTCCTC GGTGGGTGCTGTCCCAGGTGACCCTGAAGGAGTCCGGCCCCACCCTGGTGA AGCCCACCCAGACCCTGACCCTGACCTGCACCTTCAGCGGCTTTAGCCTGAG CACCTTTGGCAGCGGCGTGAGCTGGATCAGGCAGCCTCCCGGCAAGGCCCT GGAGTGGCTGGCTCACATCTATTGGGACGACGACAAGCACTATAACCCTAGC CTGAAGAGCCGGCTGACCATCACCAAGGACACCAGCAAGAACCAGGTGGTGC TGACCATCACAAACATGGACCCTGTGGATACCGCCACCTATTTTTGCGCCCGG AGGGAGAGCCACTACTATGGCAGCGGCTACTATTTCGATTATTGGGGCCAGG GCACCCTGGTGACCGTGAGCAGC WO 2024/194298 PCT/EP2024/057283 SEQ ID NO.GCCGCCACCATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGA TCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGTCCCCTTCCAGCCTGAG CGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCGGGCTTCCTCCAGCATC TCCTATATGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCCTTGGAT CTACGCTACCAGCAATCTGGCTAGCGGCGTGCCTAGCCGGTTCTCCGGCTCC GGATCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGATTT TGCTACCTACTACTGCCAGCAGTGGCGGTCCGATCCCCTGACCTTCGGCGGC GGAACCAAGGTGGAGATCAAG Accordingly, in an embodiment the nucleic acid molecule comprises a nucleotide sequence as set out in SEQ ID NO. 5 or a sequence having at least 90% sequence identity thereto, and/or a nucleotide sequence as set out in SEQ ID NO. or a sequence having at least 90% sequence identity thereto. In more particular embodiments the % sequence identity to SEQ ID NO. 5 or 6 is at least 92, 93, 94, 95, 96, 97, 98, or 99%.Such variant sequences may be degenerate sequences which encode the amino acid sequences as set out in SEQ ID NOs. 1 and 2 respectively.The antibodies and nucleic acid molecules herein may also include non- sequence-based modifications or chemical equivalents of the amino acid and nucleotide sequences, as long as these do not substantially alter the function of the antibodies or nucleic acid molecule. Thus, the antibody should retain the binding and functional properties of the corresponding unmodified antibody.The antibody may also comprise other amino acid sequences, or example tag sequences, fusion partners, or other components that do not contribute to the binding of antigen, or alterations to convert one type or format of antibody molecule or fragment to another type or format of antibody molecule or fragment (e.g. conversion from VHH to Fab or scFv or whole antibody, e.g. a full length heavy chain only antibody, or vice versa), or the conversion of an antibody molecule to a particular class or subclass of antibody molecule (e.g. the conversion of an antibody molecule to IgG or a subclass thereof, e.g. lgG2 or lgG3, for example to a camelid antibody, or to an IgA class of antibody), or the preparation of an Fc fusion, e.g. a VHH-Fc fusion. Such additional amino acid sequences may be encoded by additional nucleotide sequences present in the nucleic acid molecule.

WO 2024/194298 PCT/EP2024/057283 A person skilled in the art will appreciate that binding assays can be used to test whether any such modified antibodies and constructs retain the binding properties of the original humanised antibody.Nucleic acid fragments encoding the VH and VL sequences herein can be derived or produced by any appropriate method, e.g. by cloning or synthesis.Once nucleic acid fragments encoding the VH and VL have been obtained, these fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region fragments into full length antibody molecules with appropriate constant region domains, or into particular formats of antibody fragment discussed elsewhere herein, e.g. single domain antibodies such as VHH, Fab fragments, scFv fragments, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the VH and/or VL and optionally other sequences are generally incorporated into one or more vectors, most notably appropriate expression vectors, in order to facilitate production of the antibodies or for example to facilitate selection or screening, e.g. by incorporating into phage display vectors.Conveniently, the vectors may comprise one or more nucleotide sequences encoding other amino acid sequence(s) intended to form part of the antibodies, for example a constant region sequence.Possible expression vectors include but are not limited to cosmids, plasmids, or viral vectors, e.g. modified viruses (e.g. replication defective retroviruses, lentirviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. A variety of different vectors are available in the art for such use, including for example the pXtenl plasmid, as used in the XtenCHO™ mammalian expression system available from ProteoGenix. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain the nucleic acid molecule and regulatory sequences selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that allows expression of the nucleic acid.Accordingly, another aspect herein is a vector, particularly an expression vector, containing or comprising a nucleic acid molecule as defined or described herein, or a fragment thereof. In the case of an expression vector, this comprises also the necessary regulatory sequences for the transcription and translation of the WO 2024/194298 PCT/EP2024/057283 protein sequence encoded by the nucleic acid molecule. The vector, or expression vector, may be recombinant.For production of the antibody in host cells, it may be desirable to provide the expressed protein or protein subunit (i.e. the expressed polypeptide) with a signal or leader sequence, to direct the expressed product to a particular compartment or location, for example a secretory signal sequence, to allow the protein to be expressed extracellularly. It may then conveniently be obtained, or collected, from the supernatant or growth medium.By way of representative examples, the encoded amino acid sequences of polypeptides expressed in the Examples are set out below, comprising leader sequences: Heavy chain polypeptide- SEQ ID NO. 7 MKHLWFFLLLVAAPRWWLSQVTLKESGPTLVKPTQTLTLTCTFSGFSLSTFGSGVS WIRQPPGKALEWLAHIYWDDDKHYNPSLKSRLTITKDTSKNQVVLTITNMDPVDTA TYFCARRESHYYGSGYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Features:leader sequence SIP: [1 :19] underlined; VH Human lgG1 constant region: 144 :473] bold Light chain polypeptide- SEQ ID NO. 8 MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCRASSSISYMHWYQ QKPGKAPKPWIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWR SDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC WO 2024/194298 PCT/EP2024/057283 Features:leader sequence SIP: [1 :20] underlined; VL Human kappa constant region: [127 :233] bold Expression vectors can be introduced into host cells to produce a transformed, transduced or transfected host cell. The terms "transformed ", “transduced ”, and "transfected" are typically used to refer to different methods of introducing a nucleic acid into a cell (by plasmid, viral vector, or non-viral methods more generally). As used herein, the terms are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming, transducing and transfecting host cells are well known in the art and can be found in laboratory textbooks.Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells, or plant cell-based or fungal expression systems (e.g. Saccharomyces cerevisiae) can be used, as will be well known to a person skilled in the art. For example, the binding proteins may be expressed in yeast cells, such as Pichia or Saccharomyces, e.g. Saccharomyces cerevisiae, insect cells, or mammalian cells, such as for example CHO cells, or other cell lines. In addition, the proteins of the invention may be expressed in prokaryotic cells, such as Escherichia coli (E. coli).In vitro, cell-free protein expression is another means by which to produce recombinant proteins in solution using biomolecular translation machinery extracted from cells. The proteins may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.Where the VH and VL are provided as or in separate chains (i.e. separate polypeptides are expressed), they may be encoded by nucleotide sequences provided in separate vectors. Accordingly, a yet further aspect provides an expression construct or expression vector or expression system (e.g. a viral or bacterial or other expression construct, vector or system), e.g. one or more expression constructs or expression vectors, comprising one or more of the nucleic acid molecules herein. As noted above, such a system may be represented by a plurality of vectors comprising a first vector comprising a nucleotide sequence encoding a polypeptide comprising a VH region and a second vector comprising a nucleic acid sequence encoding a polypeptide comprising a VL region.

WO 2024/194298 PCT/EP2024/057283 Convenient constructs, vectors etc., are those which allow prolonged or sustained expression of the antibodies within the host cell. Such expression can be transient, e.g. episomal, or more permanent, e.g. via genomic integration, providing sufficient levels and length of expression are achieved.A yet further aspect provides a host cell (e.g. a mammalian or bacterial or yeast host cell) or virus, e.g. one or more host cells or viruses, comprising one or more expression constructs or expression vectors as defined herein. Also provided are host cells or viruses comprising one or more of the nucleic acid molecules as defined herein. A host cell or virus expressing an antibody as defined herein forms a yet further aspect.A yet further aspect provides a method of producing (or manufacturing) an antibody as defined herein comprising a step of culturing the host cells. More particularly, the method comprises the steps of (i) culturing a host cell comprising one or more of the expression vectors or one or more of the nucleic acid molecules under conditions suitable for the expression of the encoded antibody; and optionally (ii) isolating or obtaining the antibody from the host cell or from the growth medium/supernatant. Such methods of production (or manufacture) may also comprise a step of purification of the antibody or and/or formulating the antibody into a composition, e.g. a pharmaceutical composition, including at least one additional component, such as a pharmaceutically acceptable carrier or excipient. The method may also comprise a preceding step of introducing the vector or nucleic acid molecule into the host cell.In embodiments when the antibody is made up of more than one polypeptide chain (e.g. certain fragments such as Fab fragments or whole antibodies), then all the polypeptides are expressed in the host cell, either from the same or a different expression vector, so that the complete antibody protein can assemble in the host cell and be isolated or purified therefrom.Compositions comprising an antibody, or a nucleic acid molecule or expression vector, or a first host cell as defined herein, constitute further aspects. Formulations (compositions) comprising one or more antibodies, optionally in a mixture with a suitable diluent, carrier or excipient constitute a preferred embodiment. Such formulations may be for pharmaceutical use, and thus such compositions are preferably pharmaceutically acceptable or acceptable for administration to human or non-human animals. Suitable diluents, excipients and carriers are known to the skilled man.

WO 2024/194298 PCT/EP2024/057283 Pharmaceutical compositions may be presented in a form suitable for systemic administration such as parenteral administration including subcutaneous administration, intravenous administration, administration by infusion, intra-arterial administration, intraperitoneal administration, intramuscular administration, or administration into the brain, such as intracerebroventricular administration. The pharmaceutical compositions may be presented as conventional pharmaceutical formulations suitable for systemic administration, such as solutions, suspensions or any other preparation suitable for injection or infusion. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these pharmaceutical formulations.Injection or infusion solutions, e.g. sterile aqueous solutions, may, for example, be produced in the conventional manner, such as by the addition of excipients and preservation agents, anti-oxidants and/or stabilizers.The formulations may be presented in unit-dose or multi-dose formats, e.g. containers, for example vials or ampoules. Suitable dosage units can be determined by a person skilled in the art.A further aspect provides antibodies defined herein, or the nucleic acid molecules, expression vectors or host cells as defined herein, or the pharmaceutical compositions containing them, for use in therapy or in vivo diagnosis, in particular for use in the treatment or prevention of amyloid disease. In particular, this may be any disease or condition associated with (or characterised by) Ap42 prefibrillar oligomers with p structure (i.e. soluble 3-structured Apoligomers), or where such oligomers play a role, for example a causative (e.g. a wholly or partially causative role) or an essential role.As used herein the term “amyloid disease ” refers particularly to a disease (or condition or disorder) associated with the abnormal aggregation of protein or peptide into amyloid deposits. Although the amyloid deposits may be seen in many cases in the CNS, or more particularly in the brain (such as in the case of AD), the diseases to be treated by the binding protein herein are not limited to this, and the deposits may be found or observed in other tissues, or body sites. As indicated above, however, amyloid disease may also be associated with the abnormal aggregation of protein or peptide into soluble aggregates typically denoted “oligomers”. More specifically, the disease is associated with the abnormal aggregation of Ap, and especially Ap42, into amyloid fibrils and toxic soluble assemblies collectively referred to as Ap oligomers. These oligomers are prefibrillar, WO 2024/194298 PCT/EP2024/057283 and have structures distinct from insoluble fibrillar plaques. Such disease includes any disease which would benefit from removal or depletion of soluble Apprefibrillar oligomers with p structure, which have been recognised in the art as toxic agents. The removal of these toxic oligomers in these diseases may prevent or delay the development of disease.In an embodiment, the disease is a neurodegenerative condition, e.g. a dementia, associated with Ap, especially Ap42, and more particularly with Apprefibrillar oligomers with p structure.In an embodiment, the disease is Alzheimer’s Disease (AD) or a related disease, such as Down’s syndrome for which the AD prevalence is 90-100% in the seventh decade of life and the leading cause of death (Fortea et al., Lancet 395, 1988-1997, 2020).In another embodiment the disease is a progressive degenerative muscle disorder, such as sporadic inclusion-body myositis for which aberrant Ap aggregation into oligomers has been implicated as contributing to disease (Kitazawa etal., Journal of Neuroscience 29, 6132-6141, 2009).In accordance with the therapies herein, the antibodies may target and inhibit or reduce the toxic effect of Ap42 prefibrillar oligomers with p structure. Thus, the antibodies may be used in the treatment or prevention of any disease or condition where reduction of this toxic effect is beneficial.The administration of the antibodies or the nucleic acid molecules, expression vectors or host cells, in the therapeutic methods and uses herein is carried out in pharmaceutically, therapeutically, or physiologically effective amounts, to subjects in need of treatment. Thus, said methods and uses may involve the additional step of identifying a subject in need of treatment.The subject may be a human or non-human animal, in particular a mammalian animal.The treatment of disease includes cure of said disease, or any reduction or alleviation of disease, e.g. reduction in disease severity, or symptoms of disease, or any improvement in the condition of the subject. Exemplary parameters to assess might include any improvement in the functional, cognitive or memory, or other tests used to assess the disease.The beneficial effect may include a delay or inhibition of progress of the disease. In particular, in cases of early diagnosis, or in subjects at risk from or susceptible to the disease, the onset of the condition, or of symptoms thereof, may WO 2024/194298 PCT/EP2024/057283 be delayed, or indeed prevented. The therapies herein may thus also be prophylactic, or preventative.Such preventative aspects may be carried out on healthy or normal or at risk subjects and can include both complete prevention and significant prevention. Significant prevention can include the scenario where severity of disease or symptoms of disease is reduced (e.g. measurably or significantly reduced) compared to the severity or symptoms which would be expected if no treatment is given. Further, the progression of the disease may be delayed or slowed in subjects in the early stages of the disease.By “pharmaceutically or physiologically or therapeutically effective amount” is meant an amount sufficient to show benefit to the condition of the subject. Whether an amount is sufficient to show benefit to the condition of the subject may readily be determined by a person skilled in the art. A pharmaceutically or physiologically or therapeutically effective amount can be determined based on clinical assessment and can be readily monitored.Further, the antibodies or pharmaceutical compositions described herein may be used in conjunction with one or more other therapeutic agents (e.g. other agents known or proposed for use in the treatment of the disease in question). In other words, the antibodies herein or pharmaceutical compositions described herein may be used in combination therapy, such as combination with Standard of Care (SOC) therapy. In an embodiment, the antibodies or pharmaceutical compositions described herein are for use in combination therapy, such as combination with Standard of Care (SOC) therapy for an amyloid disease.For such combination treatments, the second agent may be administered to a subject together with the antibody. This may be simultaneously, in the same pharmaceutical composition, or in separate compositions, or separately, or sequentially.Examples of therapeutic agents for use in combination therapy with an antibody as herein described, are other antibodies proposed or developed for use in treating an amyloid disease or for lowering the amount of beta-amyloid, e.g. in the brain (often referred to as antiplaque agents), including for example lecanemab (Leqembi®), oraducanumab (Aduhelm®). Donanemab, which successfully has completed clinical phase 3 trials and is currently under review by FDA and EMA, is yet another plaque-reducing antibody which may be useful for combination therapy with an antibody as herein described.

WO 2024/194298 PCT/EP2024/057283 In an additional embodiment, the antibodies may be used in conjunction with one or more other agents or technologies to increase blood-brain-barrier penetrance of the antibody, many of which are known in the art (Ayub and Wettig, Pharmaceutics 14, 224, 2022). One such preferred embodiment utilises an antibody-transferrin fusion construct, where transferrin receptor-mediated transcytosis transports the binding protein between blood and brain.The binding proteins may also be used as molecular tools for in vivo applications, for example diagnostic in vivo methodsThus, yet further aspects of the invention provide a reagent that comprises an antibody as defined herein and the use of such antibodies as molecular tools in in vivo assays, for example for the detection of Ap42 prefibri liar oligomers with p structure in a subject, e.g. in an in vivo imaging procedure.In one embodiment the method of diagnosing the disease as described herein is an in vivo method.Such an in vivo method may for example involve administering an antibody in detectable from to the subject, e.g. in the format of an imaging agent, and imaging the subject to detect the antibody. The antibody in this embodiment may be labelled. It may be provided in the form of a conjugate of the antibody with a diagnostic agent. The diagnostic agent may be a detectable label or moiety that may be detected by imaging. For example, the antibody may be used as a PET tracer.As noted above, the parental antibody ALZ-201 is characterised by a unique, and beneficial binding profile. It is specific for soluble oligomeric Ap42 but does not bind to other forms of AP (e.g. to insoluble aggregates or plaques, or to monomeric or non-aggregated AP). It is thus truly specific, not just selective, for structured Ap42 oligomers now recognised as primarily responsible for driving AD. The humanised antibodies herein advantageously retain the same or similar binding profile.As described in the examples below, the humanised antibody (Ab11) comprising the humanised VL and VH sequences which characterise the claimed binding proteins exhibits a high affinity for the target antigen (Ap42 prefibrillar oligomers with p structure), which is in a comparable range to that of the parental antibody ALZ-201, and similar to that of a chimera of ALZ-201 (chALZ-201) variable regions with human lgG1 constant regions. Such an affinity is appropriate for an effective therapeutic agent. Binding agents as defined herein may thus exhibit a WO 2024/194298 PCT/EP2024/057283 comparable affinity for the Ap42CC antigen to that of Ab11. The affinity may conveniently be measured by a SPR assay, e.g. by a Biacore assay, according to methods well known in the art. The Ap42CC peptide is described in Sandberg et al. 2010, PNAS, 107(35), 15595-15600 and in WO2009/128772.As demonstrated in the examples below, other humanised antibodies tested in the development of the antibodies herein exhibited a slightly higher affinity. Nonetheless, Ab11 was selected based on a combination of other features. For example, as noted above, it has a high % humanness of the VH and VL sequences (88.7%). Since the VH and VL of the Ab11 antibody are retained in the antibodies herein, this beneficial property is retained.Furthermore, as further described in the Examples, Ab11 exhibits a high thermal stability, characterised by a high Tm of 71 °C, as determined by differential scanning fluorimetry (DSF) analysis. The antibodies herein demonstrate a similar thermal stability to that of Ab11. Accordingly, an antibody herein may have a Tm of at least 69, 70 or 71 °C, as determined by differential scanning fluorimetry (DSF) analysis.Still further, as demonstrated in the Examples below, Ab11 may be obtained at high yield from an expression system in mammalian cells. Accordingly, in certain embodiments, the antibody herein may be expressed at a level of at least 4, 4.5 or mg/ml from mammalian cells.As described above, the original antibody ALZ-201 has been demonstrated to cause a positive physiological and protective impact on the integrity and morphology of mouse neurons. The antibodies herein may exhibit a similar protective effect. This may be determined in a neurotoxicity assay using brain extracts from AD patients to test their toxicity in neuronal cultures of neurons from mouse embryos, and examining the effect on the toxicity of the antibody. Such an assay is described in the Sandberg et al. 2022 publication cited above.Further details of the antibodies herein, and their development are provided in the following non-limiting examples, with reference to the following figures.

Description of the FiguresFigure 1 shows the protein purification profiles of the recombinant antibodies after non-reduced SDS-PAGE analysis of the Protein A chromatography fractions. (A): chALZ-201 (Ab15) pilot batch. (B): chALZ-201 (Ab15). (C): Ab1 and Ab2. (D): Ab3. (E): Ab10. (F): Ab11. (G): Ab17. Coomassie blue staining was used. MW.

WO 2024/194298 PCT/EP2024/057283 Molecular weight marker. IN. Input. FT. Flowthrough. W. Washes. E. Eluted fractions.Figure 2 shows results from the reduced and non-reduced SDS-PAGE analysis of the final pooled samples of each purified antibody (for quality checking purposes). (A): chALZ-201 (Ab15) pilot batch. (B): chALZ-201 (Ab15). (C): Ab1, Ab2 and Ab3. (D): Ab10 and Ab11. (E): Ab17. Coomassie blue staining was used. MW. Molecular weight marker.Figure 3 shows the average molecular weight of the Ap42CC oligomers determined by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). Results shown are normalised 280-nm absorbance (a.u.) and molar mass (kDa) for the Ap42CC oligomers as they elute from the column. Figure adapted from Sandberg et al. 2022, Alz Res and Therapy 14:196.Figure 4 shows the ELISA results for the affinity of chALZ-201 or lecanemab for unstructured Ap42, fibrillar Ap42, and oligomeric Ap42CC. Figure adapted from Sandberg et al. 2022, Alz Res and Therapy 14:196.Figure 5 shows the ELISA results for the affinity of Ab1, Ab2, Ab3 and chALZ-201 for Ap42CC oligomers.Figure 6 shows the ELISA results for the affinity of Ab10, Ab11, Ab17 and chALZ-201 for Ap42CC oligomers.Figure 7 shows the thermal stability data from Differential Scanning Fluorimetry (DSF) of the antibodies as the first derivative of the F350/F330 ratio (dF/dT). (A): data for Ab1, Ab2, Ab3 and PBS (control). (B): data for chALZ-201, Ab10, Ab11, Ab17 and PBS (control).Figure 8 shows the results of the expression comparison test for Ab10, Ab11 and chALZ-201 as expression yield in mg/L culture. (A): Ab10 and Ab11. (B): Ab10 and chALZ-201. (C): Ab11 and chALZ-201.Figure 9 shows the reduced and non-reduced SDS-PAGE analysis of the final purified Ab11 from each minipool (for quality checking purposes). 2 pg of Abwas applied to each lane. Coomassie blue staining was used. MW. Molecular weight marker. 1. Pool 1.2. Pool 2.Figure 10 shows the reduced and non-reduced SDS-PAGE analysis of the final purified Ab11 from each monoclone (for quality checking purposes). 2 pg of Ab11 was applied to each lane. Coomassie blue staining was used. MW. Molecular weight marker. 1. 2D7. 2. 4H9. 3. 13F11.4. 8B6. 5. 8F9. 6. 15H5. 7. 1H10. 8. 1A3. 9. 2A6. 10. 5A9.

WO 2024/194298 PCT/EP2024/057283 Figure 11 shows a cell viability and stability assessment of the top three monoclones 4H9, 1A3, and 2A6. Cells were cultured for 15 passages (generations), and viable cell density and viability was monitored for each passage.Figure 12 shows the agarose gel electrophoresis results from PCR amplification of the VH and VL regions of Ab11 in the genome extracted from each of the top three monoclones 4H9, 1A3, and 2A6. Ethidium bromide staining was used. MW. Molecular weight marker.Figure 13 shows the reduced and non-reduced SDS-PAGE analysis of the final purified Ab11 from each monoclone subjected to the cell viability and stability assessment (for guality checking purposes). 2 pg of Ab11 from cycle 5, 10, and was applied to each lane. Coomassie blue staining was used. MW. Molecular weight marker. 5, 10, or 15 indicate samples from passage number 5, 10, or 15.Figure 14 shows the agarose gel electrophoresis results from the PCR- based mycoplasma test. Ethidium bromide staining was used. 1.4H9. 2. 1A3. 3. 2A6. +. Positive control (290 bp). -. Negative control. MW. Molecular weight marker.

ExamplesExample 1: Humanisation of Antibody ALZ-201 ALZ-201 model buildingA molecular model of the murine antibody clone ALZ-201 was constructed according to established protocols. The variable heavy (VH) and variable light (VL) chain seguence were numbered/annotate according to the Kabat and IMGT conventions to identify the framework (FW) and complementarity determining residue (CDR) seguences.The seguences were used to search solved murine antibody structures to identify structures with the most seguence identity to select templates for the VL and VH FW and CDRs, and these were used to construct partial models for the VH and VL. The best tertiary arrangement of the partial models was selected in order to construct the final model, using the PAPS (packing Angle Prediction) server: http://www.bioinf.org.uk/abs/paps/ . The PAPS server predicted a solved murine antibody structure to provide the best tertiary arrangement of VH and VL and the final model was assembled by fitting the backbone coordinates of the conserved anchor segments of the VH and VL partial models to the selected murine antibody WO 2024/194298 PCT/EP2024/057283 structure. Lastly, the coordinates of the final model were subjected to a round of energy minimisation employing GROMACS with the GROMOS96 force-field.The model was used to aid the subsequent humanisation design. Particularly, by inspecting the model, in light of the sequences of the human germline candidates for engrafting, potential design errors that may arise from a design based purely on sequence considerations can be avoided. In particular, the model was used to select residues in the human FWQ to be “back-mutated ” (substituted with the parental murine amino acid), in order to prevent loss of antibody stability or affinity due to structural incompatibility of the FWwith the engrafted murine CDR sequences. Further, the model was used to select residues from the engrafted murine CDRs for replacement with human residues from the target human germline sequence (“germ-lining”). This allows the “human-ness” of the resulting antibody to be increased without loss of affinity.A final challenge in humanisation design is the amelioration of potential sequence-based liabilities characteristic of the parental murine CDRs; certain sequences are subject to chemical degradation over time and in CDRs this can result in loss of affinity or antibody stability. The model facilitated the selection of residue substitutions to ameliorate potential liabilities.

Humanization design by CDR-qraftinq of ALZ-201 mouse monoclonal antibodyThe ALZ-201 murine antibody was humanized by grafting the three CDRs, as defined by the Kabat nomenclature, from the light chain variable region (VL) into a human germline VL that was as-homologous-as-possible to the murine antibody VL. Similarly, the three CDRs from the heavy chain variable region (VH) were grafted into a human germline VH that was as-homologous-as-possible to the murine antibody VH.Human germlines to be used as acceptor sequences for the CDR grafting were selected based on sequence database searching and sequence identity comparisons. For VH, two human germlines, IGHV2-5*09 and IGHV4-30-4*07 were selected. ForVL, four human germlines were selected: IGKV3-11*01, IGKV3- 20*01, IGKV1-39*01 and IGKV6-21*01.The CDRs of ALZ-201 were analysed for sequence liabilities (deamidation and isomerisation motifs).The murine VH/VL and corresponding human germline sequences were aligned and the CDRs were grafted. In addition, a few amino acid residues in the WO 2024/194298 PCT/EP2024/057283 framework regions of the selected human germline variable regions were back- mutated to the amino acid residues that were present in the murine variable regions. Based upon information on the structure of immunoglobulin variable regions, and with the guidance of the above homology molecular model of the Fv of the ALZ-201 murine monoclonal antibody, these few residues in the framework regions were identified as having key roles in either maintaining the CDRs in the right conformation or in VH/VL packing, and thus they were retained in one humanized version (version A) or substituted with their human germline counterparts, if possible, in the subsequent humanized versions. Under guidance of the homology molecular model, in subsequent version B, when judged possible the CDR residues, as defined by Kabat, were also substituted for their human germline counterparts (germ-lined) in order to increase the degree of humanness (i.e. percentage sequence identity for both VH and VL between the humanized versions and the closest human germline used as acceptor sequence for the CDR-grafting). The structural model thus allowed the limits of the humanization process to be expanded, taking it beyond mere CDR-grafting.Humanised versions A were designed as conservative versions that minimised/avoided CDR alternations. The subsequent versions B, C and D were designed to increase the % humanness (i.e. to reach a higher % of sequence identity with the closest human germline (a least 85% or above) by germ-lining.For the VH, 6 different humanised VH sequences were designed (based on two different human germlines, with 2 or 4 versions each, respectively). For the VL, different VL humanised sequences were designed (based on four human germlines with 2 versions each). This makes 48 possible combinations between the humanized VH and VL. The best VH and VL chain combinations between the humanised VH and VL sequences were selected.The 48 combinations were thus reduced to 18 based on various considerations, including % humanness, and the sequence modifications introduced in the different versions B, C and D.

Table 1 below shows the 18 combinations identified on the basis of the humanised VH and VL sequences selected.

Table 1 WO 2024/194298 PCT/EP2024/057283 Humanized versions Heavy chain Light chain ALZ201-25VHA-311VLA ALZ201-3-11-VLAALZ201-25VHA-139VLAALZ201-2-5-VHAALZ201-1-39-VLAALZ201-25VHA-621VLA ALZ201-6-21-VLAALZ201-4304VHA-311VLA ALZ201-3-11-VLAALZ201-4304VH A-139VLAALZ201-4-30-4-VHAALZ201-1-39-VLAALZ201-4304VH A-621VLA ALZ201-6-21-VLAALZ201-4304VHB-311VLA ALZ201-3-11-VLAALZ201-4304VHB-139VLAALZ201-4-30-4-VHCALZ201-1-39-VLAALZ201-4304VHB-621VLA ALZ201-6-21-VLAALZ201-25VHA-311VLBA ALZ201-3-11-VLBALZ201-25VHA-139VLBAALZ201-2-5-VHAALZ201-1-39-VLBALZ201-25VH A-621VLBA ALZ201-6-21-VLBALZ201-4304VHA-311VLA ALZ201-3-11-VLBALZ201-4304VH A-139VLAALZ201-4-30-4-VHAALZ201-1-39-VLBALZ201-4304VH A-621 VLA ALZ201-6-21-VLBALZ201-4304VHB-311VLA ALZ201-3-11-VLBALZ201-4304VHB-139VLAALZ201-4-30-4-VHCALZ201-1-39-VLBALZ201-4304VHB-621VLA ALZ201-6-21-VLB Of the 18 combinations, 16 were selected for further study (omitting humanised antibody variants 15 and 18)The various combinations were then subjected to assessment andexperimental testing, as described in the Examples below, based on the following criteria:Level of transient expression in mammalian cells as human lgG1/kappa antibodiesBinding capacity (EC50 by ELISA, or Kd by Biacore)- Determination of biophysical properties, particularly analysis bydifferential scanning fluorimetry (DSF) to determine Tm of FAB, CHand CH3.

Example 2: Vector design and recombinant productionVector design, chimeric ALZ-201 (chALZ-201)Starting from seguences of the Heavy Chain (HC) and Light Chain (LC) variable regions of the murine mAb20 antibody (WO2009128772A1), a chimeric full length human lgG1 antibody with kappa light chain was designed (chALZ-201) and used as a control reference for humanised variants of the parent murine antibody.

WO 2024/194298 PCT/EP2024/057283 The cDNA coding for the HC and LC were chemically synthesized with optimization for expression in CHO cells. Sequences coding for signal peptides were added in 57Nter position. The two cDNA sequences constructed for recombinant chALZ-2expression in CHO cells are set forth in SEQ ID NOs. 9 and 10. chALZ-201 HC cDNA (SEQ ID NO. 9)GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCCC CTCGGTGGGTTCTGAGCCAGGTGACCCTGAAGGAGTCCGGCCCTGGCATCAG CCAGCCTAGCCAGACCCTGAGCCTGACCTGTTCCTTTAGCGGCTTCTCCCTGA GCACCTTTGGCAGCGGCGTGTCCTGGATCAGGCAGCCTAGCGGCAAGGGCC TGGAGTGGCTGGCTCACATCTACTGGGACGACGATAAGCACTACAATCCCAG CCTGAAGTCCCGGCTGACCATCAGCAAGGATACCAGCAACAATCAGGTGTTTC TGAAGATCACCACCGTGGACACCGCCGATACCGCCACCTATTTCTGCGCCCG GCGGGAGAGCCACTACTATGGCTCCGGCTACTACTTCGATTACTGGGGCCAGGGCACCACCCTGACCGT GTCCTCCGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTA AGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTC CCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGC ATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTG GTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAA CCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGC GATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCC CACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGT GTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGT GGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATAC AACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCT GAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCT ATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCT ATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGAC ATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCA ATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGAC GGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCA GGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATA CTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGAGCGGCCGC WO 2024/194298 PCT/EP2024/057283 chALZ-201 LC cDNA (SEQ ID NO. 10)GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGT GGATCTCCGGCGCCTATGGCCAGATCGTGCTGACCCAGTCCCCTGCTATCCT GTCCTCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGGGCCAGCAGCAG CGTGAGCTATATGCACTGGTACCAGCAGAAGCCCGGCAGCAGCCCCAAGCCC TGGATCTACGCTACCAGCAACCTGGCTTCCGGCGTGCCTGCCAGGTTTAGCG GCTCCGGCTCCGGCACCTCCTATTCCCTGACCATCTCCCGGGTGGAGGCCGA GGATGCCGCTACCTACTATTGTCAGCAGTGGAGGTCCGACCCCCTGACCTTC GGCGCTGGCACCAAGCTGGAGCTGAAGCGTACGGTGGCTGCACCTTCTGTGT TCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTC TGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGA CAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCC AAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTA TGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGC CCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC The lower-case nucleic acid sequence “gccgccacc” present in SEQ ID NOs. and 10 is the Kozak sequence that functions as the protein translation initiation site, and the “GAATTC” sequence immediately preceding it is the EcoR1 restriction enzyme site. The two cDNA sequences were subcloned, using the enzyme EcoR1, into the pXtenl mammalian cell expression vector (ProteoGenix, Schiltigheim, France). The expected protein sequence for the antibody chALZ-201 HC is thus set forth in SEQ ID NO. 11. chALZ-201 HC (SEQ ID NO. 11)MKHLWFFLLLVAAPRWWLSQVTLKESGPGISQPSQTLSLTCSFSGFSLSTFGSGV SWIRQPSGKGLEWLAHIYWDDDKHYNPSLKSRLTISKDTSNNQVFLKITTVDTADT ATYFCARRESHYYGSGYYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK WO 2024/194298 PCT/EP2024/057283 In SEQ ID NO. 11, amino acids 1 to 19 is a secretion signal that is cleaved off during the translation of the protein, amino acids 20 to 143 comprises the VH domain, and amino acids 144 to 473 comprises the lgG1 constant heavy domain.Conversely, the expected protein sequence for the antibody chALZ-201 LC is set forth in SEQ ID NO. 12. chALZ-201 LC (SEQ ID NO. 12)MVLQTQVFISLLLWISGAYGQIVLTQSPAILSSSPGEKVTMTCRASSSVSYMHWYQ QKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQW RSDPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC In SEQ ID NO. 12, amino acids 1 to 20 is a secretion signal that is cleaved off during the translation of the protein, amino acids 21 to 126 comprises the VH domain, and amino acids 127 to 233 comprises the lgG1 constant light domain.

Vector design, antibody variants 10 (Ab10) and 11 (Ab11)For Ab10 and Ab11, designed using in silica CDR grafting as described above, the cDNA coding for the VH and VL chains of the antibodies were chemically synthesized with optimization for expression in CHO cells. Sequences coding for signal peptides were added in 57Nter position.The VH sequence is identical for the two antibodies. The two cDNA sequences constructed for recombinant antibody expression of Ab10 VH and AbVH in CHO cells are set forth in SEQ ID NO. 13.

Ab10 VH cDNA and Ab11 VH cDNA (SEQ ID NO. 13)GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCTGCTC CTCGGTGGGTGCTGTCCCAGGTGACCCTGAAGGAGTCCGGCCCCACCCTGG TGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCAGCGGCTTTAGCCT GAGCACCTTTGGCAGCGGCGTGAGCTGGATCAGGCAGCCTCCCGGCAAGGC CCTGGAGTGGCTGGCTCACATCTATTGGGACGACGACAAGCACTATAACCCTA GCCTGAAGAGCCGGCTGACCATCACCAAGGACACCAGCAAGAACCAGGTGGT GCTGACCATCACAAACATGGACCCTGTGGATACCGCCACCTATTTTTGCGCCC WO 2024/194298 PCT/EP2024/057283 GGAGGGAGAGCCACTACTATGGCAGCGGCTACTATTTCGATTATTGGGGCCA GGGCACCCTGGTGACCGTGAGCAGC As above, the lower-case nucleic acid sequence “gccgccacc” in SEQ ID NO. is the Kozak sequence that functions as the protein translation initiation site, and the "GAATTC" sequence immediately preceding it is the EcoR1 restriction enzyme site. The two cDNA sequences were subcloned, using the enzyme EcoR1, into the pXtenl mammalian cell expression vector (ProteoGenix, Schiltigheim, France) containing the backbone sequence for the human IgG 1 heavy chain constant region described above. The expected full protein sequence for the HC of humanised antibodies Ab10 and Ab11 is thus set forth in SEQ ID NO. 7.

Ab10 HC and Ab11 HC (SEQ ID NO. 7)MKHLWFFLLLVAAPRWWLSQVTLKESGPTLVKPTQTLTLTCTFSGFSLSTFGSGVS WIRQPPGKALEWLAHIYWDDDKHYNPSLKSRLTITKDTSKNQVVLTITNMDPVDTA TYFCARRESHYYGSGYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQG N VFSCSVM H EALH N H YTQKSLSLSPG KIn SEQ ID NO.7, amino acids 1 to 19 is a secretion signal that is cleaved off during the translation of the protein, amino acids 21 to 143 comprises the VH domain, and amino acids 144 to 473 comprises the lgG1 constant heavy domain.

The cDNA sequence constructed for recombinant antibody expression of Ab10VL is set forth in SEQ ID NO. 14.

Ab10 VL cDNA (SEQ ID NO. 14)GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGT GGATCAGCGGCGCTTATGGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCT GTCCCTGAGCCCAGGAGAGCGGGCTACCCTGAGCTGTCGGGCCTCCTCCAG CGTGTCCTACATGCACTGGTACCAGCAGAAGCCTGGCCAGGCTCCTCGGCCC TGGATCTATGCCACCAGCAACCTGGCCACCGGCATCCCCGCCAGGTTCTCCG WO 2024/194298 PCT/EP2024/057283 GAAGCGGCTCCGGAACCGATTTTACCCTGACCATCTCCAGCCTGGAGCCTGA GGACTTTGCCGTGTACTACTGCCAGCAGTGGAGGAGCGATCCTCTGACCTTT GGCGGCGGCACCAAGGTGGAGATCAAG The cDNA sequence constructed for recombinant antibody expression of Ab11 VL is set forth in SEQ ID NO. 15.

Ab11 VL cDNA (SEQ ID NO. 15) GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGT GGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGTCCCCTTCCAGCCT GAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCGGGCTTCCTCCAG CATCTCCTATATGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCCTT GGATCTACGCTACCAGCAATCTGGCTAGCGGCGTGCCTAGCCGGTTCTCCGG CTCCGGATCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCTGAG GATTTTGCTACCTACTACTGCCAGCAGTGGCGGTCCGATCCCCTGACCTTCGG CGGCGGAACCAAGGTGGAGATCAAG As mentioned above, the lower-case nucleic acid sequence “gccgccacc” in SEQ ID NOs. 14 and 15 is the Kozak sequence that functions as the protein translation initiation site, and the “GAATTC” sequence immediately preceding it is the EcoR1 restriction enzyme site. The two cDNA sequences were subcloned, using the enzyme EcoR1, into the pXtenl mammalian cell expression vector (ProteoGenix, Schiltigheim, France) containing the backbone sequence for the human lgG1 light chain constant region described above. The expected protein sequences for the LC of humanised antibodies Ab10 and Ab11 are thus set forth in SEQ ID NOs. 16 and 8.

Ab10 LC (SEQ ID NO. 16) MVLQTQVFISLLLWISGAYGEIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQ QKPGQAPRPWIYATSNLATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWR SDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Ab11 LC (SEQ ID NO. 8) WO 2024/194298 PCT/EP2024/057283 MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCRASSSISYMHWYQ QKPGKAPKPWIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWR SDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC In SEQ ID NOs. 16 and 8, amino acids 1 to 20 is a secretion signal that is cleaved off during the translation of the protein, amino acids 21 to 126 comprises the two different VL domains, and amino acids 127 to 233 comprises the lgGconstant light domain.Humanised ALZ-201 variants Ab10 and Ab11 both have 88.7% sequence identity to a human IgG homologue. All other humanised ALZ-201 candidates described herein were similarly subcloned for expression in CHO cells. These are denoted Ab1 (87.6% human residues), Ab2 (87.6% human residues), Ab3 (86.1% human residues), and Ab17 (85.6% human residues).

Transient expression in Chinese Hamster Ovary cellsXtenCHO cells were used for transient expression of the antibodies described herein (PX-XTE-004; ProteoGenix, Schiltigheim, France). This cell line is derived from the Chinese Hamster Ovary CHO-K1 cell line. Vials containing 1 mb frozen cell suspensions at 1*107 cells/mL in XtenCHO Expression Medium (PX- XTE-002; ProteoGenix, Schiltigheim, France) with 8 mM L-glutamine, 0.5% antidumping agent (0010057AE; Gibco/Thermo Fisher Scientific, USA), and 10% DMSO, were thawed for a maximum of 90 seconds by submerging and gently swirling the vial in XtenCHO Expression Medium at 37 °C until only a small amount of ice remained. Vials were decontaminated by wiping them off with 70% ethanol and moved to a laminar flow hood. Contents were transferred into 8 mL of pre- warmed XtenCHO Expression Medium supplemented with 8 mM L-glutamine and centrifuged at 300xg for 5 minutes. Supernatant solution was discarded, and the cells resuspended in 2 mL of XtenCHO Expression Medium with 8 mM L-glutamine and the viable cell number and viability determined using an automated cell counter (Countstar Biotech, Model IC 1000) using default settings of the cell mode for counting cells. In brief, a sample of cell suspension was removed from the flask and diluted 1:1 with 0.4% trypan blue stain. After mixing gently, 20 pl of the sample mixture was added into the chamber port on a cell counting chamber slide and WO 2024/194298 PCT/EP2024/057283 inserted into the automated cell counter. Three horizons (up, medium and down) were chosen to count, and the system then calculated the average cell density and viability.A 125-mL disposable sterile vented Erlenmeyer shaker flask (TAB-012-125; Guangzhou Jet Bio-Filtration Co., Guangzhou, China) containing 30 mL XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C was seeded at a density of 0.2-0.3x106 cells/mL and antidumping agent added to a final concentration of 0.5%. Cells were incubated at 37 °C with >80% relative humidity and 5% CO2 on an orbital shaker platform (IS-RDS6C5 Incubator, Crystal Technology & Industries, USA).When cells reached 1.5-2.5x106 viable cells/mL, typically at 2-3 days post- thaw, cells were subcultured. The culture was first centrifuged at 300xg for minutes and the supernatant solution discarded. Cells were then transferred to mL XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C at a density of 0.2-0.3x106 cells/mL and antidumping agent added to a final concentration of 0.5%. The culture was returned to 37 °C incubation with >80% relative humidity and 5% CO2 on an orbital shaker platform. When cells again reached 1.5-2.5x106 viable cells/mL, the process was repeated at least once before transfection. Transfection was never performed on cells that had undergone more than 20 passages.The day before transfection, the cells were subcultured in XtenCHO Expression Medium with 8 mM L-glutamine but without the antidumping agent by using the subculturing procedure described above. After 24h, cells had typically reached a density of 2-3x1 06 viable cells/mL, with a viability of >90%. The culture was centrifuged at 300xg for 5 minutes and the supernatant solution discarded.Cells were then co-transfected with the pXtenl plasmids carrying the respective HC and LC cDNA sequences of each antibody using the following protocol. Cells were first transferred to 15 mL XtenCHO Expression Medium with mM L-glutamine at 37 °C at a density of 5x106 viable cells/mL. 48 pg of each expression plasmid, pXtenl, carrying the cDNA sequences for the HC and LC ALZ- 201 antibody derivative to be expressed, was added to the cells, and 144 pL XtenFect reagent Working Solution (PX-XTE-003; ProteoGenix, Schiltigheim, France) added dropwise under gentle stirring of the cells. After incubating the cells at 37 °C with >80% relative humidity and 5% CO2 on an orbital shaker platform for 2h, an additional 15 mL XtenCHO Expression Medium with 8 mM L-glutamine at °C was added to the cells in addition to 96 pL XtenCHO Enhancer solution (PX- WO 2024/194298 PCT/EP2024/057283 XTE-003; ProteoGenix, Schiltigheim, France). Cells were then returned to 37 °C with >80% relative humidity and 5% CO2 on an orbital shaker platform.24h post-transfection, antidumping agent was added to a final concentration of 0.5% and the temperature reduced to 33 °C. Cell density and viability of the cells was checked regularly during this expression phase.For the initial expression analyses of all humanised ALZ-201 antibodies and the chALZ-201 reference described herein, cells from 30 mL cultures were harvested when viability dropped below 50% at day 14 post-transfection by centrifugation at 300xg for 5 minutes. The supernatant solution was removed and clarified by centrifugation at 5000xg for 30 minutes and passed through a Millex-GP 0.22 pm sterile filter (SLMPL25SS; Merck KGaA, Darmstadt, Germany).Antibodies were then purified using Protein A. In brief, Protein A chromatography resin (Mabselect SuRe LX #17547402; Cytiva, USA) was soaked in 0.5M NaOH for 30 minutes, then washed and equilibrated with phosphate buffered saline at pH 7.5 (PBS: 10 mM Na2HPO4, 2.8 mM KH2PO4, 137 mM NaCI, 2.7 mM KCI). Supernatant solutions from the cell cultures were added and binding allowed to take place for 4 hours at room temperature. After washing the beads with PBS pH 7.5 with 50x the bead volume, bound antibodies were eluted with 20 mM solution of 2-hydroxypropane-1,2,3-tricarboxylic acid (citric acid) buffer at pH 2.7. The eluted fractions were neutralized with 1M tris(hydroxymethyl)aminomethane with HCI (Tris-HCI) at pH 9.0.Fractions containing antibody were confirmed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) using the Mini-PROTEAN Tetra Vertical Electrophoresis System (1658000FC; Bio-Rad, USA) and Tris-HCI gels (8% and 12%) with Tris-Glycine as running buffer (25 mM Tris-HCI, 200 mM Glycine, 0.1% w/v SDS, pH 8.3). 10pl samples were mixed 4:1 with loading buffer (10% w/v SDS, 20% v/v glycerol, 0.2M Tris-HCI pH 6.8, 0.05% w/v Bromophenolblue) with or without reducing agent (10mM beta-mercapto-ethanol). Molecular weight marker (2pl per well) consisted of 11 protein bands ranging from to 250 kDa (Epizyme Biotechnology Co., Ltd, China). Gels were run at 120V and stained with Coomassie brilliant blue G-250.Fractions containing antibody were pooled and buffer changed to PBS pH 7.5 by dialysis against 200x the sample volume using 3.5K MWCO snakeskin tubes (88244; Thermo Scientific, USA). Dialysis was performed overnight (for approximately 16h) at 4°C under continuous stirring, after which the dialysis buffer WO 2024/194298 PCT/EP2024/057283 was changed and dialysis continued for additional 3h at 4°C. Samples were then filtered through a 0.22-pm Millex syringe filter (SLGV013SL or SLGV004SL depending on final sample volume; Merck KGaA, Germany) and assayed for endotoxin levels by using an endotoxin assay kit (ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit#L00350; Genscript, USA). The final, pooled, sample of each purified antibody was quality checked with SDS-PAGE as above.One 30-mL pilot batch of chALZ-201 (also denoted Ab15) indicated that the antibody expressed well in the XtenCHO cells, and Ab10 and Ab11 were subsequently expressed in parallel with a second batch of chALZ-201 in addition to humanised variants Ab1, Ab2, Ab3, and Ab17. Figure 1 shows the SDS-PAGE analyses of the Protein A chromatography fractions, and SDS-PAGE analyses of the pooled samples are shown in Figure 2. All antibodies were found to conform to the expected sizes, where IgG antibodies with reduced disulphide bonds typically migrate as approximately 50 kDa and 25 kDa species on SDS-PAGE gels, corresponding to the heavy and light chains, respectively. Under non-reducing conditions, on the other hand, IgG antibodies give rise to a single band on SDS- PAGE gels, with a size of at least 150 kDa.Based on the SDS-PAGE results, all antibodies expressed herein were estimated to have a purity of >90%. The concentration of antibody was determined by UV spectrophotometry at 280 nm using an extinction coefficient of 210,000 M1־ cm1־. Expression results from the 30-mL cell culture batches are shown in Table 2.Ab11 and Ab17 were found to express very well, whereas Ab10 was found to express poorly compared to all derivatives.

Table 2:Expression test in 30mL CHO cell cultures Antibody Yield (mg/30mL) Concentration (mg/mL) Estimated purity chALZ-201 4.05 0.90 >90%PilotchALZ-201 3.33 1.34 >90%Ab1 2.22 0.60 >90% WO 2024/194298 PCT/EP2024/057283 Antibody Yield (mg/30mL) Concentration (mg/mL) Estimated purity Ab2 2.78 0.78 >90%Ab3 2.33 0.76 >90%Ab10 1.09 0.87 >90%Ab11 5.37 1.85 >90%Ab17 5.15 1.65 >90% Example 3: Binding affinities Amyloid-beta (AB) peptide preparationsFor assessing the reactivity towards the unstructured monomeric antigen, synthetic Ap42 peptides (H-1368; Bachem, Switzerland) were reconstituted at mg/mL in 0.1 M aqueous ammonia solution (pH 9) and used within 2h of preparation. The fibrillar form of the antigen was obtained by reconstituting lyophilized peptide (H-1368; Bachem, Switzerland) at 1.0 mg/mL in PBS with 0.02% azide, and then shaking the solution for 55 h at 700 rpm and 37 °C. Fibrils were then incubated for 90 h without shaking at RT before being frozen at -20 °C. Frozen vials of fibrillar Ap42 were thawed immediately before use.The Ap42CC peptide is a non-fibrillogenic derivative of the Ap42 peptide with two alanine to cysteine replacements at amino acid positions 21 and 30, and an intramolecular disulphide bond connecting the two (WO2009128772A1; Sandberg, A. et al., 2010, Proc. Natl. Acad. Sci. USA, 107:15595-600). An oligomeric form of this peptide was used in the development of murine ALZ-2(WO2012120035A1; Sandberg, A. et al., 2022, Alz. Res. Therapy 14:196). The synthetic Ap42CC used herein was custom made by solid-state peptide synthesis and purified to 95% with reversed-phase HPLC using standard methods and practices (AmbioPharm Inc., North Augusta, SC, USA). Oligomeric peptide preparations were obtained by first dissolving peptides at pH 10.0-10.4 and then allowing them to oligomerise in PBS. Oligomers were then frozen to prevent further oligomerisation. Frozen vials of oligomeric A342CC were thawed immediately before use.The size of the A342CC oligomers used herein was determined with size- exclusion-HPLC (1100 Series; Agilent Technologies, Santa Clara, CA, USA) where a 100 pL sample was injected on a TSK-GEL® G4000SWxl 7.8 X 300 mm column WO 2024/194298 PCT/EP2024/057283 (Tosoh, Tokyo, Japan) with 20 mM sodium phosphate buffer, 150 mM NaCI, pH 7.4, as running buffer. The flow rate was 0.6 mL/min. UV at 280 nm and multi-angle light scattering (MALS; MiniDAWN Treos from Wyatt Technology Corporation, Santa Barbara, CA, USA) was used to determine the weight average molecular weight using ASTRA 6.1 Software (Wyatt Technology). The oligomers used herein had an average molecular weight of 702 ± 3.5 kDa and with an oligomer content of >94% (Figure 3).The concentrations of monomeric and fibrillar Ap42 was inferred from the net peptide content determined by the manufacturer of the peptide (Bachem, Bubendorf, Switzerland), whereas the concentration of Ap42CC was determined with ultraviolet (UV) spectroscopy using an extinction coefficient of 1401 cm1־ M1־ for the difference in absorbance at 280 nm and 300 nm.

Antibody specificity using ELISAThe antigen specificities of the chimeric and humanised derivatives of ALZ- 201 were assessed using ELISA to ensure that the unique binding characteristics of the parent antibody were preserved.96-well Nunc Maxisorp™ ELISA plates (44-2404-21; Thermo Fisher Scientific, Waltham, MA, USA) were coated with 5 pg/mL antigen (100 pL/well) for h at 37 °C. Monomeric Ap42 was coated at high pH to maintain its random coil conformation. Plates were blocked with 1% BSA in PBS (150 uL/well) for 40 min at °C. Washing between steps was carried out by washing three times with PBS supplemented with 0.05% Tween-20 (300 uL/well). Primary antibodies were diluted from 1000 ng/mLto 0.46 ng/mLwith 0.1% BSA in PBS, added at 100 uL/well, and incubated for 1.5 h at RT. After washing, an HRP-conjugated secondary mAb was added at 1000 ng/mL (100 pL/well) and the plates incubated for 45 min at 37 °C. Plates were again washed, after which 3,3',5,5'-tetramethylbenzidine substrate was added (100 pL/well), and the plates were incubated for 5-10 min at 37 °C. The reaction was stopped with 2 M HCI (50 pl per well). The difference in absorbance at 450 and 630 nm was measured using a spectrophotometer and dose-response curve analysed by fitting a 4-parameter non-linear logistic function to the measured absorbance values and extracting the half maximal effective concentration of antibody (EC50).The ELISA results for chALZ-201 against unstructured Ap42, fibrillar Ap42, and oligomeric Ap42CC are shown in Figure 4. There is no binding towards WO 2024/194298 PCT/EP2024/057283 unstructured or fibrillar Ap42, but strong binding to oligomeric Ap42CC, indicating that the chALZ-201 antibody has retained the parent antibody ’s specificity for the oligomeric antigen, as expected. In contrast, the antibody lecanemab (an exact copy of the BAN2401/lecanemab Ab sequence expressed in XtenCHO cells; PX- TA1746; ProteoGenix) was here used as a positive control and found to exhibit near-equal binding affinity to all three different forms of the Ap peptide in the direct ELISA setup used in this example.The EC50 values obtained from the best-fitted equation are shown in Table 3. Here, the EC50 is the average of two measurements. The S.D. given in the table is the average of the two standard errors for determining the individual EC50 values (calculated as S.D.=V((sd1 2+ sd2 2)/2), where sd1 and sd2 are the two standard errors).

Table 3:Half maximal effective concentrations (EC50) of chALZ-201 and lecanemab binding to different derivatives of the Ap42 peptide in ELISA Antibody EC50 ± S.D. (ng/mL) Ap42CC oligomers Ap42 monomers Ap42 fibrils chALZ-201 28±1 No binding No bindingLecanemab copy 59±17 89±11 114±26 Antibody affinities using ELISA and surface plasmon resonance (SPR)That humanised variants retained their affinities to the target antigen was assessed with ELISA analyses as well as with SPR techniques using Biacore™ The ELISA protocol was here similar to the one described above, although only Ap42CC oligomers were used as antigen in this example. Ab1, Ab2, Ab3, Ab10, Ab11, Ab17, and chALZ-201 were assessed. The results are shown in Figure 5 and Figure 6 with the EC50 values obtained in Table 4. One of the experiments on antibody Ab3 did not result in data that could be readily curve-fitted to the Hill equation, leading to substantial errors in the determination of the EC50. The affinities of chALZ-201 and the humanised derivatives for the oligomeric Ap42CC antigen are within the same range, indicating that the affinity for the antigen is retained after humanisation.

WO 2024/194298 PCT/EP2024/057283 Table 4:Half maximal effective concentrations (EC50) of antibody binding toAp42CC oligomers in ELISA Plate Antibody EC50 ± S.D. (ng/mL) Ab176.6 ±22.3 Plate 1Ab254.2 ± 20.2Ab374.7 ± 121.8chALZ-20166.4 ± 11.0Ab1072.3 ±8.7Plate 2 Ab1192.3 ±9.1chALZ-20162.6 ± 11.4 Plate 3Ab1741.6 ±7.5chALZ-20182.1 ±27.2 Affinity determination by SPR was performed using a Biacore™ 8k instrument. SPR analyses biomolecular interactions in real time, providing quantitative measurements of reaction kinetics and affinity constants. It has been shown to be the preferred method for studies that require sensitive and reliable detection of antibody binding rate constants (Yang, D. et al., 2016, Anal. Biochem. 508:78-96). An anti-human Fc IgG antibody (BR-1008-39; GE Healthcare, USA) was coupled to a CM5 sensor chip (BR100399; Cytiva, USA) using the maleimide EDC/NHS (N-ethyl-N3)-׳-(dimethylamino)propyl)carbodiimide/N- hydroxysuccinimide) coupling method (BR-1000-50; GE Healthcare, USA). Running buffer was HBS-EP+ (0.01M HEPES pH7.4, 0.15M NaCI, 3mM EDTA, 0.01% Surfactant P20), and regeneration buffer was Glycine pH 1.5. The chALZ-2antibody and humanised antibodies Ab1, Ab2, Ab10, Ab11, and Ab17 were diluted in running buffer and captured by the anti-human Fc antibody. The oligomer antigen in running buffer was flown over the sensor chip at defined oligomer concentrations (calculated using the 702 kDa Mw data obtained by SEC-MALS in Figure 3) ranging from 1.25nM up to 80nM and the response captured over time and data from a reference channel subtracted. The sensor chip was washed with regeneration buffer between each concentration. Rates for association and dissociation were obtained from the sensorgrams, and the kinetic parameters of association ('on rate', k a) and dissociation ('off rate', kd) calculated, together with the equilibrium dissociation constant ('binding constant', Kd), using the BIAevaluation Software WO 2024/194298 PCT/EP2024/057283 (Biacore™). Results are shown in Table 5, demonstrating that all ALZ-2derivatives are high-affinity binders.

Table 5:Kinetic parameters of binding determined by SPR Antibody ka (M1־ s1־) kd (s1־) Kd(M)Ab14.87 x 105 6.92 x 104־ 1.42 x 109־Ab24.89 x 105 4.30 x 104־ 8.80 x 1010־Ab105.37 x 105 3.06 x 104־ 5.70 x 1010־Ab112.99 x 105 4.67 x 104־ 1.56 x 109־Ab176.49 x 105 1.85 x 104־ 2.85 x 1010־chALZ-2014.49 x 105 6.79 x 104־ 1.51 x 109־ Example 4: Thermal stabilities The tendency of biological macromolecules to withstand thermal denaturation is commonly used as an approximative measure of their inherent stability to aggregation and denaturation. Aggregation of proteins typically requires at least partial unfolding of the native structure, which is a process that can be monitored as a function of time and/or temperature by measuring changes in intrinsic protein fluorescence as proteins unfold. Unfolding of proteins typically exposes fluorescent groups, e.g. Tryptophan and Tyrosine amino acid side chains, to water molecules that quench the fluorescence.Differential Scanning Fluorimetry (DSF) was herein used to measure the temperature at which each antibody unfolds, the Tm, taken as the inflection point of the change in the ratio of fluorescence at 350 nm and 330 nm (F350/F330) as a function of temperature. 50 pL-samples were analysed undiluted in PBS pH7.5 at the concentrations indicated in Table 2. The instrument used was a Prometheus NT.48 nanoDSF (NanoTemperTechnologies GmbH, Germany) operated ata scan rate of 1°C/min from 40°C to 90°C, and the fluorescence at 350nm and 330nm collected at a rate of 10 data points per minute. The data is shown as the first derivative of the F350/F330 ratio (dF/dT) in Figure 7A and 7B, and the extracted melting points in Table 6. All antibodies exhibit a transition, Tm1, within a temperature range of 67-71 °C. Antibodies Ab2, Ab11, and Ab17 also display a second transition, Tm2, around 76-78oC. The two distinct unfolding events of the Ab2, Ab11, and Ab17 antibodies is likely a reflection of different thermal stabilities of the Fab and Fc domains. Antibodies Ab2, Ab11, and Ab17 displayed the highest WO 2024/194298 PCT/EP2024/057283 thermostabilities, where the pre-transitional baselines for Ab11 and Ab17 indicated a somewhat later onset of denaturation compared to Ab2.

Table 6:Thermal melting points determined by differential scanning fluorimetry Antibody Tm 1 (°C) Tm 2 (°C) Ab1—Ab278Ab3—Ab10—Ab1178Ab1776chALZ-201— Example 5: Expression comparison test of Ab10, Ab11, and chALZ-201 That Ab10 had a much lower level of expression than Ab11 was unexpected and here confirmed by transient expression in CHO cells with quantitation using an Octet RED96 device from Sartorius (FortBio/Sartorius, USA).Gene synthesis, cloning, transfection and recombinant production of Ab10, Ab11, and chALZ-201 was carried out as described in Example 2 with the following changes made during transfection and expression. Before transfection, cells were transferred to 7.5 mb XtenCHO Expression Medium with 8 mM L-glutamine at °C at a density of 5x106 viable cells/mL. 24 pg of each expression plasmid (pXtenl) carrying the respective HC and LC domains of Ab10, Ab11, or chALZ-2antibodies, was added to the cells, and 106 pL XtenFect reagent Working Solution (PX-XTE-003; ProteoGenix, Schiltigheim, France) added drop-wise under gentle stirring of the cells. After incubating the cells at 37 °C with >80% relative humidity and 5% CO2 on an orbital shaker platform for 2h, an additional 7.5 mL XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C was added to the cells in addition to 48 pL XtenCHO Enhancer solution (PX-XTE-003; ProteoGenix, Schiltigheim, France). Cells were then returned to 37 °C with >80% relative humidity and 5% CO2 on an orbital shaker platform. 24h post-transfection, antidumping agent was added to a final concentration of 0.5% and the temperature reduced to 33 °C. Cell density and viability of the cells was checked regularly during this expression phase.

WO 2024/194298 PCT/EP2024/057283 Three separate co-transfections were performed in parallel for each antibody construct. 200pL of culture medium was collected on days 4, 7, 10, and 14, and lgG1 concentration determined with an Octet RED96 device equipped with a Protein G Biosensor (18-5082; FortBio/Sartorius, USA). The Octet system uses bio-layer interferometry to measure binding events in real time and can be used to directly detect specific proteins in complex mixtures. Using the quantitation experiment module of the Octet system data acquisition software, the binding rates of Ab10, Ab11, and chALZ-201 to the Protein G Biosensor were measured. Greater antibody concentrations result in faster binding rates. Data were analysed with Octet Data Analysis HT software and concentrations calculated from each rate based on the values from a human lgG1 standard curve (FHJ92850; Antibody Systems). The reference standard was prepared in PBS and analysed in parallel at 50pg/mL, 25pg/mL, 12.5pg/mL, 6.25pg/mL, 3.125pg/mL, 1.563pg/mL, 0.7813pg/mL, and 0 pg/mL. Culture medium samples were diluted 1/20 in PBS before analysis. Sample shaking speed was 1000 rpm, quantitation time set to 3sec, temperature to 30°C, and data acquisition time to 5Hz.The results are shown as expression yield in mg/L culture in Figure 8, where the antibodies are compared pairwise in different panels for clarity. The pairwise statistical comparisons for each data point are shown in Table 7. Here, the difference between the observed means in two independent samples is reported with significance values (P-values) and 95% Confidence Intervals (Cl) of the difference. The P-value is the probability of obtaining the observed difference between the samples if the null hypothesis were true. The null hypothesis is the hypothesis that the difference is 0.

Table7: Statistical comparison of the differences in antibody expression levels in CHO cells Comparison Day P Diff ± SE 95% Cl 0.0004 42 ± 3.9 32.2469 to 53.7531 Ab10-Ab11ד <0.0001 3.7 ± דד 66.7359 to 87.26410.0045 109 ± 18.9 56.4917 to 161.50830.0069 130 ±25.4 59.3960 to 200.6040 WO 2024/194298 PCT/EP2024/057283 Comparison Day P Diff ± SE 95% Cl 0.0444 27 ±9.3 1.1030 to 52.8970ד 0.0032 79 ± 12.5 44.3964 to 113.6039Ab10 - chALZ-2010.049 89 ±31.9 -0.5212 to 177.47880.0022 121 ± 17.4 72.7772 to 169.22280.1443 -16 ±8.8 -40.5209 to 8.5209ד 0.8791 2 ± 12.3 -32.2678 to 36.2678Ab11 - chALZ-2010.5337 -20 ± 29.4 -101.6262 to 61.62620.6852 -9 ± 20.6 -66.2828 to 48.2828 Example 6: Generation of stable monoclonal CHO cell lines expressing Ab11 Vector design used for stable transfectionFor development of a stable CHO cell line expressing the target antibody Ab11, the VH cDNA sequence SEQ ID NO. 13 and the VL cDNA SEQ ID NO. were combined with the respective cDNA sequences for the IgG 1 HC and LC constant regions described in Example 2. The full-length cDNA sequences used for stable recombinant Ab11 expression were: Ab11 HC cDNA (SEQ ID NO. 19)GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCTGCTC CTCGGTGGGTGCTGTCCCAGGTGACCCTGAAGGAGTCCGGCCCCACCCTGG TGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCAGCGGCTTTAGCCT GAGCACCTTTGGCAGCGGCGTGAGCTGGATCAGGCAGCCTCCCGGCAAGGC CCTGGAGTGGCTGGCTCACATCTATTGGGACGACGACAAGCACTATAACCCTA GCCTGAAGAGCCGGCTGACCATCACCAAGGACACCAGCAAGAACCAGGTGGT GCTGACCATCACAAACATGGACCCTGTGGATACCGCCACCTATTTTTGCGCCC GGAGGGAGAGCCACTACTATGGCAGCGGCTACTATTTCGATTATTGGGGCCA GGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGACCTTCTGTGTTC CCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTG TCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAG CTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTG TACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACT TACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGT GGAGCCAAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCA GAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATAC WO 2024/194298 PCT/EP2024/057283 CCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTC ATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCA TAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTG GTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACA AGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGC AAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCA GGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTT TTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAAT AATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTA TAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCT GCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAG CCTGTCCCCCGGGAAATGAGCGGCCGC Ab11 LC cDNA (SEQ ID NO. 20)GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGT GGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGTCCCCTTCCAGCCT GAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCGGGCTTCCTCCAG CATCTCCTATATGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCCTT GGATCTACGCTACCAGCAATCTGGCTAGCGGCGTGCCTAGCCGGTTCTCCGG CTCCGGATCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCTGAG GATTTTGCTACCTACTACTGCCAGCAGTGGCGGTCCGATCCCCTGACCTTCGG CGGCGGAACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCTTCTGTGTTC ATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTG TCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACA ACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAA GGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATG AAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCC CGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC The lower-case nucleic acid sequence “gccgccacc” in SEQ ID NOs. 19 and is the Kozak sequence that functions as the protein translation initiation site, and the “GAATTC” sequence immediately preceding it is the EcoR1 restriction enzyme site. The genes were chemically synthesized and subcloned in the pTXs?-GSbis expression vector for mammalian stable cell lines development (ProteoGenix, WO 2024/194298 PCT/EP2024/057283 Schiltigheim, France). Note that the HC and the LC are both subcloned in a single plasmid.

Creation of stable transfected poolsThe pTXs7-GSbis construct obtained as described above was used to develop stable CHO-K1 cell line pools expressing Ab11. This vector also contains a gene for glutamine synthetase (GS) that confers resistance to methionine sulfoxamine (MSX) toxicity. MSX is a drug similar to glutamate which binds to GS and thereby inhibiting the production of glutamine, which is necessary for cell growth. This ensures that CHO cells containing one or multiple copies of the vector, and hence the gene for Ab11, are selected as the MSX concentration is increased in the cell culture medium.After determination of the natural MSX resistance of the initial CHO-Ksuspension culture, the cells were transfected with the pTXs7-GS construction linearized with the enzyme Pvul. After 48h of incubation post-transfection, cells were seeded in six 96-well plates and the stable clones were selected by culturing in the presence of 30pM MSX for four weeks. After four weeks of screening, all wells were examined under a microscope and by ELISA using the 702 ± 3.5 kDa Ap42CC oligomer antigen described in Example 3.In parallel, three pools were also generated in 15mL T75 flasks similarly cultured in selective medium containing 30pM MSX.For the ELISA, 96-well Nunc Maxisorp™ ELISA plates (44-2404-21; Thermo Fisher Scientific, Waltham, MA, USA) were coated with 5 pg/mL antigen (1pL/well) for 2 h at 37 °C. Plates were blocked with 3% BSA in PBS (300 pl/well) at 37°C for 1.5h. Washing between steps was carried out by washing three times with PBS supplemented with 0.05% Tween-20 (300 uL/well). Medium from the cultures were diluted 1:1000 with PBS and 100 pL added per well. Plates were incubated at °C for 1h, washed, and an HRP-conjugated secondary mAb was added at 10ng/mL (100 pL/well) and the plates again incubated for 1h at 37 °C. After washing, 3,3',5,5'-tetramethylbenzidine substrate was added (100 pL/well), and the plates were incubated for 7 min at 37 °C. The reaction was stopped with 2 M HCI (50 pl per well). The difference in absorbance at 450 and 630 nm was measured using a spectrophotometer. The results of the ELISA screen identified 74 positive minipools for which the results are shown in Table 8.

WO 2024/194298 PCT/EP2024/057283 Table 8.ELISA results for the 74 positive minipools of transfected CHO cells Sample OD450 Sample OD450 Sample OD450 Sample OD450 1A4 0.23 2E8 0.21 4A7 0.23 5E4 1.411B1 0.21 2E11 0.21 4A11 0.26 5E7 0.211B6 0.26 2G3 0.22 4B9 0.24 5E10 0.711C1 2.15 2G4 0.22 4B12 0.23 5E11 0.231C9 0.22 2G7 0.24 4C3 0.22 5E12 0.241D2 0.22 2H5 0.23 4D2 0.22 5F1 0.241E4 1.75 3A5 0.22 4D11 0.22 5F6 0.261F3 0.20 3B7 0.20 4E2 0.23 6C1 0.241F4 0.22 3B9 0.21 4E10 0.23 6C4 0.711F5 0.21 3C3 0.21 4F5 0.25 6D3 0.201F6 0.21 3C5 0.22 4F11 0.24 6D6 0.361F12 0.21 3D8 0.22 4G6 0.94 6E2 0.241H2 0.20 3E9 0.24 4G9 0.21 6E6 0.231H5 0.21 3E12 0.22 4H6 0.22 6E9 0.231H9 1.88 3F3 0.23 4H10 0.23 6E11 0.532B2 0.24 3F5 0.20 5A4 0.24 6F12 0.632B9 0.24 3F10 0.20 5A12 0.24 6H3 0.222C3 0.21 3G7 0.20 5C1 0.25 - -2C9 0.20 4A2 0.24 5C3 0.24 - - Small-scale production and purification testsFive minipools (1E4, 5E10, 6C4, 1C1, and 4G6) and the 3 generated poolswere used to run fed-batch culture expression evaluations. Cells were cultured in 30mL of selective medium (30pM MSX) in 125mL shaking flasks and incubated for at least 3 generations (37OC, 5%CO2, 130 rpm). When the viability reached >95%, 30mL fed-batch expression tests were performed. Cells were seeded at 5x10 6 cells per ml in 30mL expression medium in 125mL shaking flasks and incubated aspreviously. Feeding medium was added on Day 3, Day 5, Day 7, and Day 9. Glucose was monitored and adjusted to 5-7g/L when needed. One of the three pools died during fed-batch expression.The cultures were stopped when the viability dropped <50% (11 days), and culture medium samples were purified and analysed by SDS-PAGE and UVabsorbance using the general protocols and procedures as those used for the WO 2024/194298 PCT/EP2024/057283 transient CHO expression analyses described in Example 2. The assessment of purity following purification is shown in Figure 9. The yield and purity obtained are summarized in Table 9. One stable minipool (5E10) and Pool 1 exhibited high expression and were selected for further development.

Table 9.Yield and purity obtained for small-scale expression tests Antibody Quantity* (mg)Yield** (mg/L)Purity*** Pool 1 32.5 1083 >90%Pool 2 0.93 31 >90%1E4 17.02 566 >90%5E10 20.59 687 >90%6C4 7.68 256 >90%1C1 9.77 325 >90%4G6 6.48 216 >90%*Obtained after purification from the 30mL-culture test. **Extrapolated from the 30ml test. ***Estimated from analysis of full-length antibodies observed on non-reduced SDS-PAGE analyses.

Isolation and Screening of Stable MonoclonesStarting from the stable pools 5E10 and Pool 1, isolation and screening of monoclones was performed using standard methods. Briefly, monoclones were isolated by using the limiting dilution method with a seeding density of 0.2 cells/well in 96-well plates. Monoclones were confirmed by microscope observation and expression evaluation screening was done by ELISA with anti-Fc antibodies. After two rounds of limiting dilution and expression screening by ELISA, the 10 best expressing monoclones were amplified by culture in 6-well plates and used to perform small-scale production evaluations with 30ml cultures in 125mL shaking flasks by using the same protocol as described above. Antibodies were then purified and analysed as previously described and the final samples obtained were buffer exchanged to PBS pH7.5, and then qualitatively and quantitatively analyzed by SDS-PAGE and UV analysis methods, respectively, as described above. Assessment of purity following purification is shown in Figure 10. The concentrations obtained are shown in Table 10. All 10 monoclones isolated WO 2024/194298 PCT/EP2024/057283 produced antibody with high purity and exhibited good expression levels in flask cultures (- 1 g/L), with 3 of them having high expression level (>1.5g/L) and one of them having a very high expression level (>2g/L).

Table 10.Fed-batch production yields obtained for the 10 best monoclones *Obtained after purification of the Clone Quantity* (mg) Yield** (g/L) 2D7 31.5 1.0504H9 61.2 2.04013F11 34.2 1.1408B6 35.00 1.1668F9 25.5 0.85015H5 26.25 0.8751H10 44.20 1.4731A3 48.60 1.6182A6 47.52 1.5845A9 46.20 1.540 30mL-culture test. **Extrapolated from the 30mL test.

RGB stability studyStability analysis of cell growthMonoclones 4H9, 1A3, and 2A6 were seeded at a density of 0.5x1 06cells/mL into 30mL selective medium in 125mL shaking flasks, and incubated at 37°C, 5%CO2, and 130 rpm for 15 passages (30 generations). Viable cell density and viability was monitored for each passage and the stability of the whole growth cycle was analyzed. The results are shown in Figure 11, for which the coefficient of variation (CV; in %) is shown in Table 11.

WO 2024/194298 PCT/EP2024/057283 Table 11.Cell growth stability analysis over 15 passages Clone CV% of VCD* CV% of viability 2A6 7.21% 0.28%1A3 6.82% 0.26%4H9 5.91% 0.25%*VCD: Viable cell density DNA sequence of transgeneGenomic DNA was extracted for PCR amplification and gene sequencing for each of the three monoclones from cell passages 5, 10 and 15. The DNA was amplified by PCR using the following primers:HC-Forward:GCAGTCACCGTCCTTGACACGGGATCCGCCGCCACCATGAAGCACCTGTGG (SEQ ID NO. 21)HC-Reverse:ATGGCTGATTATGATCAATCTCGAGTCATTTCCCGGGGGACAGGCTCAG (SEQ ID NO. 22)LC-Forward:CAGTCACCGTCCTTGACACGAAGCTTGCCGCCACCATGGTGCTGCAGAC (SEQ ID NO. 23)LC-Reverse:GTATGGCTGATTATGATCAATGAATTCTCAGCACTCCCCCCTATTAAAGC (SEQ ID NO. 24)The results of the PCR amplifications are illustrated in Figure 12, confirming the presence of the Ab11 gene. The PCR products were then sequenced and an alignment was performed which indicated that no point mutation has occurred in the Ab11 gene in any clone for 15 passages.

Production yield analysis30ml fed-batch expression tests were performed for each monoclone from passage 5, 10 and 15 and the antibodies were purified as described above. The assessments of purity using SDS-PAGE are shown in Figure 13, and the yields in Table 12. No stability issues were observed during the study for monoclones 1A WO 2024/194298 PCT/EP2024/057283 and 4H9. Monoclone 2A6 showed lower production yields during stability analysis with CV%>20%.

Table 12.Yield obtained over 15 cycles during the stability analysis Clone Initial yield (g/L) Yield of P5* (g/L) Yield of P10* (g/L) Yield of P15* (g/L) CV% 2A6 1.58 0.87 0.72 0.83 39.61A3 1.62 1.88 1.61 1.35 13.484H9 2.04 2.06 2.22 1.73 10.2Initial yield taken from Table 10 for comparison.*P5/P10/P15: Passage 5/10/15Mycoplasma test2-3x1 05 cells from each of the clones 2A6, 1A3, and 4H9 were collected for mycoplasma testing by a PCR mycoplasma test kit. The results are shown in Figure 14, which shows that the monoclonal cell cultures were devoid of mycoplasmainfection.

ConclusionIn conclusion, antibody Ab11 was successfully introduced into the genome of the CHO-K1 cell line and was found to be highly expressed in a robust and stablemanner, yielding up to 2g antibody per L culture with an estimated purity of >90%. Viable monoclonal cell lines were isolated and confirmed to be devoid of mycoplasma bacteria. Three monoclonal research cell banks were herein isolated, denoted 2A6, 1A3, and 4H9. Of these, clone 4H9 gave the highest expression level and also proved to be more stable over 15 passages than the other two clones.

Claims (26)

WO 2024/194298 PCT/EP2024/057283 Claims

1. An antibody comprising an antigen-binding domain capable of binding specifically to Ap42 prefibrillar oligomers with p structure, said antigen-binding domain comprising:(i) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO.1; or(ii) a light chain variable region (VL) comprising the sequence of SEQ ID NO.2; or a combination thereof.

2. The antibody of claim 1, wherein the antigen-binding domain comprises both said VH of (i) and said VL of (ii) in combination.

3. The antibody of claim 1 or claim 2, wherein said antibody comprises two said antigen-binding domains.

4. The antibody of any one of claims 1 to 3, wherein said antibody is a full- length immunoglobulin (ig) antibody, or an antigen-binding fragment thereof.

5. The antibody of claim 4, wherein said antibody is an IgG antibody, or an antigen-binding fragment thereof.

6. The antibody of claim 4 or claim 5, wherein the antibody is an IgG 1 Kappa antibody, or a fragment thereof.

7. The antibody of any one of claims 1 to 6, wherein said antigen- binding domain comprises:(i) a heavy chain sequence comprising the VH sequence of SEQ ID NO. linked to the heavy chain constant region sequence of SEQ ID NO. 3 or an amino acid sequence having at(ii) least 90% sequence identity to SEQ ID NO. 3; and/or(iii) a light chain sequence comprising the VL sequence of SEQ ID NO. 2 linked to the light chain constant region sequence of SEQ ID NO. 4 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 4. - 55 - WO 2024/194298 PCT/EP2024/057283

8. A conjugate comprising the antibody as defined in any one of claims 1 to 7, linked to at least one diagnostic agent.

9. The antibody as defined in any one of claims 1 to 7, for use in therapy.

10. The antibody as defined in any one of claims 1 to 7, for use in the treatmentof an amyloid disease.

11. The antibody for use according to claim 10, wherein the amyloid disease is a neurodegenerative condition associated with Ap.

12. The antibody for use according to claim 10 or claim 11, wherein the amyloid disease is a disease associated with soluble 3-structured Ap42 oligomers.

13. The antibody for use according to any one of claims 10 to 12, wherein the amyloid disease is Alzheimer’s Disease (AD), Down’s syndrome or Inclusion Body Myositis (IBM).

14. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 7, in admixture with at least one pharmaceutically acceptable carrier or excipient.

15. The antibody according to any one of claims 1 to 13, or the pharmaceutical composition of claim 14, for use in combination with Standard of Care (SOC) therapy.

16. The antibody or pharmaceutical composition for use according to claim 15, wherein the SOC therapy is an agent selected from an antiplaque agent, lecanemab and donanemab.

17. An antibody as defined in any one of claims 1 to 7, or a conjugate according to claim 8, for use in in vivo diagnosis of an amyloid disease in a subject.

18. The antibody for use in in vivo diagnosis according to claim 17, wherein the amyloid disease is a disease associated with soluble 3-structured Ap42 oligomers. - 56- WO 2024/194298 PCT/EP2024/057283

19. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody as defined in any one of claims 1 to 7, or a polypeptide comprising a VH and/or VL region thereof.

20. A vector comprising a nucleic acid molecule as defined in claim 19.

21. The vector of claim 20, wherein the vector is an expression vector.

22. A host cell comprising a vector as defined in 20 or 21.

23. The host cell of claim 22, which is a mammalian host cell.

24. A method for producing an antibody as defined in any one of claims 1 to 7, said method comprising culturing a host cell as defined in claim 22 or claim under conditions suitable for expression of the antibody as claimed in any one of claims 1 to 7.

25. The antibody as defined in any one of claims 1 to 7, for use in combination with a second therapeutic agent for use in therapy of an amyloid disease.

26. The antibody for use according to claim 25, where the second agent is selected from an antiplaque agent, lecanemab and donanemab. - 57-