Humanized antibodies against amyloid-beta 42

Abstract

The present invention provides a humanized antibody comprising an antigen-binding domain capable of specifically binding to an Aβ42 prefibril oligomer having a β structure, wherein the antigen-binding domain comprises (iii) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 1, (iv) a light chain variable region (VL) comprising the sequence of SEQ ID NO: 2, or a combination thereof. The invention also provides the use of the above antibody in the treatment of amyloid diseases, particularly Alzheimer's disease, conjugates and pharmaceutical compositions comprising the above antibody, nucleic acid molecules encoding the above antibody or its heavy chain or light chain polypeptide, and further vectors and host cells comprising such molecules.

Description

[Technical Field]

[0001] Field of Invention This invention relates to a novel humanized antibody and its use in the treatment of amyloid diseases such as Alzheimer's disease and related disorders. [Background technology]

[0002] background Alzheimer's disease (AD) is a progressive neurological disorder characterized by loss of synaptic function, ultimately leading to neuronal death. One of the main histopathological features of AD is the extracellular deposition of insoluble amyloid-beta (Aβ) peptide aggregates in the brain, forming plaques (essentially insoluble fibrous aggregates). The role these plaques play in the pathogenesis of AD has been the subject of intensive research. Various antibodies targeting these plaques have been developed. These antibodies are typically selective for aggregated Aβ or specific for pyroglutamine-oxidized Aβ. However, so far, the clinical utility of such antibodies has been very limited.

[0003] Recent studies have shown that plaques are not actually the most toxic form of Aβ, and that the neurotoxicity of Aβ is primarily due to soluble oligomeric aggregates of the amyloid-beta 1-42 peptide (Aβ42). Notably, of all Aβ present in the brains of AD patients, only a small fraction of soluble aggregated oligomeric Aβ42 is responsible for the majority of the neurotoxicity (Hong et al., Acta Neuropathologica 136, 19-40, 2018). In other words, the naturally occurring toxic form of Aβ is extremely rare, which has hindered the development of beneficial therapies. This is because therapeutics that target or cross-react with inactive, non-toxic forms of Aβ may never reach the truly toxic species.

[0004] Alzinova AB, the applicant in this case, has developed a monoclonal antibody (International Publication No. 2012 / 120035; Sandberg et al., Alz Res Therapy 14, 196, 2022). This antibody, named ALZ-201 (mAb20), is specific to a subset of the soluble oligomer Aβ42 but does not bind to other forms of Aβ (e.g., insoluble aggregates or plaques, or monomeric Aβ or non-aggregated Aβ). ALZ-201 was generated using Aβ42-CC, a stabilized form of the Aβ42 peptide, as an immunogen. Aβ42-CC restricts the conformation of the peptide and inhibits its fibrillation. Aβ42CC accumulates as a toxic prefibril oligomer with a β structure and is being developed by Alzinova as a vaccine candidate called ALZ-101 (International Publication No. 2009 / 128772), which is currently in clinical trials (NCT05328115). Therefore, the obtained ALZ-201 antibody recognizes the conformational epitope on the stabilized soluble oligomer antigen (i.e., on the vaccine candidate). The conformational specificity of this antibody has been confirmed, and it has been shown to neutralize the neurotoxic effects of postmortem brain extracts from AD patients in primary mouse neuronal cell cultures. Immunodeficiency of AD brain extracts using ALZ-201 resulted in reduced loss of nuclei, neurites, synapses, and branching compared to undeficiencyd extracts, comparable to the reduction in neurotoxicity achieved by immunodeficiency using a pan-specific anti-Aβ antibody (4G8) that binds to all forms of Aβ (Sandberg et al., Alz Res Therapy 14, 196, 2022). Further nonclinical efficacy assays have been conducted in learning tests in zebrafish embryos after injection of human extracts into the brain (Sandberg et al., poster no. 43003, presented at the Alzheimer's Association International Conference, USA, July 26-30, 2020). These data further demonstrate that the toxic effects of AD brain extracts can be prevented by immunodeficiency with ALZ-201.

[0005] Therefore, studies have shown that ALZ-201 has a high neutralizing effect against the neurotoxicity of patient-derived Aβ, even though it targets only a small fraction of patient-derived Aβ. Based on these promising preclinical studies, the monoclonal antibody (mAb) ALZ-201 is being developed for clinical use. For this purpose, a humanized antibody is necessary. [Overview of the Initiative]

[0006] Summary of the Invention In a first embodiment of the present invention, an antibody comprising an antigen-binding domain capable of specifically binding to an Aβ42 prefibril oligomer having a β structure, wherein the antigen-binding domain is (i) Heavy chain variable region (VH) containing the sequence of sequence number 1, (ii) A light chain variable region (VL) containing the sequence of sequence number 2, or Antibodies containing those combinations are provided.

[0007] In particular, the antibodies mentioned above, more specifically their binding domains, do not bind to Aβ (e.g., Aβ42) monomers or unstructured oligomers, or to the fibrous form of Aβ (e.g., Aβ42), or show only minimal or negligible binding.

[0008] The antibodies described above may be provided in various formats. They may be monovalent or bivalent. Furthermore, they may be in single-chain format or comprise two or more separate chains, for example, four chains. They may also contain both VH and VL regions, for example, a polypeptide (i.e., a chain) containing the VH region and a polypeptide (i.e., a chain) containing the VL region. However, single-domain antibodies or binding proteins containing single-domain antibodies are also known and are included herein. These may contain only VH or only VL.

[0009] The above-mentioned antibody may be an intact antibody or a full-length antibody, or a fragment thereof, particularly a fragment that retains the antigen-binding domain of the antibody. Therefore, the above-mentioned antibody may contain one or more, for example, two antigen-binding domains.

[0010] In one embodiment, the above heavy-chain variable region (VH) consists of the sequence of SEQ ID NO: 1. In one embodiment, the above light-chain variable region (VL) consists of the sequence of SEQ ID NO: 2.

[0011] The above antibody may be provided in an immunoglobulin (Ig) format, more specifically, an Ig format containing two heavy chains and two light chains, or may be provided as a fragment thereof. However, as described in more detail below, the above antibody may be in the form of any construct containing the above antigen-binding domain. This may include, for example, a fusion protein containing the above antibody and another protein, that is, a bispecific construct.

[0012] Therefore, in one embodiment, the above antibody is in the form of a full-length Ig antibody or in the form of its antigen-binding fragment (i.e., a fragment containing the antigen-binding domain). In a specific embodiment, the above antibody is an IgG antibody or a fragment thereof.

[0013] Therefore, the above antibody may include a first chain (heavy chain) having a VH region and a heavy-chain constant region, and a second chain (light chain) having a VL region and a light-chain constant region. In a specific embodiment, the light-chain constant region is a kappa (κ) constant region. In another specific embodiment, the heavy-chain constant region is an IgG constant region, more specifically an IgG1 constant region. The above antibody may contain one or more of each of the first and second chains, for example, two heavy chains and two light chains.

[0014] In a specific embodiment, the above antibody is an IgG1 kappa antibody or a fragment thereof.

[0015] In another aspect, a conjugate is provided that comprises an antibody as defined herein conjugated to at least one diagnostic agent.

[0016] In a further aspect, an antibody as defined herein for use in a method of treatment is provided.

[0017] In another aspect, an antibody or conjugate thereof for use in the diagnosis of a human or animal body (i.e., in vivo diagnosis) is provided.

[0018] In a further aspect, a pharmaceutical composition is provided that comprises an antibody as defined herein admixed with at least one pharmaceutically acceptable carrier or excipient.

[0019] In yet a further aspect, an antibody or pharmaceutical composition as defined herein for use in the treatment of an amyloid disease is provided.

[0020] In yet a further aspect, a conjugate as defined herein for use in the in vivo diagnosis of an amyloid disease is provided.

[0021] A related aspect provides for the use of an antibody as defined herein in the manufacture of a medicament for use in a method of treatment of an amyloid disease.

[0022] A further related aspect provides a method of treatment of an amyloid disease, the method comprising administering to a subject in need thereof an antibody or pharmaceutical composition as defined herein in a therapeutically effective amount.

[0023] The various medical uses and methods described above are particularly for the treatment or prevention of amyloid diseases.

[0024] In some embodiments of the various uses and methods described above, the amyloid disease may be any one of Alzheimer's disease (AD), Down syndrome, or inclusion body myositis (IBM).

[0025] The subject mentioned above may be any human or animal, and is particularly human.

[0026] Another embodiment provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or its VH region and / or VL region as defined herein.

[0027] In this regard, it will be understood that the above antibodies may consist of or contain one or more (e.g., two or more) protein chains or subunits, and in such a form, individual chains or subunits, for example, individual molecules (i.e., polypeptides) containing the VH region and / or VL region, may be encoded by separate nucleic acid molecules.

[0028] Therefore, this embodiment can be considered to provide one or more nucleic acid molecules comprising a nucleotide sequence encoding an antibody as defined herein.

[0029] Furthermore, nucleic acid molecules as defined herein or vectors comprising one or more nucleic acid molecules are also provided. Similarly, this embodiment can be considered to provide one or more expression vectors comprising one or more nucleic acid molecules as defined herein.

[0030] In one embodiment, the vector is an expression vector.

[0031] The vector described above may be a viral vector. Therefore, viruses comprising one or more nucleic acid molecules as defined herein are also provided.

[0032] Another embodiment provides a host cell containing a vector (e.g., an expression vector) or a nucleic acid molecule (more specifically, one or more vectors or nucleic acid molecules as defined herein) as defined herein, or a host cell expressing an antibody as defined herein.

[0033] The host cell described above may be a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell is a mammalian host cell.

[0034] Further embodiments provide a method for producing an antibody as defined herein, comprising culturing a host cell as defined herein under conditions suitable for the expression of the antibody.

[0035] The above method may further include obtaining the antibody from the host cells or from the culture (e.g., the growth medium or supernatant after culturing) (e.g., by collection or isolation).

[0036] The above method may further include the step of introducing one or more nucleic acid molecules or vectors as defined herein into the host cell.

[0037] Detailed description of the invention The mouse antibody ALZ-201 was humanized using the following method: Three complementarity-determining regions (CDRs), defined by Kabat nomenclature, were obtained from the light chain variable region (VL) and heavy chain variable region (VH), respectively. These were then transplanted into human germline VL and VH cells, selected to approximate the corresponding mouse VL and VH cells as closely as possible in terms of sequence homology, thereby humanizing the antibody.

[0038] The term "VH" (or VH domain), or "heavy chain variable region," refers to the variable region of the heavy chain of an antibody molecule. VH contains three heavy chain CDRs (VHCDRs) called VHCDR1, VHCDR2, and VHCDR3, arranged from the amino terminus to the carboxyl terminus.

[0039] The term "VL" (or VL domain), or "light chain variable region," refers to the variable region of the light chain in an antibody molecule. The VL contains three light chain CDRs (VLCDRs) called VLCDR1, VLCDR2, and VLCDR3, arranged from the amino terminus to the carboxyl terminus.

[0040] CDR is a hyper-variable region within the variable region.

[0041] Furthermore, the variable regions of the heavy and light chains each possess four framework regions (FR1, FR2, FR3, and FR4, from the amino terminus to the carboxyl terminus). These framework regions are more conserved and demarcate the CDR.

[0042] Designing functional humanized antibodies requires considering several factors, and humanization is not a simple process.

[0043] As described above, and in more detail in Example 1 below, a molecular model of the antibody ALZ-201 was constructed in preparation for humanization. This construction itself was a difficult task requiring considerable human effort. This molecular model was then used as a guide for humanization design. Using this molecular model as a guide, the first step of humanization design was carried out in silico. This step also involved various design considerations and choices, including the selection of specific human germline sequences and the selection of residues within the human framework region to be "back-mutated," that is, substituted with amino acids derived from parental mice. Furthermore, during the design process, residues within the CDR were selected for "germ-lining" (i.e., substitution with their human germline counterparts). As a result, six humanized heavy chain variable region (VH) sequences were designed based on two human germline VH sequences, and eight humanized light chain variable region (VL) sequences were designed based on four human germline VL sequences. In this way, various different humanized VH / VL combinations were derived that could serve as alternative humanized variants of ALZ-201. In the next step, a list of 48 combinations was narrowed down to 18 using various selection criteria, and from these, 16 combinations were selected for experimental testing along with chimeric variants of ALZ-201 (hereinafter referred to as chALZ-201 or mAb15). Candidate combinations were iteratively ranked based on their degree of humanness, more specifically, percentage (%) (% identity with human amino acid residues, in other words, % sequence identity between humanized VH and VL sequences and human V gene sequences; % sequence identity with human homologous antibodies ranged from 81.0% to 88.7%), as well as the results of various experimental tests regarding expression yield, affinity, and thermal stability.More specifically, six candidates were selected for further testing of affinity (EC50 measured by ELISA) and temperature stability. These selections and tests were further narrowed down to five candidates for affinity testing by SPR (Kd(nM) measured by Biacore), then to the top two candidates for expression yield testing, and finally, the lead candidate antibody identified as Ab11 in the example described below was selected.

[0044] The resulting humanized antibody is defined by specific VH and VL sequences. It is well known in the art that antibodies can be provided in a variety of formats and configurations, including various constructs, beyond the classical format of immunoglobulins and their fragments. Not only antibodies and constructs containing both VH and VL regions, but also so-called single-domain antibodies containing only VH or only VL can be prepared. Accordingly, this specification provides antibodies defined by the humanized VH and / or VL sequences of the resulting humanized ALZ-201 derivative. The antibody comprises one or at least one antibody-binding domain (or, in other words, antigen-binding unit). Since the antigen-binding domain is obtained from or derived from the antibody, the antibody may be referred to or considered as an antibody-based binding protein, or a binding protein containing an antibody-derived binding domain.

[0045] The antigen-binding domain of the antibody described above includes at least one VH region or at least one VL region. In one embodiment, the antigen-binding domain includes at least one VH region and at least one VL region.

[0046] The above-mentioned binding protein may contain one or more antigen-binding domains. Each antigen-binding domain may contain one (or at least one) VH region, one (or at least one) VL region, or both one VH domain and one VL domain (or at least one VH region and at least one VL region). In a typical antibody format, the above-mentioned binding protein contains two antigen-binding domains (in other words, it is bivalent), and more specifically, each antigen-binding domain contains a VH region and a VL region. However, as will be further explained below, both monovalent antibodies and antibody fragments are known, which contain a single antigen-binding domain. In such embodiments, the single antigen-binding domain may contain both a VH region and a VL region. Furthermore, as described above, the binding protein may contain an antigen-binding domain that contains only a VH region or only a VL region, for example, as a single-domain antibody, such as a VHH antibody or a camelid antibody.

[0047] In this specification, the terms "a" and "an" are used to mean "at least one," "at least one first," "one or more," or "more than one" of the mentioned components or processes, unless an upper limit is explicitly provided thereafter.

[0048] Furthermore, where the terms “comprise,” “comprises,” “has,” or “having,” or other equivalent terms are used herein, in some more specific embodiments these terms may include “consists of,” “consists essentially of,” or other equivalent terms.

[0049] As described above, the VH region contains the amino acid sequence described in SEQ ID NO: 1. VH (Sequence ID 1) QVTLKESGPTLVKPTQTLTLTCTFS GFSLSTFGSGVSWIRQPPGKALEWLA HIYWDDDKH YNPSLKSRLTITKDTSKNQVVLTITNMDPVDTATYFCAR RESHYYGSGYYFDY WGQGTLVTVSS

[0050] The VL region contains the amino acid sequence described in Sequence ID No. 2. VL (Sequence ID 2) DIQLTQSPSSLSASVGDRVTITC RASSSISYMH WYQQKPGKAPKPWIY ATSNLAS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQWRSDPLT FGGGTKVEIK

[0051] As mentioned above, each of these variable region sequences contains three complementarity determination regions (CDRs, underlined in the sequences above) and four framework regions (FRs).

[0052] The Kabat numbering scheme is a widely adopted standard for numbering and assigning CDR residues in antibodies based on sequence alignment data (Johnson G. and Wu TT, Nucleic Acids Res. 28, 214-18, 2000). In this scheme, VHCDR1 is assigned to residues 31-35B (position 35 is a specified insertion point, and the notation "35B" is understood to indicate this in the art), VHCDR2 is assigned to residues 50-65, and VHCDR3 is assigned to residues 95-102. On the other hand, VLCDR1 is assigned to residues 24-34, VLCDR1 is assigned to residues 50-56, and VLCDR3 is assigned to residues 89-97.

[0053] Chothia's definition of CDRs was later introduced based on available antibody structures (Chothia, C. and Lesk, AM, J. Mol. Biol. 196, 910, 1987). The AbM method (KR Abhinandan KR and Andrew CR Martin ACR, Molecular Immunology 45, 3832-3839, 2008) utilizes a modified version of the Chothia scheme, assigning VHCDR1 to residues 26-35B, VHCDR2 to residues 50-58, 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 the above SEQ ID NOs: 1 and 2.

[0054] The AbM scheme was used to define the CDR sequence of the parent antibody (International Publication No. 2012 / 120035A1). These CDR sequences, with the exception of VLCDR1, correspond to the CDR sequence of the parent antibody ALZ-201. In the selected humanized antibody, VLCDR1 consists of the residue RASSSISYMH (SEQ ID NO: 17), while in the unhumanized parent antibody ALZ-201, it consists of the residue RASSSVSYMH (SEQ ID NO: 18). Therefore, the amino acid at position 29 of the humanized derivative is isoleucine, not valine.

[0055] An alternative method for assigning CDRs is called the IMTG scheme (Lefranc MP et al., Developmental & Comparative Immunology 27, 55-77, 2003). Using this scheme, VHCDR1 is assigned to residues 26-35B, VHCDR2 to residues 51-56, and VHCDR3 to residues 93-102. On the other hand, VLCDR1 is assigned to residues 27-32, VLCDR2 to residues 50-51, and VLCDR3 to residues 89-97.

[0056] Antibodies can take various forms, including antibody fragments, as described above. All such forms are encompassed. As described above, an antibody may contain one or more antigen-binding domains, or one or more VH regions and / or one or more VL regions. The VH and / or VL regions may be contained in a single chain (polypeptide) or in separate chains (polypeptides). Accordingly, an antibody may contain one or more polypeptide chains, for example, two or four polypeptides. Each polypeptide may contain one or more VH regions and / or one or more VL regions, for example, both VH and VL regions.

[0057] Therefore, the antibody-binding domain may contain one or more polypeptides (chains) that include one or more VH regions and / or one or more VL regions.

[0058] Furthermore, the polypeptide (chain) containing the variable region sequence may contain all or part of the constant region sequence. For example, in the case of VH, it may contain one, two, or all three of the CH1, CH2, and CH3 of the heavy chain constant region, and in the case of VL, it may contain all or part of the light chain constant region (CL). Since VH and VL in this specification are humanized, the constant region sequence is a human sequence or a humanized sequence, and is particularly a human sequence.

[0059] Therefore, the term “antibody” encompasses all known antibody forms, including whole antibodies or full-length antibodies, or any antigen-binding fragments thereof, or single-chain or single-chain derivatives thereof, as well as synthetic or artificial antibody constructs comprising at least one VH region or at least one VL region as defined herein, and their multimers (e.g., dimers, trimers, or higher-order multimer antibodies). It will be understood that this term also encompasses recombinant antibodies and engineered antibodies. More broadly, the term “antibody” can be considered to include any binding protein (sometimes called an immunological binding protein) that contains an antigen-binding domain, specifically an antigen-binding domain derived from an antibody. Therefore, the term “antibody” is used to refer to any antibody-like molecule having an antigen-binding region obtained from or derived from an antibody. In a preferred embodiment, the antibody or fragment thereof includes at least one VH region and at least one VL region.

[0060] In one embodiment, the antibody is an immunoglobulin antibody, more specifically, an antibody comprising at least two heavy chains and at least two light chains, or a fragment thereof.

[0061] In any format described herein, if the antibody contains a constant region, the heavy chain comprises all or part of the VH and the heavy chain constant region as defined herein, and the light chain comprises all or part of the VL and the light chain constant region as defined herein. If the antibody comprises all of the constant regions derived from the heavy chain and the light chain, the antibody is called a full-length antibody. Such a full-length antibody is one preferred embodiment. Antibodies are classified into one of five major classes, IgA, IgD, IgE, IgG, and IgM, depending on the type of constant domain in the heavy chain. While all of these are included, IgA and IgG are preferred, and IgG is particularly preferred. Some of these are further classified into subclasses or isotypes such as IgG1, IgG2, IgG3, and IgG4. For example, camelid antibodies are IgG antibodies and often have a constant domain of IgG2 or IgG3. All subclasses are included herein. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of each different class of immunoglobulin are well known. Preferred heavy chain constant regions are known and available in the art. The light chains of mammalian antibodies are classified into two distinct types: kappa (κ) and lambda (λ), and either type can be used. Again, suitable light chain constant region sequences are known and available in the art.

[0062] In one embodiment, the heavy chain constant region is all or part of a human IgG1 constant region having the amino acid sequence described in Sequence ID No. 3, or an amino acid sequence having at least 90% sequence identity with said sequence. Sequence ID 3, Human IgG1 constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0063] In another embodiment, the light chain constant region is all or part of a human kappa constant region having the amino acid sequence described in Sequence ID No. 4, or an amino acid sequence having at least 90% sequence identity with said sequence. Sequence ID 4, Human Kappa Steady Region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0064] In each more specific embodiment, the above amino acid sequences have at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 3 or 4, respectively.

[0065] Therefore, some degree of variation is permissible in the constant region sequence of the binding proteins / antibodies described herein.

[0066] A sequence that exhibits at least 90% sequence identity with a specified reference sequence is sometimes referred to as substantially identical or substantially similar. Such a sequence may contain one or more changes (additions, substitutions, insertions, or deletions) of a base or amino acid relative to the given sequence. Amino acid substitutions may be conservative substitutions. Such sequence modifications may be made for a variety of reasons, including, for example, ease of antibody production, stability, and pharmacokinetics.

[0067] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As another example, families of amino acid residues can be grouped based on whether they have hydrophobic or hydrophilic side chains.

[0068] Sequence identity can be evaluated by any convenient method. However, computer programs that perform multiple alignments of sequences are useful for determining the degree of identity between sequences, such as ClustalW (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If necessary, the ClustalW algorithm can be used in combination with the BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap start penalty of 10 and a gap extension penalty of 0.1, which yields the highest degree of agreement between two sequences, where at least 50% of the total length of one sequence is included in the alignment. Another method that can be used to align sequences is the alignment method by Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970), which was improved by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981). This improvement allows for the highest degree of agreement between two sequences and also determines the number of identical amino acids between the two sequences. Other methods for calculating the percentage of identity between two amino acid sequences are generally recognized in the art and include, for example, the method described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988), and the methods described in "Computational Molecular Biology" (edited by Lesk, Oxford University Press, New York, 1988) and "Biocomputing: Informatics and Genomics Projects".

[0069] Generally, computer programs are used for such calculations. Programs that compare and align pairs of sequences, such as 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 et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984), are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics Institute provides structure-based protein sequence alignment (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.

[0070] Sequences according to the present invention having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity can be determined using any of these available programs, for example, the LALIGN program with default parameters.

[0071] In one embodiment, the antibody is an IgG antibody, more specifically an IgG1 antibody, or a fragment thereof.

[0072] In one particular embodiment, the antibody is an IgG kappa antibody or a fragment thereof.

[0073] In another embodiment, the antigen-binding domain is (i) A heavy chain sequence including the VH sequence of Sequence ID No. 1 linked to the heavy chain constant region sequence of Sequence ID No. 3 or an amino acid sequence having at least 90% sequence identity with Sequence ID No. 3, and / or (ii) A light chain sequence comprising the constant region sequence of the light chain of Sequence ID No. 4 or the VL sequence of Sequence ID No. 2 linked to an amino acid sequence having at least 90% sequence identity with Sequence ID No. 4.

[0074] Antibody fragments that retain the antigen-binding ability of the full-length parent antibody, in other words, antibody fragments containing an antigen-binding domain, include Fab', Fab, F(ab')2, Fd, Fv, single-domain antibodies (DABs), such as those consisting of a VH domain (also called VHH antibodies), and nanobodies.

[0075] Other antibody formats known in the art and included herein include scFv (single-chain Fv), dsFv, ds-scFv, linear antibodies, TandAb, minibody, diabody, biobody, and tribody (scFv-Fab fusions, each having bispecificity or triplicity); sc-diabody; kappa (lambda)body (scFv-CL fusion); BiTE (Bispecific T-cell Engager, a T-cell-attracting scFv-scFv tandem); DVD-Ig (bivariable domain antibody, bispecificity format); scFv-Fc dimer; and DART (disulfide-stabilized diabody "Dual Affinity ReTargeting")).

[0076] Examples of single-domain antibodies include camelid antibodies, vNAR (shark) antibodies, VH antibodies, or VL antibodies.

[0077] Techniques for producing various antibody formats, fragments, and constructs are well known in the relevant field.

[0078] Antibodies can be fragmented using conventional techniques. For example, an F(ab')2 fragment can be produced by treating an antibody with pepsin. The resulting F(ab')2 fragment can be subjected to a disulfide bond reduction treatment to produce a Fab' fragment. A Fab fragment can be formed by papain digestion. Fab, Fab', and F(ab')2, scFv, Fv, dsFv, Fd, dAb, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be produced by recombinant techniques, cell-free expression systems, or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.

[0079] As described above, in some embodiments, the antibody or antibody fragment comprises all or part of the heavy chain constant region. Furthermore, the antibody or antibody fragment may comprise all or part of the kappa light chain constant region or the lambda light chain constant region. All or part of such constant regions may be naturally occurring or fully or partially synthesized. Suitable sequences for such constant regions are well known in the art, described in the literature, and exemplified above.

[0080] In other embodiments, there is no constant region (e.g., a constant region of the heavy or light chain), and for example, a variable domain or heavy chain variable domain (VH) is the sole part of the antibody.

[0081] The antibodies and nucleic acids described herein are artificial (e.g., manipulated or synthesized) constructs that do not exist in nature and do not correspond to naturally occurring molecules. In other words, the antibodies and nucleic acids described herein are non-natural. This is implied by their composition.

[0082] In one embodiment, the antibody may be provided in a form or conjugate comprising a binding protein linked to or attached to another entity (i.e., conjugated). The other entity may be another protein or polypeptide component and may be conjugated to the binding protein via a peptide bond or peptide linker. In this case, the resulting conjugate can be considered a fusion protein comprising a binding partner linked to (or fused with) the fusion partner. Alternatively, the other entity may be non-proteinogenic, for example, a small molecule or other chemical or physical entity (e.g., polymer, label, etc.) and may be chemically linked to the binding protein by 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.

[0083] Those skilled in the art will understand that the antibodies and antibody fragments may be prepared by any of several methods known and described in the art, but can be easily prepared using recombinant methods.

[0084] For this purpose, nucleic acid molecules (e.g., one or more nucleic acid molecules) comprising a nucleotide sequence encoding an antibody or a part or fragment thereof as defined herein are other embodiments herein.

[0085] In this regard, appropriate coding nucleotide sequences can be devised based on the VH and VL amino acid sequences of SEQ ID NOs: 1 and 2. These can be codon-optimized for expression in a desired host cell according to well-known techniques and principles in the art. In one embodiment, the above nucleotide sequences are codon-optimized for expression in mammalian cells.

[0086] An exemplary nucleotide sequence encoding the VH sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 5 below, and an exemplary nucleotide sequence encoding the VL sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 6 below. Here, the first nine nucleotides GCCGCCACC constitute the protein translation initiation site. Both SEQ ID NOs: 5 and 6 are optimized for expression in CHO cells and contain a signal peptide for protein secretion (underlined nucleotides). Sequence ID 5 GCCGCCACC ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCTGCTCTCGGTGGGTGCTGTCC CAGGTGACCCTGAAGGAGTCCGGCCCCACCCTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCAGCGGCTTTAGCCTGAGCACCTTTGGCAGCGGCGTGAGCTGGATCAGGCAGCCTCCCGGCAAGGCCCTGGAGTGGCTGGCTCACATCTATTGGGACGACGACAAGCACTATAAC CCTAGCCTGAAGAGCCGGCTGACCATCACCAAGGACACCAGCAAGAACCAGGTGGTGCTGACCATCACAAACATGGACCCTGTGGATACCGCCACCTATTTTTGCGCCCGGAGGGAGAGCCACTACTATGGCAGCGGCTACTATTTCGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC Sequence ID 6 GCCGCCACC ATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGTCCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCGGGCTTCCTCCAGCATCTCCTATATGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCCTTGGATCTACGCTACCAGCAATCTG GCTAGCGGCGTGCCTAGCCGGTTCTCCGGCTCCGGATCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTGGCGGTCCGATCCCCTGACCTTCGGCGGCGGAACCAAGGTGGAGATCAAG

[0087] Therefore, in one embodiment, the nucleic acid molecule includes the nucleotide sequence described in SEQ ID NO: 5 or a sequence having at least 90% sequence identity with respect to said nucleotide sequence, and / or the nucleotide sequence described in SEQ ID NO: 6 or a sequence having at least 90% sequence identity with respect to said nucleotide sequence. In each more specific embodiment, the % sequence identity with respect to SEQ ID NO: 5 or 6 is at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0088] Such variant sequences may be degenerate sequences that encode the amino acid sequences described in SEQ ID NOs. 1 and 2, respectively.

[0089] The antibodies and nucleic acid molecules described herein may contain sequence-based modifications or chemical equivalents to the amino acid and nucleotide sequences, provided that these modifications do not substantially alter the function of the antibody or nucleic acid molecule. Therefore, the antibody must retain the binding and functional properties of the corresponding unmodified antibody.

[0090] The antibody may include other amino acid sequences that do not contribute to antigen binding, such as tag sequences, fusion partners, or other components; it may include changes for converting 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 a whole antibody, e.g., an antibody consisting only of the full-length heavy chain, or vice versa); it may include changes for converting one antibody molecule to a specific class or subclass of antibody molecule (e.g., conversion from one antibody molecule to IgG or its subclasses, e.g., IgG2 or IgG3, e.g., a camelid antibody, or an IgA class antibody); and it may include changes for creating 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.

[0091] Those skilled in the art will understand that a binding assay can be used to test whether any such modified antibody and construct retains the binding properties of the original humanized antibody.

[0092] The nucleic acid fragments encoding the VH and VL sequences described herein can be derived or produced by any suitable method, such as cloning or synthesis.

[0093] After obtaining nucleic acid fragments encoding VH and VL, these fragments can be further manipulated using standard recombinant DNA techniques. For example, variable region fragments can be manipulated to convert them into full-length antibody molecules with appropriate constant region domains, or into antibody fragments of specific formats as described elsewhere herein, such as single-domain antibodies like VHH, Fab fragments, scFv fragments, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding VH and / or VL are, if necessary, incorporated into one or more vectors, in particular, appropriate expression vectors, to facilitate antibody production or to facilitate selection or screening, such as by incorporation into a phage display vector.

[0094] Conveniently, the vector may contain one or more nucleotide sequences that encode other amino acid sequences, such as a constant region sequence, intended to form part of the antibody.

[0095] Possible expression vectors include, but are not limited to, cosmids, plasmids, or viral vectors, such as modified viruses (e.g., replication-deficient retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses), provided that the vector is compatible with the host cell in which it will be used. Various vectors are available in the art for such applications, including, for example, the pXten1 plasmid used in the XtenCHO® mammalian expression system available from ProteoGenix. An expression vector is "suitable for transformation of host cells," meaning it contains a nucleic acid molecule and a regulatory sequence selected based on the host cell used for expression, the regulatory sequence being operably ligated to the nucleic acid molecule. "Operatably ligated" means that the nucleic acid is ligated to the regulatory sequence in a manner that enables the expression of that nucleic acid.

[0096] Accordingly, another aspect herein is a vector, particularly an expression vector, containing or comprising a nucleic acid molecule or fragment thereof as defined or described herein. If it is an expression vector, it also includes regulatory sequences necessary for the transcription and translation of the protein sequence encoded by the nucleic acid molecule. The vector or expression vector may be recombinant.

[0097] For antibody production in host cells, it may be desirable to conjugate a signal sequence or leader sequence to the expressed protein or protein subunit (i.e., the expressed polypeptide) to guide the expression product to a specific compartment or site, such as a secretion signal sequence for extracellular expression of the protein. The expression product can then be easily collected or recovered from the supernatant or growth medium.

[0098] As a representative example, the encoded amino acid sequences of the polypeptides expressed in the examples are shown below. These include the leader sequence. Heavy chain polypeptide - SEQ ID NO: 7

number

[0099] Light chain polypeptide - SEQ ID NO: 8

number

[0100] Expression vectors can be introduced into host cells to produce transformed, transduction, or transfected host cells. The terms “transformed,” “transduction,” and “transfection” are typically used to refer to different methods of introducing nucleic acids into cells (more generally, by plasmids, viral vectors, or nonviral methods). In this specification, these terms encompass the introduction of nucleic acids (e.g., vectors) into cells by one of the many possible techniques known in the art. Preferred methods for transforming, transduction, and transfecting host cells are well known in the art and are described in textbooks on experimental techniques.

[0101] Suitable host cells include a wide variety of eukaryotic and prokaryotic cells, but as is well known to those skilled in the art, it is also possible to use plant cell-based or fungal expression systems (e.g., Saccharomyces cerevisiae). For example, the binding protein may be expressed in yeast cells of the genera Pichia or Saccharomyces (e.g., Saccharomyces cerevisiae), insect cells, mammalian cells (e.g., CHO cells), or other cell lines. Furthermore, the protein of the present invention can also be expressed in prokaryotic cells such as Escherichia coli (E. coli).

[0102] In vitro cell-free protein expression is another method for producing recombinant proteins in solution using biomolecular translation mechanisms extracted from cells. Furthermore, proteins can also be synthesized through chemical synthesis using well-known techniques in protein chemistry, such as solid-phase synthesis.

[0103] When VH and VL are provided as separate chains or in separate chains (i.e., separate polypeptides are expressed), they may be encoded by nucleotide sequences provided in separate vectors. Therefore, further embodiments provide expression constructs, expression vectors, or expression systems (e.g., viral, bacterial, or other expression constructs, vectors, or systems), such as one or more expression constructs or vectors, comprising one or more nucleic acid molecules described herein. As described above, such a system may be represented by a plurality of vectors, including a first vector containing a nucleotide sequence encoding a polypeptide comprising a VH region and a second vector containing a nucleic acid sequence encoding a polypeptide comprising a VL region.

[0104] A suitable construct or vector is one that can express antibodies in host cells for a long period or sustainedly. Such expression may be transient (e.g., episomal) or more permanent (e.g., by genomic integration), provided that sufficient expression levels and durations are achieved. Further embodiments provide a host cell (e.g., a mammalian, bacterial, or yeast host cell) or virus containing one or more expression constructs or expression vectors as defined herein, for example, one or more host cells or viruses. Also provided are a host cell or virus containing one or more nucleic acid molecules as defined herein. A host cell or virus expressing an antibody as defined herein constitutes a further embodiment.

[0105] Further embodiments provide a method for producing (or manufacturing) an antibody as defined herein, comprising the step of culturing host cells. More specifically, the method comprises (i) culturing host cells containing one or more expression vectors or one or more nucleic acid molecules under conditions suitable for the expression of an encoded antibody, and optionally (ii) isolating or collecting the antibody from the host cells or from the growth medium / supernatant. Such a production (or manufacturing) method may also include the steps of purifying the antibody and / or formulating the antibody into a composition, such as a pharmaceutical composition, containing at least one additional component, such as a pharmaceutically acceptable carrier or excipient. The method may also include a prior step of introducing the vector or nucleic acid molecule into the host cells.

[0106] In embodiments in which the antibody is composed of multiple polypeptide chains (e.g., a specific fragment such as a Fab fragment or the entire antibody), all polypeptides are expressed in the host cell from the same or different expression vectors, thereby allowing the complete antibody protein to assemble in the host cell and be isolated or purified from that cell. Compositions comprising antibodies, nucleic acid molecules or expression vectors, or a first host cell as defined herein constitute further embodiments. Formulations (compositions) comprising one or more antibodies in a mixture with a suitable diluent, carrier, or excipient as needed constitute preferred embodiments. Such formulations may be for pharmaceutical use, and therefore, it is preferable that such compositions be pharmaceutically acceptable or acceptable for administration to humans or non-human animals. Suitable diluents, excipients, and carriers are known to those skilled in the art.

[0107] Pharmaceutical compositions may be provided in forms suitable for systemic administration, such as subcutaneous, intravenous, infusion, intra-arterial, intraperitoneal, intramuscular, or parenteral administration, including intracerebral (intraventricular) administration. Pharmaceutical compositions may also be provided as conventional pharmaceutical formulations suitable for systemic administration, such as solutions, suspensions, or other preparations suitable for injection or infusion. Conventional pharmaceutical excipients and conventional manufacturing methods can be used to prepare these pharmaceutical formulations.

[0108] Solutions for injection or intravenous infusion, such as sterile aqueous solutions, can be manufactured by conventional methods, for example, by adding excipients and preservatives, antioxidants, and / or stabilizers.

[0109] The formulation may be provided in a form containing a single dose or multiple doses, for example, in a container such as a vial or ampoule. A suitable dosage unit can be determined by those skilled in the art.

[0110] Further embodiments provide antibodies as defined herein, or nucleic acid molecules, expression vectors, or host cells as defined herein, or pharmaceutical compositions containing them, for use in therapeutic or in vivo diagnostic purposes, particularly for use in the treatment or prevention of amyloid disease. In particular, amyloid disease may be any disease or condition associated with (or characterized by) Aβ42 prefibril oligomers having a β structure (i.e., soluble Aβ42 oligomers having a β structure), or in which such oligomers play a role, e.g., a causative role (e.g., a complete or partial causative role) or an essential role.

[0111] In this specification, the term “amyloid disease” refers, in particular, to a disease (or condition or disorder) associated with the abnormal aggregation of proteins or peptides to form amyloid deposits. Amyloid deposits are often found in the central nervous system, more specifically in the brain (e.g., in the case of AD), but the diseases treated with the binding proteins described herein are not limited to this, and such deposits may be present or observed in other tissues or body parts. However, as stated above, amyloid disease may also be associated with the abnormal aggregation of proteins or peptides to form soluble aggregates commonly called “oligomers.” More specifically, the disease is associated with the abnormal aggregation of Aβ, particularly Aβ42, to form amyloid fibrils and toxic soluble aggregates (collectively referred to as Aβ oligomers). These oligomers are prefibrils and have a structure distinct from insoluble fibrous plaques. Such diseases include any disease that benefits from the removal or depletion of soluble Aβ42 prefibril oligomers having a β structure, and these oligomers are recognized as toxic substances in the art. By removing these toxic oligomers in these diseases, the onset of the disease may be prevented or delayed.

[0112] In one embodiment, the disease is a neurodegenerative disease related to Aβ, particularly Aβ42, and more specifically, Aβ42 prefibril oligomers having a β structure, such as dementia.

[0113] In one embodiment, the disease is Alzheimer's disease (AD) or a related disorder, such as Down syndrome, in which the prevalence of AD is 90% to 100% in people in their 70s and it is considered a major cause of death (Fortea et al., Lancet 395, 1988-1997, 2020).

[0114] In another embodiment, the disease is a progressive degenerative muscle disease, such as sporadic inclusion body myositis, in which abnormal Aβ aggregation has been suggested to contribute to the disease (Kitazawa et al., Journal of Neuroscience 29, 6132-6141, 2009).

[0115] According to the therapies described herein, antibodies can target, inhibit, or reduce the toxic effects of Aβ42 prefibril oligomers having a β structure. Therefore, antibodies can be used to treat or prevent any disease or condition in which the reduction of this toxic effect is beneficial.

[0116] In the therapeutic methods and uses described herein, the administration of antibodies, nucleic acid molecules, expression vectors, or host cells is carried out in a pharmaceutically, therapeutically, or physiologically effective amount to the subject requiring treatment. Therefore, such methods and uses may include additional steps to identify the subject requiring treatment.

[0117] The subjects may be humans, or non-human animals, especially mammals.

[0118] Treatment of a disease includes curing the disease, or reducing or alleviating it, such as reducing the severity or symptoms of the disease, or improving the patient's condition. Examples of parameters to be evaluated may include improvements in functional, cognitive, or memory tests, or other tests used to assess the disease.

[0119] Beneficial effects include delaying or inhibiting the progression of the disease. In particular, in cases of early diagnosis, or in individuals at risk of or susceptible to the disease, the onset of the condition or the manifestation of its symptoms may be delayed or even prevented. Therefore, the treatments described herein may be prophylactic or preventative.

[0120] Such preventative measures may be implemented for healthy subjects, normal subjects, or subjects at risk, and may include both complete prevention and significant prevention. Significant prevention may include scenarios in which the severity or symptoms of the disease are reduced (e.g., by a measurable degree or significantly) compared to the severity or symptoms that would be expected without treatment. Furthermore, in subjects in the early stages of the disease, the progression of the disease may be delayed or slowed.

[0121] A "pharmaceutical, physiological, or therapeutically effective amount" means an amount sufficient to demonstrate benefit for the condition in question. Whether an amount is sufficient to demonstrate benefit for the condition in question can be easily determined by those skilled in the art. A pharmacological, physiological, or therapeutically effective amount can be determined based on clinical evaluation and is easily monitorable.

[0122] Furthermore, the antibodies or pharmaceutical compositions described herein may be used in combination with one or more other therapeutic agents (for example, other agents known or proposed for use in the treatment of the disease). In other words, the antibodies or pharmaceutical compositions described herein may be used in combination therapy, such as in combination with standard of care (SOC) therapy. In one embodiment, the antibodies or pharmaceutical compositions described herein are intended for use in combination therapy, such as in combination with standard of care (SOC) therapy for amyloid disease.

[0123] In such combination therapy, a second drug may be administered to the target patient together with the antibody. This administration may be carried out simultaneously, separately, or sequentially, in the same pharmaceutical composition or in separate compositions.

[0124] Examples of therapeutic agents used in combination therapy with the antibodies described herein include other antibodies proposed or developed for use in the treatment of amyloid diseases, or other antibodies proposed or developed to reduce the amount of β-amyloid in the brain, for example (often called anti-plaque agents), such as lecanemab (Leqembi®) or aducanumab (Aduhelm®). Donanemab has successfully completed Phase 3 clinical trials and is currently under review by the FDA and EMA. This is yet another plaque-reducing antibody that may be useful in combination therapy with the antibodies described herein.

[0125] In additional embodiments, the antibody may be used in combination with one or more other agents or techniques to enhance the blood-brain barrier penetrance of the antibody, many of which are known in the art (Ayub and Wetig, Pharmaceuticals 14, 224, 2022). One such preferred embodiment utilizes an antibody-transferrin fusion construct, in which the binding protein is transported between the blood and the brain via transcytosis mediated by the transferrin receptor.

[0126] The binding protein can also be used for in vivo applications, such as as a molecular tool for in vivo diagnostics.

[0127] Therefore, in further aspects of the present invention, reagents comprising antibodies as defined herein and the use of such antibodies as molecular tools in in vivo assays are provided. For example, this includes use for detecting Aβ42 prefibril oligomers having a β structure in a target in an in vivo imaging procedure.

[0128] In one embodiment, the method for diagnosing a disease described herein is an in vivo method.

[0129] Such an in vivo method may include, for example, administering an antibody in a detectable form (e.g., in the format of an imaging agent) to a subject and imaging the subject to detect the antibody. In this embodiment, the antibody may be labeled. The antibody may be provided in the form of a conjugate with a diagnostic agent. The diagnostic agent may be a detectable label or portion that can be detected by imaging. For example, the antibody may be used as a PET tracer.

[0130] As described above, the parent antibody ALZ-201 is characterized by a unique and beneficial binding profile. This antibody is specific to the soluble oligomer Aβ42 but does not bind to other forms of Aβ (e.g., insoluble aggregates or plaques, or monomers or non-aggregated Aβ). Therefore, this antibody is not only selective but truly specific to the structured Aβ42 oligomer, which is currently recognized as the primary cause of AD. The humanized antibodies described herein advantageously retain the same or similar binding profiles.

[0131] As described in the examples below, the humanized antibody (Ab11) containing the humanized VL and VH sequences characterizing the binding protein described in the claims exhibits high affinity for the target antigen (Aβ42 prefibril oligomer having a β structure), and this affinity is in a range comparable to that of the parent antibody ALZ-201 and similar to that of the chimera (chALZ-201) of the ALZ-201 variable region and the human IgG1 constant region. Such affinity is suitable for an effective therapeutic agent. Therefore, the binding agents defined herein may exhibit affinity for the Aβ42CC antigen equivalent to that of Ab11. Affinity can be readily measured by an SPR assay, such as the Biacore assay, according to methods well known in the art. The Aβ42CC peptide is described in Sandberg et al., 2010, PNAS, vol. 107(35), pp. 15595-15600 and International Publication No. 2009 / 128772.

[0132] As demonstrated in the examples described below, other humanized antibodies tested in the development of the antibody described herein showed slightly higher affinity. Nevertheless, Ab11 was selected based on a combination of other characteristics. For example, as stated above, Ab11 has a high percentage of humanity (88.7%) in its VH and VL sequences. This beneficial characteristic is also retained because the VH and VL of the Ab11 antibody are retained in the antibody described herein.

[0133] Furthermore, as detailed in the examples, Ab11 exhibits high thermal stability, characterized by a high Tm of 71°C as measured by differential scanning fluorescence (DSF) analysis. The antibodies described herein exhibit similar thermal stability to Ab11. Therefore, the antibodies described herein may have a Tm of at least 69°C, 70°C, or 71°C as measured by differential scanning fluorescence (DSF) analysis.

[0134] Furthermore, as demonstrated in the examples described below, Ab11 can be obtained in high yield from expression systems in mammalian cells. Therefore, in some embodiments, the antibodies described herein can be expressed from mammalian cells at levels of at least 4 mg / ml, 4.5 mg / ml, or 5 mg / ml.

[0135] As described above, the original antibody ALZ-201 has been demonstrated to have positive physiological and protective effects on the integrity and morphology of mouse neurons. The antibodies described herein may exhibit similar protective effects. This can be determined in a neurotoxicity assay that examines the toxicity of these antibodies in neuronal cell cultures of mouse embryo-derived neurons using brain extracts from AD patients. Such assays are described in the 2022 publication by Sandberg et al. cited above. Further details regarding the antibodies described herein and their development are provided in the non-limiting examples below with reference to the following figures. [Brief explanation of the drawing]

[0136] [Figure 1] Figure 1 shows the protein purification profiles of each recombinant antibody after non-reducing SDS-PAGE analysis of the protein A chromatography fraction. (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: Molecular weight marker. IN: Input. FT: Flow-through. W: Wash fraction. E: Elute fraction. [Figure 2-1] Figure 2 shows the results of reduced and unreduced SDS-PAGE analysis (performed for quality assurance) for the final pooled samples of each purified antibody. (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 2-2] Figure 2 continued [Figure 3] Figure 3 shows the average molecular weight of Aβ42CC oligomers measured by a combination of size exclusion chromatography and multi-angle light scattering (SEC-MALS). The results shown are the normalized 280 nm absorbance (au) and molar mass (kDa) of Aβ42CC oligomers upon elution from the column. The figure is an adaptation from the 2022 paper by Sandberg et al. (Alz Res and Therapy 14:196). [Figure 4] Figure 4 shows the ELISA results for the affinity of chALZ-201 or lecanemab to unstructured Aβ42, fibrous Aβ42, and oligomeric Aβ42CC. The figure is a modification of the 2022 paper by Sandberg et al. (Alz Res and Therapy 14:196). [Figure 5-1] Figure 5 shows the ELISA results for the affinity of Ab1, Ab2, Ab3, and chALZ-201 to the Aβ42CC oligomer. [Figure 5-2] Figure 5 continued [Figure 6-1] Figure 6 shows the ELISA results for the affinity of Ab10, Ab11, Ab17, and chALZ-201 to the Aβ42CC oligomer. [Figure 6-2] Figure 6 continued [Figure 6-3] Figure 6 continued [Figure 7-1] Figure 7 shows the thermal stability data for each antibody obtained by differential scanning fluorescence (DSF) 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 7-2] Figure 7 continued [Figure 8] Figure 8 shows the results of the expression comparison test of Ab10, Ab11, and chALZ-201 as expression yield (mg / L) per liter of culture medium. (A): Ab10 and Ab11. (B): Ab10 and chALZ-201. (C): Ab11 and chALZ-201. [Figure 9] Figure 9 shows the reduced and unreduced SDS-PAGE analyses (performed for quality control purposes) of the final purified Ab11 obtained from each minipool. 2 μg of Ab11 was applied to each lane. Coomassie blue staining was used. MW: Molecular weight marker. 1: Pool 1. 2: Pool 2. [Figure 10] Figure 10 shows the reduced and unreduced SDS-PAGE analyses (performed for quality control) of the final purified Ab11 from each monoclonal. 2 μg 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. [Figure 11] Figure 11 shows the evaluation of cell viability and stability for the top three monoclonal types: 4H9, 1A3, and 2A6. The cells were cultured for 15 passages (30 generations), and viable cell density and viability were monitored at each passage. [Figure 12] Figure 12 shows the agarose gel electrophoresis results of PCR amplification products of the VH and VL regions of Ab11 from genomes extracted from the top three monoclonal strains: 4H9, 1A3, and 2A6. Ethidium bromide staining was used. MW: Molecular weight marker. [Figure 13] Figure 13 shows the reduced and unreduced SDS-PAGE analyses (performed for quality assurance) of the final purified Ab11 from each monoclonal after evaluation of cell viability and stability. Each lane contained 2 μg of Ab11 from cycles 5, 10, and 15, respectively. Coomassie blue staining was used. MW: Molecular weight marker. 5, 10, and 15 represent samples from passage numbers 5, 10, and 15, respectively. [Figure 14] Figure 14 shows the agarose gel electrophoresis results in a 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. [Examples]

[0137] Example 1: Humanization of the antibody ALZ-201 ALZ-201 Model Construction A molecular model of the mouse antibody clone ALZ-201 was constructed according to an established protocol. The sequences of the heavy chain variable region (VH) and light chain variable region (VL) were numbered / annotated according to Kabat and IMGT rules to identify the framework (FW) and complementarity-determining residue (CDR) sequences. Using these sequences, we searched for structurally analyzed mouse antibody structures and identified the structures with the highest sequence identity to select templates for the VL FW and CDR, as well as the VH FW and CDR. Using these templates, we constructed partial models of VH and VL. To construct the final model, we selected the optimal tertiary configuration of the partial models using the PAPS (Packing Angle Prediction) server (http: / / www.bioinf.org.uk / abs / paps / ). The PAPS server predicted the structurally analyzed mouse antibody structures that provided the optimal tertiary configuration for VH and VL. The final model was assembled by fitting the backbone coordinates of the conserved anchor segments in the VH and VL partial models to the selected mouse antibody structures. Finally, we performed a GROMACS energy minimization using the GROMOS96 force field on the coordinates of the final model. This model was used to support subsequent humanization design. Specifically, by examining this model against the sequences of candidate human germline cells for transplantation, potential design errors that may occur when designing solely based on sequence considerations can be avoided. In particular, this model was used to select residues in the human FWQ to be "reverse mutations" (substituted with parental mouse amino acids) to prevent loss of antibody stability or affinity due to structural incompatibility of the FW with the transplanted mouse CDR sequence. Furthermore, this model was also used to select residues from the transplanted mouse CDR to be replaced ("germ-derived") with human residues derived from the target human germline sequence. This makes it possible to enhance the "humanity" of the resulting antibody without losing affinity. One of the ultimate challenges in humanization design is mitigating the sequence-dependent potential risks specific to parental mouse CDRs. Certain sequences are susceptible to chemical degradation over time, which can result in a loss of affinity or antibody stability in CDRs. This model facilitated the selection of residue substitutions to mitigate these potential risks.

[0138] Humanization design of ALZ-201 mouse monoclonal antibody by CDR transplantation. The ALZ-201 mouse antibody was humanized by transplanting three CDRs of the light chain variable region (VL), as defined by Kabat nomenclature, into human germline VL cells with the highest possible homology to the mouse antibody VL. Similarly, three CDRs of the heavy chain variable region (VH) were transplanted into human germline VH cells with the highest possible homology to the mouse antibody VH. The human germline cells used as acceptor sequences for CDR transplantation were selected based on sequence database searches and sequence identity comparisons. For VH, IGHV2-5 was selected. * 09 and IGHV4-30-4 * Two types of human germ cell lineages were selected from 07. For VL, IGKV3-11 was selected. * 01, IGKV3-20 * 01, IGKV1-39 * 01, and IGKV6-21 * Four types of human germ cell lineages were selected (01). Each CDR of ALZ-201 was analyzed from the perspective of sequence-related risks (deamidation and isomerization motifs). Mouse VH / VL sequences were aligned with corresponding human germline sequences, and each CDR was transplanted. Furthermore, a small number of amino acid residues in the framework region of selected human germline variable regions were reversed to amino acid residues present in the mouse variable region. Based on information regarding the structure of immunoglobulin variable regions, and guided by the homology molecular model of Fv of the ALZ-201 mouse monoclonal antibody described above, these few residues in the framework region were identified as playing a crucial role in maintaining the correct conformation of the CDR or in VH / VL packing. Therefore, these residues were retained in one humanized version (version A), and in subsequent humanized versions, they were replaced with corresponding human germline residues where possible. Under the guidance of the homology molecular model, in subsequent version B, where deemed possible, CDR residues defined by Kabat were also replaced with corresponding residues from the human germline (germ-line-based) to increase the degree of humanness (i.e., the percentage of sequence identity for both VH and VL between the humanized version and the nearest human germline used as the acceptor sequence for CDR transplantation). Thus, this structural model extends the limits of the humanization process, making it possible to go beyond simple CDR transplantation. Humanized version A was designed as a conservative version to minimize / avoid changes in the CDR. Subsequent versions B, C, and D were designed to increase % humanity through germlineization (i.e., to increase sequence identity with the nearest human germline to a higher percentage (at least 85%)). For VH, six different humanized VH sequences were designed (based on two different human germ cell lineages, each having two or four versions). For VL, eight different humanized VL sequences were designed (based on four different human germ cell lineages, each having two versions). This results in 48 possible combinations between humanized VH and VL sequences. The optimal combination of VH and VL strands between the humanized VH and VL sequences was selected. As a result, the above 48 combinations were narrowed down to 18 based on various considerations, such as % humanity and sequence modifications introduced in different versions B, C, and D. Table 1 below shows the 18 combinations identified based on the selected humanized VH and VL sequences. [Table 1] Of the 18 combinations listed above, 16 were selected for further investigation (humanized antibody variants 15 and 18 were excluded). Next, these various combinations were evaluated and experimentally tested based on the following criteria, as will be explained in the examples below. • Transient expression levels in mammalian cells as a human IgG1 / kappa antibody. • Binding capacity (EC50 by ELISA, or Kd by Biacore) • Measurement of biophysical properties, particularly differential scanning fluorescence (DSF) analysis to measure the Tm of FAB, CH2, and CH3.

[0139] Example 2: Vector Design and Recombinant Production Vector design, Chimera ALZ-201 (chALZ-201) A chimeric full-length human IgG1 antibody with a kappa light chain (chALZ-201) was designed using the sequences of the variable regions of the heavy chain (HC) and light chain (LC) of the mouse mAb20 antibody (International Publication No. 2009 / 128772A1) as a starting point. This antibody was used as a control reference for the humanized variant of the parental mouse antibody. The cDNAs encoding the HC and LC were chemically synthesized and optimized for expression in CHO cells. The sequence encoding the signal peptide was added at the 5' / N-terminus. These two cDNA sequences constructed for recombinant chALZ-201 expression in CHO cells are shown in SEQ ID NOs: 9 and 10. chALZ-201 HC cDNA (SEQ ID NO: 9) GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCCCCTCGGTGGGTTCTGAGCCAGGTGACCCTGAAGGAGTCCGGCCCTGGCATCAGCCAGCCTAGCCAGACCCTGAGCTGACCTGTTCCTTTAGCGGCTTCTCCCTGAGCACCTTTGGCAGCGGCGTGTCCTGGATCAGG CAGCCTAGCGGCAAGGGCCTGGAGTGGCTGGCTCACATCTACTGGGACGACGATAAGCACTACAATCCCAGCCTGAAGTCCCGGCTGACCATCAGCAAGGATACCAGCAACAATCAGGTGTTTCTGAAGATCACCACCGTGGACACCGCCGATACCGCCACCTATTTCTGCGCCCGGCGGGAGAGCCACTACT ATGGCTCCGGCTACTACTTCGATTACTGGGGCCAGGGCACCACCCTGACCGTGTCCTCCGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTG TCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGAGCGGCCGC chALZ-201 LC cDNA (SEQ ID NO: 10) GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCTCCGGCGCCTATGGCCAGATCGTGCTGACCCAGTCCCCTGCTATCCTGTCCTCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGGGCCAGCAGCAGCGTGAGCTATATGCACTGGTACC AGCAGAAGCCCGGCAGCAGCCCCAAGCCCTGGATCTACGCTACCAGCAACCTGGCTTCCGGCGTGCCTGCCAGGTTTAGCGGCTCCGGCTCCGGCACCTCCTATTCCCTGACCATCTCCCGGGTGGAGGCCGAGGATGCCGCTACCTACTATTGTCAGCAGTGGAGGTCCGACCCCCTGAC CTTCGGCGCTGGCACCAAGCTGGAGCTGAAGCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGT AATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC The lowercase nucleic acid sequence "gccgccacc" present in SEQ ID NOs. 9 and 10 is a Kozak sequence that functions as a protein translation initiation site, and the "GAATTC" sequence immediately preceding it is an EcoR1 restriction enzyme site. These two cDNA sequences were subcloned into the mammalian cell expression vector pXten1 (ProteoGenix, France, Schiltigheim) using the EcoR1 enzyme. Therefore, the expected protein sequence of the antibody chALZ-201 HC is as shown in SEQ ID NO. 11. chALZ-201 HC (SEQ ID NO: 11) MKHLWFFLLLVAAPRWVLSQVTLKESGPGISQPSQTLSLTCSFSGFSLSTFGSGVSWIRQPSGKGLEWLAHIYWDDDKHYNPSLKSRLTISKDTSNNQVFLKITTVDTADTATYFCAR RESHYYGSGYYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In Sequence ID No. 11, amino acids 1-19 are secretory signals that are cleaved during protein translation, amino acids 20-143 constitute the VH domain, and amino acids 144-473 constitute the IgG1 constant heavy chain domain. On the other hand, the expected protein sequence of the antibody chALZ-201 LC is as shown in SEQ ID NO: 12. chALZ-201 LC (Sequence ID 12) MVLQTQVFISLLLWISGAYGQIVLTQSPAILSSSPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWRSDPLT FGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In Sequence ID No. 12, amino acids 1-20 are secretory signals that are cleaved during the protein translation process, amino acids 21-126 constitute the VH domain, and amino acids 127-233 constitute the IgG1 constant light chain domain.

[0140] Vector design, antibody variant 10 (Ab10) and antibody variant 11 (Ab11) As described above, for Ab10 and Ab11, which were designed using in silico CDR transplantation, the cDNA encoding the VH and VL chains of each antibody was chemically synthesized and optimized for expression in CHO cells. The sequence encoding the signal peptide was added at the 5' / N-terminus. The VH sequences are identical in both antibodies. The two cDNA sequences constructed for recombinant antibody expression of Ab10 VH and Ab11 VH in CHO cells are shown in SEQ ID NO: 13. Ab10 VH cDNA and Ab11 VH cDNA (SEQ ID NO: 13) GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCTGCTCCTCGGTGGGTGCTGTCCCAGGTGACCCTGAAGGAGTCCGGCCCCACCCTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCAGCGGCTTTAGCCTGAGCACCTTTGGCAGCGGCGTGAGCTGGATCAGGCAGCCTCCCGGCAAGGCCCTGGAGTGGCTG GCTCACATCTATTGGGACGACGACAAGCACTATAACCCTAGCCTGAAGAGCCGGCTGACCATCACCAAGGACACCAGCAAGAACCAGGTGGTGCTGACCATCACAAACATGGACCCTGTGGATACCGCCACCTATTTTTGCGCCCGGAGGGAGAGCCACTACTATGGCAGCGGCTACTATTTCGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC As described above, the lowercase nucleic acid sequence "gccgccacc" in Sequence ID No. 13 is a Kozak sequence that functions as a protein translation initiation site, and the sequence "GAATTC" immediately preceding it is an EcoR1 restriction enzyme site. These two cDNA sequences were subcloned using the EcoR1 enzyme into the mammalian cell expression vector pXten1 (ProteoGenix, France, Siltikaim), which contains the backbone sequence of the human IgG1 heavy chain constant region described above. Therefore, the expected complete protein sequences of the HC of humanized antibodies Ab10 and Ab11 are shown in Sequence ID No. 7. Ab10 HC and Ab11 HC (SEQ ID NO: 7) MKHLWFFLLLVAAPRWVLSQVTLKESGPTLVKPTQTLTLTCTFSGFSLSTFGSGVSWIRQPPGKALEWLAHIYWDDDKHYNPSLKSRLTITKDTSKNQVVLTITNMDPVDTATYFCAR RESHYYGSGYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In Sequence ID No. 7, amino acids 1-19 are secretory signals that are cleaved during protein translation, amino acids 21-143 constitute the VH domain, and amino acids 144-473 constitute the IgG1 constant heavy chain domain. The cDNA sequence constructed for recombinant antibody expression of Ab10 VL is shown in SEQ ID NO: 14. Ab10 VL cDNA (SEQ ID NO: 14) GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGTCCCTGAGCCCAGGAGAGCGGGCTACCCTGAGCTGTCGGGCCTCCTCCAGCGTGTCCTACATGCACTGGTACCAGCAGAAGCCTGGCCAGGCTCCTCGGCCCTGGATCTATGCCACCAGCAACCTGGCCACCGGCATCCCCGCCAGGTTCTCCGGAAGCGGCTCCGGAACCGATTTTACCCTGACCATCTCCAGCCTGGAGCCTGAGGACTTTGCCGTGTACTACTGCCAGCAGTGGAGGAGCGATCCTCTGACCTTTGGCGGCGGCACCAAGGTGGAGATCAAG The cDNA sequence constructed for the recombinant antibody expression of Ab11 VL is shown in SEQ ID NO: 15. Ab11 VL cDNA (SEQ ID NO: 15) GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGTCCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCGGGCTTCCTCCAGCATCTCCTATATGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCCTTGGATCTACGCTACCAGCAATCTGGCTAGCGGCGTGCCTAGCCGGTTCTCCGGCTCCGGATCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTGGCGGTCCGATCCCCTGACCTTCGGCGGCGGAACCAAGGTGGAGATCAAG As described above, the lowercase nucleic acid sequence "gccgccacc" in SEQ ID NOs. 14 and 15 is a Kozak sequence that functions as a protein translation initiation site, and the "GAATTC" sequence immediately preceding it is an EcoR1 restriction enzyme site. These two cDNA sequences were subcloned using the EcoR1 enzyme into the mammalian cell expression vector pXten1 (ProteoGenix, France, Siltikaim), which contains the human IgG1 light chain constant region backbone sequence described above. Therefore, the expected protein sequences of the LC of humanized antibodies Ab10 and Ab11 are as shown in SEQ ID NOs. 16 and 8. Ab10 LC (Sequence ID 16) MVLQTQVFISLLLWISGAYGEIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPWIYATSNLATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSDPLT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ab11 LC (Sequence ID 8) MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCRASSSISYMHWYQQKPGKAPKPWIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWRSDPLT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In Sequence IDs 16 and 8, amino acids 1-20 are secretory signals that are cleaved during protein translation, amino acids 21-126 constitute two different VL domains, and amino acids 127-233 constitute the IgG1 constant light chain domain. Both Ab10 and Ab11, humanized ALZ-201 variants, exhibit 88.7% sequence identity with the human IgG homolog. All other humanized ALZ-201 candidates described herein were similarly subcloned for expression in CHO cells. These are designated Ab1 (87.6% human residues), Ab2 (87.6% human residues), Ab3 (86.1% human residues), and Ab17 (85.6% human residues).

[0141] Transient expression in Chinese hamster ovarian cells XtenCHO cells were used for the transient expression of the antibodies described herein (PX-XTE-004; ProteoGenix, France, Siltikaim). This cell line is derived from the Chinese hamster ovary CHO-K1 cell line. 1 × 10⁶ cells were expressed in XtenCHO expression medium (PX-XTE-002; ProteoGenix, France, Siltikaim) containing 8 mM L-glutamine, 0.5% anticlumping agent (0010057AE; Gibco / Thermo Fisher Scientific, USA), and 10% DMSO. 7Each vial containing 1 mL of the frozen cell suspension at cells / mL was thawed at 37°C for a maximum of 90 seconds by immersing the vial in XtenCHO expression medium and gently swirling until a small amount of ice remained. The vial was wiped with 70% ethanol for decontamination and transferred to a laminar flow hood. The contents were transferred to 8 mL of pre-warmed XtenCHO expression medium containing 8 mM L-glutamine and centrifuged at 300×g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 2 mL of XtenCHO expression medium containing 8 mM L-glutamine. The viable cell count and viability were measured using an automated cell counter (Countstar BioTech, model: IC 1000) with the default settings for the cell mode for cell counting. Briefly, a sample of the cell suspension was taken from the flask and diluted 1:1 with 0.4% trypan blue staining solution. After gentle mixing, 20 μl of the sample mixture was added to the chamber port of a cell counting chamber slide and inserted into the automated cell counter. Three layers (upper, middle, and lower) were selected for counting, and the system calculated the average cell density and viability. 30 mL of XtenCHO expression medium containing 8 mM L-glutamine was placed at 37°C in a 125 mL disposable sterilized vented-cap Erlenmeyer flask (TAB-012-125; Guangzhou Jet Bio-Filtration Co., Guangzhou, China) and seeded at a density of 0.2 - 0.3×10 6 cells / mL, and an anticlumping agent was added to a final concentration of 0.5%. The cells were incubated at 37°C, relative humidity of 80% or more, and 5% CO2 on an orbital shaker platform (IS-RDS6C5 incubator, Crystal Technology & Industries, USA). When the cells reached 1.5 - 2.5×10 6 viable cells / mL (usually on the 2nd to 3rd day after thawing), the cells were passaged. First, the culture was centrifuged at 300×g for 5 minutes, and the supernatant was discarded. Next, the cells were resuspended in 30 mL of XtenCHO expression medium containing 8 mM L-glutamine at 37°C at a density of 0.2 - 0.3×10 6The cells were transferred at a density of cells / mL, and an anti-lumping agent was added to a final concentration of 0.5%. The culture was returned to incubation on an orbital shaker platform at a relative humidity of 80% or higher, 5% CO2, and 37°C. The cells returned to 1.5–2.5 × 10⁶ 6 Once the viable cell count / mL was reached, this process was repeated at least once before transfection. Transfection was not performed on cells that had been passaged more than 20 times. The day before transfection, cells were subcultured in XtenCHO expression medium containing 8 mM L-glutamine but without anticlumping agents, using the subculture procedure described above. After 24 hours, the cells were typically 2–3 × 10⁶. 6 The cell density reached a viable cell density of 1 / mL, and the viability rate exceeded 90%. The culture was centrifuged at 300×g for 5 minutes, and the supernatant was discarded. Next, the cells were co-transfected with pXten1 plasmids containing the HC and LC cDNA sequences of each antibody according to the following protocol. First, the cells were placed in 15 mL of 8 mM L-glutamine-containing XtenCHO expression medium and incubated at 37°C for 5 × 10⁶ cells. 6 Cells were transferred at a density of viable cells / mL. 48 μg each of the expression plasmid pXten1 containing the cDNA sequences of the HC and LC ALZ-201 antibody derivatives to be expressed was added to the cells, and 144 μL of XtenFect reagent standard solution (PX-XTE-003; ProteoGenix, France, Siltikaim) was added dropwise while gently agitating the cells. The cells were incubated on an orbital shaker platform at 80% relative humidity, 5% CO2, and 37°C for 2 hours, then 96 μL of XtenCHO Enhancer solution (PX-XTE-003; ProteoGenix, France, Siltikaim) was added to the cells, followed by 15 mL of XtenCHO expression medium containing 8 mM L-glutamine at 37°C. The cells were then returned to the orbital shaker platform at 80% relative humidity, 5% CO2, and 37°C. Twenty-four hours after transfection, an anti-lumping agent was added to a final concentration of 0.5%, and the temperature was reduced to 33°C. Cell density and cell viability were periodically monitored during this expression period. For the initial expression analysis of all humanized ALZ-201 antibodies and the reference antibody chALZ-201 described herein, cells were harvested from 30 mL of culture by centrifugation at 300 × g for 5 minutes, 14 days after transfection, when viability had decreased to less than 50%. The supernatant was removed, clarified by centrifugation at 5000 × g for 30 minutes, and passed through a Millex-GP 0.22 μm sterile filter (SLMPL25SS; Merck KGaA, Darmstadt, Germany). Next, the antibody was purified using Protein A. Briefly, Protein A chromatography resin (Mabselect SuRe LX #17547402; Cytiva, USA) was immersed in 0.5 M NaOH for 30 minutes, then washed and equilibrated with pH 7.5 phosphate-buffered saline (PBS: 10 mM Na2HPO4, 2.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl). Cell culture supernatant was added, and binding was allowed to occur at room temperature for 4 hours. After washing the beads with 50 times the volume of beads in PBS (pH 7.5), the bound antibody was eluted with a 20 mM solution of pH 2.7 2-hydroxypropane-1,2,3-tricarboxylic acid (citric acid) buffer. The eluted fraction was neutralized with pH 9.0 1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl). The antibody-containing fractions were identified by electrophoresis (PAGE) using a Mini-PROTEAN Tetra vertical electrophoresis system (1658000FC; Bio-Rad, USA) and Tris-HCl gels (8% and 12%), with Tris-Glycine as the running buffer (25 mM Tris-HCl, 200 mM Glycine, 0.1% w / v SDS, pH 8.3). 10 μl of the sample was mixed with loading buffer (10% w / v SDS, 20% v / v glycerol, 0.2 M Tris-HCl (pH 6.8), 0.05% w / v bromophenol blue) in a 4:1 ratio, with or without a reducing agent (10 mM β-mercaptoethanol). The molecular weight marker (2 μl per well) consisted of 11 protein bands ranging from 10 to 250 kDa (Epizyme Biotechnology Co., Ltd., China). The gel was run at 120 V and stained with Coomassie Brilliant Blue G-250. The antibody-containing fraction was pooled, and the buffer was replaced with PBS (pH 7.5) by dialyzing against 200 times the sample volume using a 3.5K MWCO snakeskin tube (88244; Thermo Scientific, USA). Dialysis was performed overnight (approximately 16 hours) with continuous agitation at 4°C, after which the dialysis buffer was replaced and dialysis was continued for another 3 hours at 4°C. Subsequently, the sample was filtered through a 0.22 μm Millex syringe filter (SLGV013SL or SLGV004SL depending on the final sample volume; Merck KGaA, Germany), and the endotoxin level was measured 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 using SDS-PAGE as described above. Good antibody expression in XtenCHO cells was confirmed with one 30 mL pilot batch of chALZ-201 (also known as Ab15). Subsequently, Ab10 and Ab11 were expressed in parallel with a second batch of chALZ-201, along with humanized variants Ab1, Ab2, Ab3, and Ab17. Figure 1 shows the SDS-PAGE analysis of the protein A chromatography fraction, and Figure 2 shows the SDS-PAGE analysis of the pooled samples. All antibodies were confirmed to be consistent with the expected size. Disulfide-depleted IgG antibodies typically migrate on SDS-PAGE as seeds of approximately 50 kDa and 25 kDa, corresponding to the heavy and light chains, respectively. In contrast, under non-reducing conditions, IgG antibodies produce a single band on the SDS-PAGE gel, with a size of at least 150 kDa. Based on SDS-PAGE results, all antibodies expressed in this specification were estimated to have a purity exceeding 90%. The antibody concentration was 210,000 M. -1 cm -1 The extinction coefficient was used to measure the expression levels by UV spectrophotometric analysis at 280 nm. The expression results obtained from a 30 mL cell culture batch are shown in Table 2. Ab11 and Ab17 were found to be expressed very well, but Ab10 was found to be poorly expressed compared to all the derivatives. [Table 2]

[0142] Example 3: Binding affinity Amyloid-beta (Aβ) peptide preparation To evaluate the reactivity to unstructured monomer antigens, synthetic Aβ42 peptide (H-1368; Bachem, Switzerland) was prepared at a concentration of 1 mg / mL in 0.1 M aqueous ammonia (pH 9) and used within 2 hours of preparation. The fibrous form of the antigen was obtained by preparing a solution of 1.0 mg / mL of lyophilized peptide (H-1368; Bachem, Switzerland) in PBS containing 0.02% azide, and then shaking the solution at 700 rpm for 55 hours at 37°C. The fibrils were then incubated at room temperature for 90 hours without shaking and then frozen at -20°C. The frozen fibrous Aβ42 vials were thawed immediately before use. Aβ42CC peptide is a non-fibrillating derivative of Aβ42 peptide, possessing two alanine-cysteine ​​substitutions at the 21st and 30th amino acid positions, further linked by an intramolecular disulfide bond (International Publication No. 2009 / 128772A1; Sandberg, A. et al., 2010, Proc. Natl. Acad. Sci. USA, 107:15595-600). The oligomeric form of this peptide was used in the development of mouse ALZ-201 (International Publication No. 2012 / 120035A1; Sandberg, A. et al., 2022, Alz. Res. Therapy 14:196). The synthetic Aβ42CC used herein was custom-manufactured by solid-phase peptide synthesis and purified to 95% purity by reverse-phase HPLC using standard methods and techniques (AmbioPharm Inc., North Augusta, USA). The oligomeric peptide preparation was obtained by first dissolving the peptide at pH 10.0-10.4 and then oligomerizing it in PBS. The oligomer was then frozen to prevent further oligomerization. The vial of frozen oligomeric Aβ42CC was thawed immediately before use. The size of the Aβ42CC oligomers used herein was measured using size exclusion HPLC (1100 series; Agilent Technologies, Santa Clara, California, USA). 100 μL of the sample was injected into a TSK-GEL® G4000SWxl 7.8 × 300 mm column (Tosoh Corporation, Tokyo, Japan) using 20 mM sodium phosphate buffer and 150 mM NaCl (pH 7.4) as running buffers. The flow rate was 0.6 mL / min. The weight-average molecular weight was measured using 280 nm UV and multi-angle light scattering (MALS; MiniDAWN Treos, Wyatt Technology Corporation, Santa Barbara, California, USA) with ASTRA 6.1 software (Wyatt Technology). The oligomers used herein had an average molecular weight of 702 ± 3.5 kDa and an oligomer content of over 94% (Figure 3). The concentrations of monomer Aβ42 and fibrous Aβ42 were estimated from the net peptide content measured by the peptide manufacturer (Bachem, Bubendorf, Switzerland). Meanwhile, the concentration of Aβ42CC was estimated based on the difference in absorbance at 280 nm and 300 nm, which was 1401 cm⁻¹. -1 M -1 The absorption coefficient was measured by ultraviolet (UV) spectroscopy.

[0143] Evaluation of antibody specificity by ELISA The antigen specificity of chimeric and humanized derivatives of ALZ-201 was evaluated by ELISA, and it was confirmed that the unique binding characteristics of the parent antibody were preserved. 96-well Nunc Maxisorp® ELISA plates (44-2404-21; Thermo Fisher Scientific, Waltham, Massachusetts, USA) were coated with 5 μg / mL antigen (100 μL / well) at 37°C for 2 hours. Monomer Aβ42 was coated at a high pH to maintain its random coil conformation. The plates were blocked at 37°C for 40 minutes with PBS containing 1% BSA (150 μL / well). Washing between steps was performed by washing three times with PBS containing 0.05% Tween-20 (300 μL / well). Primary antibodies were diluted from 1000 ng / mL to 0.46 ng / mL using PBS containing 0.1% BSA, added at 100 μL / well, and incubated at room temperature for 1.5 hours. After washing, HRP-labeled (HRP-conjugated) secondary monoclonal antibody (mAb) was added at 1000 ng / mL (100 μL / well), and the plate was incubated at 37°C for 45 minutes. After washing the plate again, 3,3',5,5'-tetramethylbenzidine substrate was added (100 μL / well), and the plate was incubated at 37°C for 5-10 minutes. The reaction was stopped with 2 M HCl (50 μL / well). The difference in absorbance at 450 nm and 630 nm was measured using a spectrophotometer, and the dose-response curve was analyzed by fitting a 4-parameter nonlinear logistic function to the measured absorbance values ​​to extract the antibody's maximum effective concentration (EC50). Figure 4 shows the ELISA results of chALZ-201 against unstructured Aβ42, fibrous Aβ42, and oligomeric Aβ42CC. No binding was observed to unstructured Aβ42 or fibrous Aβ42, but strong binding was observed to oligomeric Aβ42CC. This indicates, as expected, that the chALZ-201 antibody retained the specificity of the parent antibody towards the oligomeric antigen. In contrast, the antibody lecanemab (a complete copy of the BAN2401 / lecanemab Ab sequence expressed in XtenCHO cells; PX-TA1746; ProteoGenix) was used as a positive control and showed nearly equal binding affinity to all three different forms of Aβ peptides in the direct ELISA method used in this example. Table 3 shows the EC50 values ​​obtained from the most fitting formula. Here, EC50 is the mean of the two measurements. The standard deviation (SD) shown in the table is the mean of the two standard errors used to determine the individual EC50 values ​​(SD = √((sd1) 2 +sd2 2 It was calculated as () / 2), where sd1 and sd2 are the two standard errors. [Table 3]

[0144] Evaluation of antibody affinity by ELISA and surface plasmon resonance (SPR) Whether humanized variants retain their affinity for target antigens was evaluated using ELISA analysis and SPR technology with Biacore®. Here, the ELISA protocol was the same as described above, except that only the Aβ42CC oligomer was used as the antigen in this example. Ab1, Ab2, Ab3, Ab10, Ab11, Ab17, and chALZ-201 were evaluated. The results are shown in Figures 5 and 6, and the obtained EC50 values ​​are shown in Table 4. In one experiment for antibody Ab3, data that could not be easily curve-fitted to the Hill formula was not obtained, resulting in a large error in the determination of EC50. The affinity of chALZ-201 and its humanized derivative for the oligomeric Aβ42CC antigen was within the same range, indicating that the affinity for the antigen is maintained even after humanization. [Table 4] Affinity measurements by SPR were performed using a Biacore® 8k instrument. SPR analyzes biomolecular interactions in real time and provides quantitative measurements of reaction kinetics and affinity constants. SPR has been shown to be a preferred method in studies where the antibody binding rate constant needs to be detected with high sensitivity and reliability (Yang, D. et al., 2016, Anal. Biochem. 508:78-96). Anti-human Fc IgG antibody (BR-1008-39; GE Healthcare, USA) was conjugated to a CM5 sensor chip (BR100399; Cytiva, USA) using the maleimide EDC / NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide / N-hydroxysuccinimide) coupling method (BR-1000-50; GE Healthcare, USA). The running buffer used was HBS-EP. +The running buffer consisted of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.01% Surfactant P20, and the regeneration buffer was glycine pH 1.5. chALZ-201 antibody and humanized antibodies Ab1, Ab2, Ab10, Ab11, and Ab17 were diluted in the running buffer and captured with anti-human Fc antibody. The oligomeric antigens in the running buffer were flowed onto the sensor chip at specified oligomer concentrations ranging from 1.25 nM to 80 nM (calculated using 702 kDa Mw data obtained by SEC-MALS shown in Figure 3). Responses were acquired over time, and data from the reference channel were subtracted. Between measurements at each concentration, the sensor chip was washed with the regeneration buffer. The rates of association and dissociation were obtained from the sensorgram, and the association rate ("on rate") was determined using BIAevaluation software (Biacore®). a ) and dissociation ("off rate", k d The dynamic parameters of each of the ) and the equilibrium dissociation constant ("coupling constant", K D The following were calculated. The results are shown in Table 5, demonstrating that all ALZ-201 derivatives are high-affinity binders. [Table 5]

[0145] Example 4: Thermal Stability The tendency of biomacromolecules to withstand thermal denaturation is commonly used as an approximate measure of their intrinsic stability against aggregation and denaturation. Protein aggregation typically requires at least partial unfolding of the native structure, a process that can be monitored as a function of time and / or temperature by measuring the change in the protein's intrinsic fluorescence during unfolding. Protein unfolding typically involves exposure of fluorescent groups, such as the amino acid side chains of tryptophan and tyrosine, to water molecules, which quenches the fluorescence. In this specification, the temperature at which each antibody unfolds T mDifferential scanning fluorescence (DSF) was used to measure T. m This is considered the inflection point of the change in the fluorescence ratio (F350 / F330) at 350 nm and 330 nm as a function of temperature. 50 μL of sample was analyzed undiluted in PBS (pH 7.5) at the concentrations shown in Table 2. The instrument used was a Prometheus NT.48 nanoDSF (NanoTemper Technologies GmbH, Germany), operated at a scanning rate of 1 °C / min from 40 °C to 90 °C. Fluorescence at 350 nm and 330 nm was collected at a rate of 10 data points per minute. The obtained data are shown as the first derivative (dF / dT) of the F350 / F330 ratio in Figures 7A and 7B, and the extracted melting points are shown in Table 6. All antibodies exhibited a transition (T) in the temperature range of 67 °C to 71 °C. m 1) is shown. Antibodies Ab2, Ab11, and Ab17 undergo a second translocation (T) around 76°C to 78°C. m 2) also indicates that the two distinct unfolding events in these Ab2, Ab11, and Ab17 antibodies may reflect differences in the thermal stability of the Fab and Fc domains. Antibodies Ab2, Ab11, and Ab17 showed the highest thermal stability, and the pre-transfer baseline of Ab11 and Ab17 suggested a slightly later onset of denaturation compared to Ab2. [Table 6]

[0146] Example 5: Expression comparison test of Ab10, Ab11, and chALZ-201 The significantly lower expression level of Ab10 compared to Ab11 was unexpected, but this was confirmed here by transient expression in CHO cells and quantification using the Octet RED96 instrument from Sartorius (ForteBio / Sartorius, USA). Gene synthesis, cloning, transfection, and recombinant production of Ab10, Ab11, and chALZ-201 were carried out as described in Example 2, but with the following modifications to transfection and expression: Prior to transfection, cells were incubated at 37°C for 5 × 10⁶ days in 7.5 mL of 8 mM L-glutamine-containing XtenCHO expression medium. 6 Cells were transferred at a density of viable cells / mL. 24 μg each of expression plasmids (pXten1) containing HC and LC domains corresponding to the Ab10, Ab11, or chALZ-201 antibodies were added to the cells. 106 μL of XtenFect reagent standard solution (PX-XTE-003; ProteoGenix, France, Siltikaim) was added dropwise while gently agitating the cells. The cells were incubated on an orbital shaker platform at 80% relative humidity, 5% CO2, and 37°C for 2 hours. Then, 48 μL of XtenCHO Enhancer solution (PX-XTE-003; ProteoGenix, France, Siltikaim) was added to the cells, along with 7.5 mL of XtenCHO expression medium containing 8 mM L-glutamine at 37°C. The cells were then returned to the orbital shaker platform at 80% relative humidity, 5% CO2, and 37°C. Twenty-four hours after transfection, an anti-lumping agent was added to a final concentration of 0.5%, and the temperature was reduced to 33°C. Cell density and cell viability were periodically monitored during this expression period. Three parallel co-transfections were performed for each antibody construct. 200 μL of culture medium was collected on days 4, 7, 10, and 14, and the IgG1 concentration was measured using an Octet RED96 instrument equipped with a Protein G biosensor (18-5082; ForteBio / Sartorius, USA). The Octet system uses biolayer interferometry to measure binding events in real time and can be used to directly detect specific proteins in complex mixtures. The binding rates of Ab10, Ab11, and chALZ-201 to the Protein G biosensor were measured using the quantitative experiment module in the Octet system's data acquisition software. Higher antibody concentrations resulted in faster binding rates. The data were analyzed using Octet Data Analysis HT software, and the concentration was calculated from each rate based on the values ​​of the human IgG1 standard curve (FHJ92850; Antibody Systems). Standards were prepared in PBS and analyzed in parallel at concentrations of 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.563 μg / mL, 0.7813 μg / mL, and 0 μg / mL. Culture medium samples were diluted 20-fold in PBS before analysis. The sample shaking speed was set to 1000 rpm, the quantification time to 300 seconds, the temperature to 30°C, and the data acquisition time to 5 Hz. The results are shown in Figure 8 as expression yield (mg / L) per liter of culture, and antibodies are compared bilaterally in different panels for clarity. Table 7 shows the bilateral statistical comparison for each data point. Here, the difference in observed mean values ​​between two independent samples is reported, along with the significance value (P-value) and the 95% confidence interval (CI) of the difference. The P-value is the probability that the observed difference between samples is obtained if the null hypothesis is true. The null hypothesis is the hypothesis that the difference is 0. [Table 7]

[0147] Example 6: Preparation of a stable monoclonal CHO cell line expressing Ab11 Vector design used for stable transfection To develop a stable CHO cell line expressing the target antibody Ab11, the VH cDNA sequence (SEQ ID NO: 13) and VL cDNA sequence (SEQ ID NO: 15) were combined with the cDNA sequences corresponding to the HC and LC constant regions of IgG1 described in Example 2. The full-length cDNA sequences used for stable recombinant Ab11 expression are as follows: Ab11 HC cDNA (SEQ ID NO: 19) Ab11 LC cDNA (SEQ ID NO: 20) GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGTCCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCGGGCTTCCTCCAGCATCTCCTATATGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCCTTGGATCTACGCTACCAGCAATCTGGCTAGCGGCGTGCCTAGCCGGTTCTCCGGCTCCGGATCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTGGCGGTCCGATCCCCTGACCTTCGGCGGCGGAACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC The lowercase nucleic acid sequence "gccgccacc" in SEQ ID NOs. 19 and 20 is a Kozak sequence that functions as a protein translation initiation site, and the "GAATTC" sequence immediately preceding it is an EcoR1 restriction enzyme site. These genes were chemically synthesized and subcloned into a pTXs7-GSbis expression vector (ProteoGenix, France, Siltikaim) for the development of stable mammalian cell lines. Both HC and LC were subcloned into single plasmids.

[0148] Creating a stable transfection pool As described above, the pTXs7-GSbis construct was used to develop a stable CHO-K1 cell line pool expressing Ab11. This vector also contains a glutamine synthase (GS) gene that confers resistance to methionine sulfoxamine (MSX) toxicity. MSX is a drug similar to glutamate and, by binding to GS, inhibits the production of glutamine necessary for cell proliferation. As a result, as the concentration of MSX in the cell culture medium increases, CHO cells containing the vector, and consequently one or more copies of the Ab11 gene, are reliably selected. After confirming innate MSX resistance in initial CHO-K1 suspension cultures, cells were transfected with a pTXs7-GS construct linearized with PvuI enzyme. Following 48 hours of incubation after transfection, cells were seeded into six 96-well plates and selected for stable clones by culturing in the presence of 30 μM MSX for 4 weeks. After 4 weeks of screening, all wells were observed under a microscope and evaluated by ELISA using the 702±3.5 kDa Aβ42CC oligomeric antigen described in Example 3. In parallel, three pools were also prepared in 15 mL T75 flasks, where the cells were similarly cultured in a selective medium containing 30 μM MSX. For the ELISA, 96-well Nunc Maxisorp® ELISA plates (44-2404-21; Thermo Fisher Scientific, Waltham, Massachusetts, USA) were coated with 5 μg / mL antigen (100 μL / well) at 37°C for 2 hours. The plates were blocked in PBS containing 3% BSA (300 μL / well) at 37°C for 1.5 hours. Washing between steps was performed by washing three times with PBS supplemented with 0.05% Tween-20 (300 μL / well). The culture medium was diluted 1:1000 with PBS and 100 μL was added to each well. After incubating the plates at 37°C for 1 hour, they were washed, and HRP-labeled secondary monoclonal antibody was added at 1000 ng / mL (100 μL / well). The plates were then incubated again at 37°C for 1 hour. After washing, 3,3',5,5'-tetramethylbenzidine substrate was added (100 μL / well), and the plates were incubated at 37°C for 7 minutes. The reaction was stopped with 2 M HCl (50 μL / well). The difference in absorbance at 450 nm and 630 nm was measured using a spectrophotometer. ELISA screening identified 74 positive minipools. These results are shown in Table 8. [Table 8]

[0149] Small-scale production and purification trials Fed batch culture expression evaluation was performed using five mini-pools (1E4, 5E10, 6C4, 1C1, and 4G6) and three prepared pools. Cells were cultured in 30 mL of selective medium (30 μM MSX) in a 125 mL shaking flask and incubated for at least three generations (37°C, 5% CO2, 130 rpm). When the viability reached over 95%, a 30 mL fed batch expression test was performed. 5 × 10⁶ cells were placed in 30 mL of expression medium in a 125 mL shaking flask. 6Cells were seeded at 1 / ml and incubated as described above. Feeding medium was added on days 3, 5, 7, and 9. Glucose was monitored and adjusted to 5-7 g / L as needed. One of the three pools died during fed batch expression. The culture was stopped when the viability rate fell to less than 50% (day 11), and the culture medium sample was purified and analyzed by SDS-PAGE and UV absorbance using the same general protocol and procedure as used for transient CHO expression analysis described in Example 2. The purity evaluation after purification is shown in Figure 9. The obtained yield and purity are summarized in Table 9. One stable minipool (5E10) and pool 1 showed high expression and were selected for further development. [Table 9]

[0150] Isolation and screening of stable monoclones Starting from stable pools 5E10 and pool 1, monoclonal isolation and screening were performed using standard methods. Briefly, monoclones were isolated in 96-well plates at a seeding density of 0.2 cells / well using the limiting dilution method. Monoclones were confirmed by microscopic observation, and expression screening was performed using ELISA with anti-Fc antibodies. After two limiting dilutions and ELISA expression screening, the top 10 monoclones with high expression levels were cultured and grown in 6-well plates and subjected to small-scale production evaluation using 30 mL cultures in 125 mL shaking flasks with the same protocol as described above. Subsequently, the antibodies were purified and analyzed according to the method described above. The resulting final samples were buffered with PBS (pH 7.5), qualitatively analyzed by SDS-PAGE as described above, and quantitatively analyzed by UV analysis. The purity evaluation after purification is shown in Figure 10. The obtained concentrations are shown in Table 10. All 10 isolated monoclones produced high-purity antibodies and showed good expression levels (approximately 1 g / L) in flask culture. Of these, three showed high expression levels (>1.5 g / L), and one showed a very high expression level (>2 g / L). [Table 10]

[0151] RCB Stability Test Stability analysis of cell proliferation Monoclones 4H9, 1A3, and 2A6 were mixed in 30 mL of selective medium in a 125 mL shaking flask at a rate of 0.5 × 10⁶ 6 Cells were seeded at a density of cells / mL and incubated for 15 passages (30 generations) at 37°C, 5% CO2, and 130 rpm. Cell viability and survival rate were monitored at each passage, and the overall stability of the growth cycle was analyzed. The results are shown in Figure 11, and the coefficient of variation (CV;%) is shown in Table 11. [Table 11]

[0152] DNA sequence of the introduced gene For each of the three monoclones, genomic DNA was extracted from cell passages 5, 10, and 15, and PCR amplification and gene sequencing were performed. The following primers were used to PCR-amplify the DNA. HC-Forward: GCAGTCACCGTCCTTGACACGGGATCCGCCGCCACCATGAAGCACCTGTGG (Sequence No. 21) HC-Reverse: ATGGCTGATTATGATCAATCTCGAGTCATTTCCCGGGGGACAGGCTCAG (Sequence No. 22) LC-Forward: CAGTCACCGTCCTTGACACGAAGCTTGCCGCCACCATGGTGCTGCAGAC (Sequence No. 23) LC-Reverse: GTATGGCTGATTATGATCAATGAATTCTCAGCACTCCCCCCTATTAAAGC (Sequence No. 24) The results of PCR amplification are shown in Figure 12. This confirmed the presence of the Ab11 gene. Subsequently, the PCR products were sequenced and aligned. As a result, it was shown that no point mutations occurred in the Ab11 gene in any of the clones over 15 passages.

[0153] Production yield analysis For each monoclone obtained in passages 5, 10, and 15, a 30 ml fed-batch expression test was performed, and the antibodies were purified as described above. The purity evaluation using SDS-PAGE is shown in Figure 13, and the yield is shown in Table 12. No stability issues were observed for monoclones 1A3 and 4H9 during the test period. Monoclone 2A6 had a CV% exceeding 20% ​​and a low production yield during the stability analysis period. [Table 12]

[0154] Mycoplasma test 2-3 × 10¹⁶ units each from clones 2A6, 1A3, and 4H9. 5 Cells were collected and tested for mycoplasma using a PCR mycoplasma test kit. The results are shown in Figure 14. This figure shows that no mycoplasma infection was detected in the monoclonal cell cultures.

[0155] conclusion In conclusion, antibody Ab11 was successfully introduced into the genome of the CHO-K1 cell line and was found to be robustly and stably expressed at high levels. Up to 2 g of antibody was obtained per liter of culture, with an estimated purity of over 90%. Viable monoclonal cell lines were isolated, and the absence of Mycoplasma bacteria was confirmed. Three monoclonal research cell banks, referred to herein as 2A6, 1A3, and 4H9, were isolated. Of these, clone 4H9 showed the highest expression level and demonstrated higher stability over 15 passages compared to the other two clones.

Claims

1. An antibody comprising an antigen-binding domain capable of specifically binding to an Aβ42 prefibril oligomer having a β structure, wherein the antigen-binding domain is (i) Heavy chain variable region (VH) containing the sequence of sequence number 1, (ii) A light chain variable region (VL) containing the sequence of sequence number 2, or Antibodies, including combinations of those.

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

3. The antibody according to claim 1 or claim 2, comprising two antibody-binding domains.

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

5. The antibody according to claim 4, wherein the antibody is an IgG antibody or an antigen-binding fragment thereof.

6. The antibody according to claim 4 or 5, which is an IgG1 kappa antibody or a fragment thereof.

7. The antigen-binding domain is (i) A heavy chain sequence including the VH sequence of SEQ ID NO: 1 linked to the heavy chain constant region sequence of SEQ ID NO: 3 or an amino acid sequence (ii) having at least 90% sequence identity with SEQ ID NO: 3, and / or (iii) The antibody according to any one of claims 1 to 6, comprising a light chain sequence including the constant region sequence of the light chain of SEQ ID NO: 4 or the VL sequence of SEQ ID NO: 2 linked to an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO:

4.

8. A conjugate comprising an antibody according to any one of claims 1 to 7, linked to at least one diagnostic agent.

9. An antibody according to any one of claims 1 to 7, for use in therapeutic purposes.

10. An antibody according to any one of claims 1 to 7, for use in the treatment of amyloid disease.

11. The antibody for use according to claim 10, wherein the amyloid disease is a neurodegenerative disease related to Aβ.

12. The antibody for use according to claim 10 or claim 11, wherein the amyloid disease is a disease related to soluble Aβ42 oligomers having a β structure.

13. The antibody for use according to any one of claims 10 to 12, wherein the amyloid disease is Alzheimer's disease (AD), Down syndrome, or inclusion body myositis (IBM).

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

15. An antibody according to any one of claims 1 to 13, or a pharmaceutical composition according to 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 selected from an antiplaque agent, recanemab, and donanemab.

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

18. The antibody for use in vivo diagnosis according to claim 17, wherein the amyloid disease is a disease related to soluble Aβ42 oligomers having a β structure.

19. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide including the antibody or its VH region and / or VL region according to any one of claims 1 to 7.

20. A vector comprising the nucleic acid molecule described in claim 19.

21. The vector according to claim 20, which is an expression vector.

22. A host cell containing the vector described in 20 or 21.

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

24. A method for producing an antibody according to any one of claims 1 to 7, comprising culturing a host cell according to claim 22 or claim 23 under conditions suitable for the expression of the antibody according to any one of claims 1 to 7.

25. An antibody according to any one of claims 1 to 7, for use in combination with a second therapeutic agent for use in the treatment of amyloid disease.

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

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