Antibodies, conjugate, pharmaceutical composition, nucleic acid molecule, vector, host cell and method for producing an antibody

Description

"ANTIBODIES, CONJUGATE, PHARMACEUTICAL COMPOSITION, NUCLEIC ACID MOLECULE, VECTOR, HOST CELL AND METHOD FOR PRODUCING AN ANTIBODY" Field of Invention

[001] The present invention relates to a novel humanized antibody and its use in the treatment of amyloid disease, such as Alzheimer's disease and related disorders. Background of the Invention

[002] Alzheimer's disease (AD) is a progressive neurological disease characterized by the loss of synaptic function, eventually leading to neuronal death. One of the main histopathological features of AD is the extracellular deposition of insoluble amyloid aggregates of amyloid-β (Αβ) peptide in plaques, essentially insoluble fibrillar clusters, in the brain, and the role of these plaques in the etiology of AD has been the subject of intense study. Several antibodies have been developed targeting amyloid plaques. These antibodies are typically selective for aggregated forms of Αβ or specific for a pyroglutamate form of Αβ. However, to date, the clinical benefit of these antibodies has been very limited.

[003] Recent studies have shown that plaques are not, in fact, the most toxic forms of Aβ, and that Aβ neurotoxicity mainly stems from soluble oligomeric aggregates of the amyloid peptide β-1-42 (β42). Notably, of all Aβ present in the brains of AD patients, only a very small fraction of soluble aggregated oligomeric Aβ42 carries most of the neurotoxic potential (Hong et al., Acta Neuropathologica 136, 19-40, 2018). In other words, the naturally occurring toxic form of Aβ is very abundant, and this has hindered the development of beneficial therapies, as therapies that target or react to it... Petition 870250084592, dated 09 / 19 / 2025, pp. 95 / 194 2 / 75 cross-contamination with inert and non-toxic forms of Αβ may never reach the true toxic species.

[004] A monoclonal antibody was developed by applicant Alzinova AB (WO 2012 / 120035; Sandberg et al., Alz Res Therapy 14, 196, 2022). This antibody, named ALZ-201 (mAb20), is specific for a subset of soluble oligomeric Aβ42, but does not bind to other forms of Aβ (e.g., insoluble aggregates or plaques, or monomeric or non-aggregated Aβ). ALZ-201 was generated using a stabilized form of the Aβ42 peptide as an immunogen, Aβ42CC, which restricts the peptide conformation by inhibiting its fibrillation. Aβ42CC accumulates in the form of toxic prefibrillar oligomers with a β structure, and this form is being developed by Alzinova as a vaccine candidate, designated ALZ-101 (WO 2009 / 128772), which is currently in clinical trials (NCT05328115). The resulting ALZ-201 antibody therefore recognizes a conformational epitope on the stabilized soluble oligomeric antigen (i.e., on the vaccine candidate).The conformational specificity of the antibody was confirmed, and its ability to neutralize the neurotoxic effect of post-mortem brain extracts from AD patients was demonstrated in primary cultures of mouse neuronal cells; when AD brain extracts were immunodepleted using ALZ-201, the loss of nuclei, neurites, synapses, and branching was reduced compared to non-depleted extracts. This result is comparable to the reduction in neurotoxicity achieved by immunodepletion using a pan-specific anti-Aβ antibody (4G8) that binds to all forms of Aβ (Sandberg et al., Alz Res Therapy 14, 196, 2022). Other non-clinical efficacy assays were performed 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). Petition 870250084592, dated 09 / 19 / 2025, pp. 96 / 194 3 / 75 of 2020). These data demonstrate once again that the toxic effects of DA brain extracts can be prevented by immunosuppression with ALZ201.

[005] Thus, despite targeting only a small fraction of patient-derived Aβ, studies have shown that ALZ-201 has a high neutralizing effect on the neurotoxicity of patient-derived Aβ. In light of these promising preclinical studies, the monoclonal antibody (mAb) ALZ201 is being developed for clinical use. For this, a humanized antibody is needed. Brief Description of the Invention

[006] In a first aspect of the invention, an antibody is provided comprising an antigen-binding domain capable of specifically binding to prefibrillar Aβ42 oligomers with a β structure, wherein said antigen-binding domain comprises: (i) a variable heavy chain (VH) region comprising the sequence with SEQ ID NO: 1; or (ii) a variable light chain (VL) region comprising the sequence with SEQ ID NO: 2; or a combination thereof.

[007] In particular, the antibody, or more particularly its binding domain, does not bind, or exhibits substantially no or insignificant binding, to unstructured monomers or oligomers of Aβ (e.g., of Aβ42), nor to fibrillar forms of Aβ (e.g., of Aβ42).

[008] The antibody can be presented in different formats. It can be monovalent or bivalent. Furthermore, the antibody can be presented in single-chain form or it can comprise two or more separate chains, for example, 4 chains. It can comprise both VH and VL regions, for example, a polypeptide, or chain, Petition 870250084592, dated 09 / 19 / 2025, pp. 97 / 194 4 / 75 comprising a VH region, and a polypeptide, or chain, comprising a VL region. However, binding proteins in the form of, or comprising, single-domain antibodies are also known and included in the present invention, which comprise either only VH or only VL.

[009] The antibody may be an intact antibody or full-length antibody, or a fragment thereof, notably a fragment that retains the antibody's antigen-binding domain. Consequently, the antibody may contain one or more, for example, two, antigen-binding domains.

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

[011] The antibody may be provided in the form of immunoglobulin (Ig), and more particularly in the form of Ig comprising 2 heavy chains and 2 light chains, or as a fragment thereof. However, as described in more detail below, the antibody may be in the form of any construct comprising the antigen-binding domain. This may include, for example, fusion proteins comprising the antibody and another protein, or bispecific constructs.

[012] Consequently, in one embodiment, the antibody is in the form of a full-length Ig antibody, or an antigen-binding fragment thereof (i.e., a fragment comprising the antigen-binding domain). In a specific embodiment, the antibody is an IgG antibody, or a fragment thereof.

[013] Thus, the antibody may comprise a first chain (a heavy chain) with the VH region and a constant chain region. Petition 870250084592, dated 09 / 19 / 2025, pp. 98 / 194 5 / 75 heavy chain, and a second chain (a light chain) with the VL region and a constant light chain region. In one specific embodiment, the constant light chain region is a kappa (κ) constant region. In another specific embodiment, the constant heavy chain region is an IgG constant region, more particularly an IgG1 constant region. The antibody may comprise one or more of each of the aforementioned chains (first and second chains), for example, two heavy chains and two light chains.

[014] In a specific embodiment, the antibody is an IgG1Kappa antibody, or a fragment thereof.

[015] In another aspect, the present invention provides a conjugate comprising the antibody as defined in the present invention linked to at least one diagnostic agent.

[016] In a further aspect, an antibody as defined in the present invention is provided for use in therapy.

[017] In another aspect, the present invention provides an antibody or a conjugate thereof for use in diagnostics performed on a human or animal body (i.e., in vivo diagnostics).

[018] In a further aspect, the present invention provides a pharmaceutical composition comprising an antibody as defined in the present invention, mixed with at least one pharmaceutically acceptable carrier or excipient.

[019] In another further aspect, the present invention provides an antibody or a pharmaceutical composition, as defined in the present invention, for use in the treatment of an amyloid disease.

[020] In a further aspect, a conjugate, as defined in the present invention, is provided for use in the in vivo diagnosis of an amyloid disease. Petition 870250084592, dated 09 / 19 / 2025, pp. 99 / 194 6 / 75

[021] A related aspect provides for the use of an antibody as defined in the present invention in the manufacture of a medicament for use in the therapy of an amyloid disease.

[022] Another related aspect provides a method of treating an amyloid disease, comprising administering to a subject with such need a therapeutically effective amount of an antibody, or pharmaceutical composition, as defined in the present invention.

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

[024] In some examples of the implementation of the various uses and methods above, amyloid disease can be any of the following: Alzheimer's Disease (AD), Down Syndrome or Inclusion Body Myositis (IBM).

[025] The subject can be any human being or animal, particularly a human.

[026] Another aspect provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody as defined in the present invention, or a VH and / or VL region thereof.

[027] It is understood in this respect that the antibody may be composed of, or may comprise, one or more protein chains, or subunits, for example, 2 or more, and in such a way the individual chains or subunits, for example, the individual molecules (i.e., polypeptides) comprising the VH and / or VL regions may be encoded by separate nucleic acid molecules.

[028] Consequently, this aspect can be seen as providing one or more nucleic acid molecules comprising nucleotide sequences that encode the antibody as defined in Petition 870250084592, dated 09 / 19 / 2025, pp. 100 / 194 7 / 75 present invention.

[029] Also provided in the present invention is a vector comprising the nucleic acid molecule, or one or more nucleic acid molecules, as defined in the present invention. Analogously, this aspect can also be viewed as providing one or more expression vectors comprising one or more nucleic acid molecules, as defined in the present invention.

[030] In an example of realization, the vector(s) is / are expression vector(s).

[031] The vector may be a viral vector. Consequently, a virus comprising one or more nucleic acid molecules is also provided, as defined in the present invention.

[032] Another aspect provides a host cell comprising a vector (e.g., an expression vector) or a nucleic acid molecule as defined in the present invention (or more particularly one or more vectors or nucleic acid molecules as defined in the present invention), or a host cell expressing an antibody as defined in the present invention.

[033] The host cell can be prokaryotic or eukaryotic. In one embodiment, the host cell is a mammalian host cell.

[034] An additional aspect provides a method for producing an antibody as defined in the present invention, wherein said method comprises culturing a host cell as defined in the present invention under conditions suitable for antibody expression.

[035] The method may also include obtaining (e.g., collecting or isolating) the antibody from the host cell or culture (e.g., growth medium or supernatant after Petition 870250084592, dated 09 / 19 / 2025, pp. 101 / 194 8 / 75 culture).

[036] The method may further comprise the step of introducing one or more nucleic acid molecules or vectors as defined in the present invention into the host cell. Detailed Description of the Invention

[037] The murine antibody ALZ-201 was humanized by grafting the 3 complementarity-determining regions (CDRs), as defined by Kabat nomenclature, of each variable light chain (VL) region and variable heavy chain (VH) region into a human germline VL and VH, respectively, that was selected to be as close as possible, in terms of sequence homology, to the corresponding murine VL and VH.

[038] The term “VH” (or VH domain) or “variable heavy chain region” refers to the variable region of a heavy chain of an antibody molecule. The VH comprises three heavy chain CDRs (VHCDRs) designated VHCDR1, VHCDR2, and VHCDR3, from the amino-terminal end to the carboxy-terminal end.

[039] The term “VL” (or VL domain) or “variable light chain region” refers to the variable region of a light chain of an antibody molecule. The VL comprises three heavy chain CDRs (VLCDRs) designated VLCDR1, VLCDR2, and VLCDR3, from the amino-terminal end to the carboxy-terminal end.

[040] CDRs are regions of hypervariability within variable regions.

[041] The variable regions of the heavy and light chains also have four structural regions (FR1, FR2, FR3, and FR4, from the amino-terminal end to the carboxy-terminal end). These structural regions (FW or FR, from the English Framework Region) are more Petition 870250084592, dated 09 / 19 / 2025, pp. 102 / 194 9 / 75 are preserved and separate the CDRs.

[042] Several factors need to be considered when designing a functional humanized antibody, and humanization is not a simple process.

[043] As noted above and described in more detail in Example 1 below, in preparation for humanization, a molecular model of the ALZ-201 antibody was constructed, which is no trivial task requiring extensive human intervention, and this was used to guide the humanization design. Guided by the molecular model, the first stage of the humanization design was performed in silico, again involving several design considerations and choices, including the selection of specific human germline sequences and residues in human structural regions to be “retromutated,” i.e., replaced by parental murine amino acids. Furthermore, during the development process, residues in the CDRs were selected for the process known as germline sequence-based humanization called “germ-lining” (i.e., replacement by their human germline counterparts).This led to the design of 6 different humanized heavy chain variable region (HV) sequences based on 2 different HV sequences from the human germline and 8 different humanized light chain variable region (LV) sequences based on 4 different LV sequences from the human germline. Thus, several possible humanized HV / LV combinations were derived, representing different options for humanized ALZ-201 variants. In the next step, several selection criteria were used to reduce the list of 48 combinations to 18, of which 16 combinations were selected for experimental testing, along with a chimeric variant of ALZ-201, herein referred to as 'chALZ201' or 'mAb15'. The candidate combinations were subjected to iterative classification based on the degree of, or more particularly, % of. Petition 870250084592, dated 09 / 19 / 2025, pp. 103 / 194 10 / 75 humanity (% identity with human amino acid residues, or, in other words, the % sequence identity of the humanized VH and VL sequences with the human V gene sequences; the % sequence identity with homologous human antibodies ranged from 81.0% to 88.7%) and in the results of experimental tests on expression yield, affinity, and thermal stability. More specifically, 6 candidates were selected for additional affinity (EC50 as determined by ELISA) and thermal stability tests, and the selections and tests were further reduced to 5 with affinity tests by SPR (Kd (nM) as determined by Biacore), then to two main candidates for expression yield tests, ultimately leading to the selection of the main candidate antibody, identified as Ab11 in the Examples below.

[044] The humanized antibody obtained is defined by specific VH and VL sequences. It is well known in the state of the art that antibodies can be provided in various formats and configurations, including various artificial constructs, in addition to the classic format of immunoglobulins and their fragments. Besides antibodies and constructs comprising a VH and VL region, so-called single-domain antibodies can be prepared, comprising only VH or only VL. Thus, in the present invention, an antibody defined by the humanized VH and / or VL sequences of the obtained humanized ALZ-201 derivative is generally provided. The antibody comprises one, or at least one, antigen-binding domain (or, alternatively, an antigen-binding unit).Since the antigen-binding domain is obtained or derived from an antibody, the antibody may be termed, or considered, as an antibody-based binding protein, or as a binding protein comprising an antibody-derived binding domain.

[045] The antibody antigen-binding domain Petition 870250084592, dated 09 / 19 / 2025, pp. 104 / 194 11 / 75 comprises at least one VH region or at least one VL region. In one embodiment, the antigen-binding domain comprises at least one VH region and at least one VL region.

[046] The binding protein may comprise one or more antigen-binding domains. Each antigen-binding domain may comprise one (or at least one) VH region, one (or at least one) VL region, or both regions, a VH domain and a VL domain (or at least one VH region and at least one VL region). In a typical antibody form, the binding protein comprises two antigen-binding domains (in other words, it is bivalent) and, more particularly, each antigen-binding domain comprises a VH region and a VL region. However, as discussed later, monovalent antibodies or antibody fragments comprising a single antigen-binding domain are known. In such an embodiment, the single antigen-binding domain may comprise a VH region and a VL region.Furthermore, and as noted above, the binding protein may comprise an antigen-binding domain comprising 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.

[047] As used in the present invention, the terms “a” and “an” are used to mean “at least one”, “at least one first”, “one or more” or “a plurality” of the referenced components or steps, except where an upper limit is subsequently specifically stated.

[048] Furthermore, when the terms “comprises”, “comprises”, “has” or “having” or other equivalent terms are used in this document, then, in some more specific embodiments, these terms include the term “consists of” or “consists essentially of”, Petition 870250084592, dated 09 / 19 / 2025, pp. 105 / 194 12 / 75 or other equivalent terms.

[049] As noted above, the VH region comprises an amino acid sequence as set out in SEQ ID NO: 1.

[050] VH (SEQ ID NO: 1): QVTLKESGPTLVKPTQTLTLTCTFSGFSLSTFGSGVSWIRQPPG KALEWLAHIYWDDDKHYNPSLKSRLTITKDTSKNQVVLTITNMDPVDTATYFCA RRESHYYGSGYYFDYWGQGTLVTVSS.

[051] The VL region comprises an amino acid sequence as set out in SEQ ID NO: 2.

[052] VL (SEQ ID NO: 2): DIQLTQSPSSLSASVGDRVTITCRASSSISYMHWYQQKPGKAPK PWIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWRSDPLTF GGGTKVEIK.

[053] As also noted above, each of these variable region sequences comprises 3 complementarity-determining regions (CDRs, which are underlined in the sequences above) and four structural regions (framework regions - FR).

[054] The Kabat numbering scheme is a widely adopted standard for numbering and assigning CDR residues in an antibody based on sequence alignment data (Johnson G. and Wu TT, Nucleic Acids Res. 28, 214-218, 2000). It assigns VHCDR1 to residues 31-35B (position 35 is a defined insertion point, and the terminology 35B is understood in the art as such), VHCDR2 to residues 50-65, and VHCDR3 to residues 95-102. Conversely, it assigns VLCDR1 to residues 24-34, VLCDR1 to residues 50-56, and VLCDR3 to residues 89-97.

[055] The CDR definitions of Chothia were subsequently introduced based on the available antibody structures (Chothia, C. and Petition 870250084592, dated 09 / 19 / 2025, pp. 106 / 194 13 / 75 (Lesk, AM, J. Mol. Biol. 196, 910, 1987). The AbM method (Abhinandan KR and Andrew CR Martin ACR, Molecular Immunology 45, 3832-3839, 2008) uses a modified version of the Chothia scheme and assigns 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 SEQ ID NO: 1 and SEQ ID NO: 2 above.

[056] The AbM scheme was used to define the CDR sequences of the parental antibody (WO2012120035A1). These CDR sequences correspond to the CDR sequences of the parental antibody ALZ-201, except for VLCDR1, which, for the chosen humanized antibody, is composed of the residues RASSSISYMH (SEQ ID NO: 17), while for the non-humanized parental antibody ALZ-201, it is composed of the residues RASSSVSYMH (SEQ ID NO: 18). Therefore, the amino acid position 29 of the humanized derivative is an isoleucine instead of a valine.

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

[058] The antibody, as noted above, can take various forms, including antibody fragments. All of these forms are included. As noted above, the antibody may comprise 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 comprised in a single chain (polypeptide) or in separate chains (polypeptides). The Petition 870250084592, dated 09 / 19 / 2025, pp. 107 / 194 14 / 75 An antibody can therefore comprise one or more polypeptide chains, for example, 2 or 4 polypeptides. An individual polypeptide can comprise one or more VH regions and / or one or more VL regions, for example, a VH region and a VL region, etc.

[059] Consequently, an antigen-binding domain may comprise one or more polypeptides (chains), each comprising one or more VH regions and / or one or more VL regions.

[060] Furthermore, a polypeptide (chain) comprising a variable region sequence may comprise all or part of a constant region sequence, for example, one, two or all 3 CH1, CH2 and CH3 of a heavy chain constant region in the case of VH, and all or part of the light chain (LC) constant region in the case of VL. Since VH and VL have been humanized, in particular, the constant region sequence is a human sequence, or humanized, especially a human sequence.

[061] Thus, the term “antibody” includes all known forms of antibodies, including whole or full-length (complete) antibodies, or any fragments thereof that bind to antigens, or single chains or derivatives of single chains thereof, as well as synthetic or artificial antibody constructs comprising at least one VH region or at least one VL region, as defined in this invention, and multimers thereof, for example, dimeric, trimeric or higher-order multimers. It is understood that the term includes recombinant and genetically modified (engineered) antibodies.

[062] More broadly, the term “antibody” can be seen as including any binding protein (which may be called an immune binding protein) comprising a binding domain to Petition 870250084592, dated 09 / 19 / 2025, pp. 108 / 194 Λ5Γ75 antigen, specifically an antigen-binding domain derived from an antibody. Consequently, the term “antibody” is used to refer to any antibody-like molecule that possesses an antigen-binding region obtained from or derived from an antibody. In a preferred embodiment, the antibody or fragment thereof comprises at least one VH region and at least one VL region.

[063] In one embodiment, the antibody is an immunoglobulin antibody, and more specifically an antibody comprising at least 2 heavy chains and at least 2 light chains, or a fragment thereof.

[064] In any format described in the present invention, wherein the antibody comprises constant regions, the heavy chain comprises the VH and all or part of a constant region of the heavy chain, and the light chain comprises the VL and all or part of a constant region of the light chain. When the antibody comprises a complete complement of the constant regions of the heavy and light chains, it is termed a complete antibody or full-length antibody. Such full-length / whole antibodies represent a preferred embodiment.

[065] Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five main classes: IgA, IgD, IgE, IgG, and IgM, and any of these are included, although IgA and IgG are preferred, particularly IgG. Several of them are divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like; for example, camelid antibodies are IgG antibodies that frequently have IgG2 or IgG3 constant domains. All subclasses are included in the present invention. The heavy chain constant domains corresponding to the different immunoglobulin classes are designated α, δ, ε, γ, and μ, respectively. The subunit structures and configurations Petition 870250084592, dated 09 / 19 / 2025, pp. 109 / 194 16 / 75 three-dimensional structures of the different classes of immunoglobulins are well known. The appropriate heavy chain constant regions are known and available in the state of the art.

[066] Mammalian antibody light chains are assigned to one of two clearly distinct types: kappa (κ) and lambda (λ), and either of them can be used in the present invention. Again, suitable light chain constant region sequences are known and available in the prior art.

[067] In one embodiment, the heavy chain constant region is either comprises all or part of the human IgG1 constant region having the amino acid sequence set out in SEQ ID NO: 3 or an amino acid sequence with at least 90% sequence identity with that sequence.

[068] SEQ ID NO: 3, human IgG1 constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK.

[069] In another embodiment, the constant light chain region is either comprises all or part of the human kappa constant region with the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence with at least 90% sequence identity with that sequence.

[070] SEQ ID NO: 4 - Human kappa constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD Petition 870250084592, dated 09 / 19 / 2025, pp. 110 / 194 17 / 75 NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC.

[071] In more specific embodiments, the amino acid sequence has at least 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 3 or 4, respectively.

[072] Thus, some variation in the sequences of the constant region of the antibodies or binding proteins used here is permitted.

[073] Sequences that exhibit at least 90% sequence identity with a declared reference sequence may be called substantially identical or substantially similar sequences. These sequences may include alterations of one or multiple bases or amino acids (additions, substitutions, insertions, or deletions) in the sequences. Amino acid substitutions may be conservative substitutions. Such sequence modifications may be made for various reasons, including, for example, to facilitate production, stability, antibody pharmacokinetics, etc.

[074] A “conservative amino acid substitution” is one in which the amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the state of the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In other examples, the Petition 870250084592, dated 09 / 19 / 2025, pp. 111 / 194 18 / 75 families of amino acid residues can be grouped based on hydrophobic side groups or hydrophilic side groups.

[075] Sequence identity can be evaluated by any convenient method. However, to determine the degree of identity between sequences, computer programs that perform multiple sequence alignments are useful, for example, Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used in conjunction with the BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest-order match is obtained between two sequences, where at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that can be used to align sequences are the Needleman and Wunsch alignment method (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by. Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981) so that the highest-order correspondence is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods for calculating the percentage of identity between two amino acid sequences are generally recognized in the state of the art and include, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) and those described in Computational Molecular Biology, Lesk, Ed. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

[076] Generally, computer programs will be used for such calculations. Programs that compare and align pairs of sequences, such as ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA Petition 870250084592, dated 09 / 19 / 2025, pp. 112 / 194 19 / 75 (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST 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. In addition, the Dali server at the European Bioinformatics Institute offers structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998). The LALIGN pairwise sequence alignment program is available on EMBL-EBI.

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

[078] In one embodiment, the antibody is an IgG antibody, and more particularly an IgG1 antibody, or a fragment thereof.

[079] In a specific embodiment, the antibody is an IgGkappa antibody, or a fragment thereof.

[080] In another embodiment, the antigen-binding domain comprises: (i) a heavy chain sequence comprising 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 with at least 90% sequence identity with SEQ ID NO: 3; and / or (ii) a light chain sequence comprising the VL sequence of SEQ ID NO: 2 linked to the light chain constant region sequence of SEQ ID NO: 4; or an amino acid sequence with at least Petition 870250084592, dated 09 / 19 / 2025, pp. 113 / 194 20 / 75 minus 90% sequence identity with SEQ ID NO: 4.

[081] Antibody fragments that retain the ability of their original full-length antibody to bind to the antigen or that, in other words, comprise an antigen-binding domain, include: Fab', Fab, F(ab')2, Fd, Fv, single-domain antibodies (DABs), for example, consisting of a VH domain (also known as VHH antibodies) and nanobodies.

[082] Other antibody formats known in the art and covered by the present invention include single-chain formats, including scFv (single-chain Fv), dsFv, ds-scFv, linear antibodies, TandAbs, minibody, diabody, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); scdiabody; kappa(lambda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); scFv-Fc dimer; DART (ds-stabilized diabody “Dual Affinity ReTargeting”) and the like.

[083] Single-domain antibodies include camelid antibodies, vNAR (shark) antibodies, VH antibodies, or VL antibodies.

[084] The techniques for preparing the various antibody formats, fragments and constructs are well known in the state of the art.

[085] Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be produced by recombinant techniques. Petition 870250084592, dated 09 / 19 / 2025, pp. 114 / 194 21 / 75 cell-free expression systems or can be chemically synthesized. Techniques for the production of antibody fragments are well known and described in the state of the art.

[086] As noted above, in certain embodiments, the antibody, or antibody fragment, comprises all or part of a heavy chain constant region. In addition, the antibody or antibody fragment may comprise all or part of a kappa light chain constant region or a lambda light chain constant region, or parts thereof. All or part of these constant regions may be naturally produced or may be wholly or partially synthetic. The appropriate sequences for such constant regions are well known and documented in the art, and exemplified above.

[087] In other embodiments, there are no constant regions, for example, no constant heavy or light chain region is present, for example, a variable domain or variable heavy chain (VH) domain is the only part of an antibody that is present.

[088] The antibodies described in the present invention and the nucleic acids that encode them are artificial constructs (e.g., engineered or synthetic) that do not occur in nature and do not correspond to naturally occurring molecules. In other words, the antibodies and nucleic acids are not native. This is implied by their composition.

[089] In one embodiment, the antibody may be provided in the form of a conjugate comprising the binding protein linked or attached (i.e., conjugated) to another entity. This may be another protein or polypeptide component, which may be conjugated to the binding protein by means of a peptide bond or peptide linker, in which case the conjugate may be considered a fusion protein comprising the binding partner linked to (or fusion to) a fusion partner. Alternatively, Petition 870250084592, dated 09 / 19 / 2025, pp. 115 / 194 22 / 75 The other entity may be of a non-protein nature, for example, it may be a small molecule or other chemical or physical entity (e.g., polymer, marker, etc.) and may be chemically linked to the binding protein by means of procedures and binding groups well known in the state of the art. As will be described in more detail below, such a fusion partner or conjugate entity may be a therapeutic or diagnostic agent.

[090] A person skilled in the art will understand that antibodies and antibody fragments can be prepared in a variety of ways that are well known and described in the art, but are conveniently prepared using recombinant methods.

[091] For this purpose, nucleic acid molecules (for example, one or more nucleic acid molecules) comprising nucleotide sequences encoding antibodies as defined in the present invention or parts or fragments thereof represent other aspects of the invention.

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

[093] An example nucleotide sequence encoding the VH sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 5 below, and an example 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 initiation site. Petition 870250084592, dated 09 / 19 / 2025, pp. 116 / 194 23 / 75 of protein translation. Both SEQ ID NO: 5 and SEQ ID NO: 6 were optimized for expression in CHO cells and include signaling peptides for protein secretion (underlined nucleotides).

[094] SEQ ID NO: 5: GCCGCCACCATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTG GCTGCTCCTCGGTGGGTGCTGTCCCAGGTGACCCTGAAGGAGTCCGGCC CCACCCTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCAGC GGCTTTAGCCTGAGCACCTTTGGCAGCGGCGTGAGCTGGATCAGGCAGC CTCCCGGCAAGGCCCTGGAGTGGCTGGCTCACATCTATTGGGACGACGA CAAGCACTATAACCCTAGCCTGAAGAGCCGGCTGACCATCACCAAGGACA CCAGCAAGAACCAGGTGGTGCTGACCATCACAAACATGGACCCTGTGGAT ACCGCCACCTATTTTTGCGCCCGGAGGGAGAGCCACTACTATGGCAGCGG CTACTATTTCGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC.

[095] SEQ ID NO: 6: GCCGCCACCATGGTGCTGCAGACCCAGGTGTTCATCAGCCT GCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGTCC CCTTCCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTC GGGCTTCCTCCAGCATCTCCTATATGCACTGGTATCAGCAGAAGCCCGGC AAGGCTCCCAAGCCTTGGATCTACGCTACCAGCAATCTGGCTAGCGGCGT GCCTAGCCGGTTCTCCGGCTCCGGATCCGGCACCGACTTCACCCTGACCA TCTCCTCCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTGG CGGTCGATCCCCTGACCTTCGGCGGCGGAACCAAGGTGGAGATCAAG.

[096] Thus, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence as defined in SEQ ID NO: 5 or a sequence with at least 90% sequence identity therewith, and / or a nucleotide sequence as defined in SEQ ID NO: 6 or a sequence with at least 90% sequence identity therewith. In more embodiments Petition 870250084592, dated 09 / 19 / 2025, pp. 117 / 194 24 / 75 specific, the sequence identity percentage with SEQ ID NO: 5 or 6 is at least 92, 93, 94, 95, 96, 97, 98, or 99%.

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

[098] The antibodies and nucleic acid molecules described in the present invention may also include non-sequence-based modifications or chemical equivalents of amino acid and nucleotide sequences, provided that they do not substantially alter the function of the antibodies or nucleic acid molecule. Thus, the antibody must maintain the binding and functional properties of the corresponding unmodified antibody.

[099] The antibody may also comprise other amino acid sequences, or marker sequences (called tags or labels), fusion partners or other components that do not contribute to antigen binding, or alterations to convert one type or shape of antibody molecule or fragment into another type or shape of antibody molecule or fragment (for example, conversion of VHH to Fab or scFv or whole antibody, for example, a full-length heavy chain-only antibody, or vice versa), or the conversion of an antibody molecule into a specific class or subclass of antibody molecule (for example, the conversion of an antibody molecule into IgG or a subclass thereof, for example, IgG2 or IgGa, for example, to a camelid antibody, or to an IgA class antibody), or the preparation of an Fc fusion, for example, a VHH-Fc fusion.These additional amino acid sequences can be encoded by additional nucleotide sequences present in the nucleic acid molecule.

[100] A technician skilled in the subject will understand that tests of Petition 870250084592, dated 09 / 19 / 2025, pp. 118 / 194 25 / 75 binding can be used to test whether such antibodies and modified constructs retain the binding properties of the original humanized antibody.

[101] Nucleic acid fragments that encode the VH and VL sequences can be derived or produced by any appropriate method, for example, by cloning or synthesis.

[102] Once the nucleic acid fragments encoding VH and VL have been obtained, these fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert variable region fragments into full-length antibody molecules with appropriate constant region domains, or into specific antibody fragment formats discussed elsewhere in the present invention, for example, single-domain antibodies such as VHH, Fab fragments, scFv fragments, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding VH and / or VL and, optionally, other sequences are generally incorporated into one or more vectors, most notably appropriate expression vectors, in order to facilitate antibody production or, for example, to facilitate selection or screening, for example, by incorporation into phage display vectors.

[103] Conveniently, vectors may comprise one or more nucleotide sequences that encode another amino acid sequence(s) intended to form part of the antibodies, for example, a constant region sequence.

[104] Some possible expression vectors include, among others, cosmids, plasmids or viral vectors, for example, modified viruses (e.g., replication-defective retroviruses, lentiviruses, adenoviruses and adeno-associated viruses), provided that the vector is compatible with the host cell used. A variety of different vectors are Petition 870250084592, dated 09 / 19 / 2025, pp. 119 / 194 26 / 75 available in the art for such use, including, for example, the pXtenl plasmid, as used in the XtenCHO® mammalian expression system, available from ProteoGenix. Expression vectors are “suitable for transformation in a host cell,” meaning that the expression vectors contain the nucleic acid molecule and regulatory sequences selected based on the host cells to be used for expression, which are operationally linked to the nucleic acid molecule. “Operationally linked” expression means that the nucleic acid is linked to the regulatory sequences in a way that allows expression of the nucleic acid.

[105] Consequently, another aspect described in the present invention is a vector, particularly an expression vector, containing or comprising a nucleic acid molecule as defined or described herein, or a fragment thereof. In the case of an expression vector, it also comprises the 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.

[106] For antibody production in host cells, it may be desirable to provide the expressed protein or protein subunit (i.e., the expressed polypeptide) with a signaling or leader sequence, to direct the expressed product to a specific compartment or site, for example, a secretory signaling sequence, to allow the protein to be expressed extracellularly. It can then be conveniently obtained, or collected, from the supernatant or growth medium.

[107] By way of representative examples, the amino acid sequences coded for polypeptides expressed in the Examples are presented below, comprising leader sequences:

[108] Heavy chain polypeptide - SEQ ID NO: 7: Petition 870250084592, dated 09 / 19 / 2025, pp. 120 / 194 27 / 75 MKHLWFFLLLVAAPRWVLSQVTLKESGPTLVKPTQTLTLTCTFS GFSLSTFGSGVSWIRQPPGKALEWLAHIYWDDDKHYNPSLKSRLTITKDTSKN QVVLTITNMDPVDTATYFCARRESHYYGSGYYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK.

[109] Characteristics: SIP leader sequence: [1:19] underlined; VH. Human IgG1 constant region: [144:473] bold.

[110] Light chain polypeptide - SEQ ID NO: 8: MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCRA SSSISYMHWYQQKPGKAPKPWIYATSNLASGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQWRSDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[111] Characteristics: SIP leader sequence: [1:20] underlined; VL; Human kappa constant region: [127:233] bold.

[112] Expression vectors can be introduced into host cells to produce a transformed, transduced, or transfected host cell. The terms “transformed,” “transduced,” and “transfected” are typically used to refer to different methods of introducing a nucleic acid into a cell (by plasmid, viral vector, or more generally, nonviral methods). As used in the present Petition 870250084592, dated 09 / 19 / 2025, pp. 121 / 194 28 / 75 invention, the terms are intended to encompass the introduction of nucleic acid (e.g., a vector) into a cell by one of the many possible techniques known in the art. The appropriate methods for transforming, transducing, and transfecting host cells are well known in the art and can be found in laboratory textbooks.

[113] Suitable host cells include a wide variety of eukaryotic and prokaryotic host cells, or plant or fungal cell-based expression systems (e.g., Saccharomyces cerevisiae) may be used, as will be well known to one skilled in the art. For example, the binding proteins may be expressed in yeast cells, such as Pichia or Saccharomyces, e.g., Saccharomyces cerevisiae, insect cells or mammalian cells, such as, for example, CHO cells, or other cell lines. In addition, the proteins of the invention may be expressed in prokaryotic cells, such as Escherichia coli (E. coli).

[114] The (in vitro) expression of cell-free proteins is another means of producing recombinant proteins in solution using biomolecular translation mechanisms extracted from cells. Proteins can also be prepared by chemical synthesis, using well-known techniques in protein chemistry, such as solid-phase synthesis.

[115] When VH and VL are provided as or in separate chains (i.e., separate polypeptides are expressed), they can be encoded by nucleotide sequences provided in separate vectors. Consequently, an additional aspect provides an expression construct, an expression vector or an expression system (e.g., a viral, bacterial or other expression construct, vector or system), for example, one or more expression constructs or expression vectors comprising one or more of the nucleic acid molecules Petition 870250084592, dated 09 / 19 / 2025, pp. 122 / 194 29 / 75 described in the present invention. As noted above, such a system can be represented by a plurality of vectors comprising a first vector comprising a nucleotide sequence encoding a polypeptide comprising a VH region and a second vector comprising a nucleic acid sequence encoding a polypeptide comprising a VL region.

[116] Convenient constructs, vectors, etc., are those that allow prolonged or sustained expression of antibodies within the host cell. This expression can be transient, for example, episomal, or more permanent, for example, via genomic integration, provided that sufficient levels and duration of expression are achieved.

[117] A further aspect provides a host cell (for example, a mammalian, bacterial or yeast host cell) or virus, for example, one or more host cells or viruses comprising one or more expression constructs or expression vectors as defined in the present invention. Host cells or viruses comprising one or more of the nucleic acid molecules as defined in the present invention are also provided. A host cell or virus expressing an antibody as defined in the present invention constitutes a further aspect.

[118] An additional aspect provides a method for producing (or manufacturing) an antibody as defined in the present invention, comprising a host cell culture step. More particularly, the method comprises the steps of (i) culturing a host cell comprising one or more expression vectors or one or more nucleic acid molecules under conditions suitable for the expression of the encoded antibody; and, optionally, (ii) isolating or obtaining the antibody from the host cell or growth medium / supernatant. These Petition 870250084592, dated 09 / 19 / 2025, pp. 123 / 194 30 / 75 Production (or manufacturing) methods may also comprise a purification step of the antibody and / or formulation of the antibody into a composition, for example, a pharmaceutical composition, including at least one additional component, such as a pharmaceutically acceptable vehicle / carrier or excipient. The method may also comprise a preceding step of introducing the vector or nucleic acid molecule into the host cell.

[119] In embodiments where the antibody is composed of more than one polypeptide chain (for example, certain fragments, such as Fab fragments or whole antibodies), all polypeptides are expressed in the host cell, from the same expression vector or from a different expression vector, so that the complete antibody protein can be assembled in the host cell and isolated or purified from it.

[120] Compositions comprising an antibody, or a nucleic acid molecule or expression vector, or a first host cell as defined in the present invention, constitute further aspects. Formulations (compositions) comprising one or more antibodies, optionally in a mixture with a suitable diluent, vehicle or excipient, constitute a preferred embodiment. Such formulations may be for pharmaceutical use and, therefore, such compositions are preferably pharmaceutically acceptable or acceptable for administration to human or non-human animals. Suitable diluents, excipients and vehicles are known to those skilled in the art.

[121] Pharmaceutical compositions may be presented in a form suitable for systemic administration, such as parenteral administration, including subcutaneous administration, intravenous administration, infusion administration, intra-arterial administration, intraperitoneal administration, intramuscular administration or administration into the brain, such Petition 870250084592, dated 09 / 19 / 2025, pp. 124 / 194 31 / 75 as intracerebroventricular administration. Pharmaceutical compositions may be presented as conventional pharmaceutical formulations suitable for systemic administration, such as solutions, suspensions, or any other preparation suitable for injection or infusion. Conventional pharmaceutical excipients, as well as usual methods of production, may be used for the preparation of these pharmaceutical formulations.

[122] Injection or infusion solutions, for example, sterile aqueous solutions, can, for example, be produced in the conventional manner, such as by adding excipients and preservatives, antioxidants and / or stabilizers.

[123] Formulations may be presented in unit dose or multidose formats, for example, containers such as vials or ampoules. The appropriate dosage units may be determined by a person skilled in the art.

[124] A further aspect provides the antibodies defined herein, or nucleic acid molecules, expression vectors or host cells as defined in the present invention, or pharmaceutical compositions containing them, for use in in vivo therapy or diagnosis, in particular for use in the treatment or prevention of amyloid disease. In particular, it may be any disease or condition associated with (or characterized by) prefibrillar β42 oligomers with β structure (i.e., soluble β42 oligomers with β structure), or where such oligomers play a role, for example, a causal role (e.g., a wholly or partially causal role) or an essential role.

[125] As used in this invention, the term “amyloid disease” refers particularly to a disease (or condition or disorder) associated with the abnormal aggregation of proteins or peptides into amyloid deposits. Although amyloid deposits can be observed in many Petition 870250084592, dated 09 / 19 / 2025, pp. 125 / 194 32 / 75 cases in the CNS, or more particularly in the brain (as in the case of AD), the diseases to be treated by the binding protein described in the present invention are not limited to these, and deposits can be found or observed in other tissues or locations of the body. As indicated above, however, amyloid disease can also be associated with the abnormal aggregation of proteins or peptides into soluble aggregates, typically termed “oligomers”. More specifically, the disease is associated with the abnormal aggregation of Aβ, and especially Aβ42, into amyloid fibrils and toxic soluble assemblies, collectively termed Aβ oligomers. These oligomers are prefibrillar and have structures distinct from insoluble fibrillar plaques. This disease includes any disease that would benefit from the removal or depletion of soluble prefibrillar Aβ42 oligomers with β structure, which have been recognized in the art as toxic agents.Removing these toxic oligomers in these diseases can prevent or delay the development of the disease.

[126] In one embodiment, the disease is a neurodegenerative condition, for example, a dementia, associated with Aβ, especially Aβ42, and more particularly with prefibrillar oligomers of Aβ42 with β structure.

[127] In one example of realization, the disease is Alzheimer's Disease (AD) or a related disease, such as Down Syndrome, for which the prevalence of AD is 90 to 100% in the seventh decade of life and the leading cause of death (Fortea et al., Lancet 395, 1988-1997, 2020).

[128] In another embodiment, the disease is a progressive degenerative muscle disorder, such as sporadic inclusion body myositis, for which aberrant aggregation of Aβ into oligomers has been implicated as a contributor to the disease (Kitazawa et al., Journal of Neuroscience 29, 6132-6141, 2009). Petition 870250084592, dated 09 / 19 / 2025, pp. 126 / 194 33 / 75

[129] According to the therapies described here, antibodies can target and inhibit or reduce the toxic effect of prefibrillar β42 oligomers with a β structure. Thus, antibodies can be used in the treatment or prevention of any disease or condition where reducing this toxic effect is beneficial.

[130] The administration of antibodies or nucleic acid molecules, expression vectors or host cells, in the therapeutic methods and uses described in the present invention, is carried out in pharmaceutically, therapeutically or physiologically effective quantities to individuals requiring treatment. Thus, said methods and uses may involve the additional step of identifying a subject requiring treatment.

[131] The subject may be a human or non-human animal, in particular a mammal.

[132] Treatment of the disease includes curing the disease, or any reduction or relief of the disease, for example, reducing the severity of the disease, or the symptoms of the disease, or any improvement in the subject's condition. Exemplary parameters that may be evaluated may include any improvement in functional, cognitive or memory tests, or other tests used to assess the disease.

[133] The beneficial effect may include a delay or inhibition of disease progression. In particular, in cases of early diagnosis, or in individuals at risk or susceptible to the disease, the onset of the condition, or its symptoms, may be delayed or even prevented. The therapies described here may also be prophylactic or preventive.

[134] These preventive measures can be carried out in healthy, normal or at-risk individuals, and can include both comprehensive prevention and significant prevention. Significant prevention can Petition 870250084592, dated 09 / 19 / 2025, pp. 127 / 194 34 / 75 includes the scenario in which the severity of the disease or the symptoms of the disease are reduced (e.g., reduced measurably or significantly) compared to the severity or symptoms that would be expected without treatment. Additionally, disease progression may be slowed or delayed in individuals in its early stages.

[135] By “pharmaceutically, physiologically or therapeutically effective amount” is meant an amount sufficient to demonstrate benefit to the subject’s condition. A person skilled in the art can easily determine whether an amount is sufficient to demonstrate benefit to the subject’s condition. A pharmaceutically, physiologically or therapeutically effective amount can be determined based on clinical assessment and can be easily monitored.

[136] Furthermore, the antibodies or pharmaceutical compositions described in the present invention can be used in conjunction with one or more additional therapeutic agents (for example, other agents known or proposed for use in the treatment of the disease in question). In other words, the antibodies or pharmaceutical compositions described in the present invention can be used in combination therapy, such as combination with standard of care (SOC). In one embodiment, the antibodies or pharmaceutical compositions described in the present invention are intended for use in combination therapy, such as combination with Standard of Care (SOC) therapy for an amyloid disease.

[137] For such combined treatments, the second agent may be administered to a subject together with the antibody. This may be done simultaneously, in the same pharmaceutical composition, or in separate compositions, either separately or sequentially.

[138] Examples of therapeutic agents for use in combination therapy with an antibody, as described in the present invention, are Petition 870250084592, dated 09 / 19 / 2025, pp. 128 / 194 35 / 75 other antibodies proposed or developed for use in the treatment of an amyloid disease or to reduce the amount of beta-amyloid, for example, in the brain (often called anti-plaque agents), including, for example, lecanemab (Leqembi®) or aducanumab (Aduhelm®). Donanemab, which has successfully completed phase 3 clinical trials and is currently under review by the FDA and EMA, is another plaque-reducing antibody that may be useful for combination therapy with an antibody of the present invention.

[139] In a further embodiment, antibodies can be used in conjunction with one or more additional agents or technologies to increase antibody penetrance across the blood-brain barrier, many of which are known in the state of the art (Ayub and Wettig, Pharmaceutics 14, 224, 2022). One such preferred embodiment utilizes an antibody-transferrin fusion construct, where transferrin receptor-mediated transcytosis transports the binding protein between the blood and the brain.

[140] Binding proteins can also be used as molecular tools for in vivo applications, for example, in vivo diagnostic methods.

[141] Thus, other aspects of the invention provide a reagent comprising an antibody as defined in the present invention and the use of such antibodies as molecular tools in in vivo assays, for example, for the detection of prefibrillar Αβ42 oligomers with β structure in a subject, for example, in an in vivo imaging procedure.

[142] In one embodiment, the disease diagnosis method as described in the present invention is an in vivo method.

[143] Such an in vivo method may, for example, involve administering an antibody in a detectable form to the subject, by Petition 870250084592, dated 09 / 19 / 2025, pp. 129 / 194 36 / 75 example, in the form of an imaging agent (agent for imaging), and obtaining images of the subject to detect the antibody. The antibody, in this embodiment, can be labeled. It can be provided in the form of an antibody conjugate with a diagnostic agent. The diagnostic agent can be a marker or detectable fraction that can be detected by imaging. For example, the antibody can be used as a tracer used in PET.

[144] As noted above, the parental antibody ALZ-201 is characterized by a unique and beneficial binding profile. It is specific for soluble oligomeric Aβ42, but does not bind to other forms of Aβ (e.g., it does not bind to insoluble aggregates or plaques, or to monomeric or non-aggregated Aβ). Therefore, it is truly specific, and not merely selective, for structured Aβ42 oligomers, now recognized as the main culprits in AD. The humanized antibodies described in the present invention advantageously maintain the same or a similar binding profile.

[145] As described in the examples below, the humanized antibody (Ab11) comprising the humanized VL and VH sequences, which characterize the claimed binding proteins, exhibits a high affinity for the target antigen (prefibrillar oligomers of Aβ42 with β structure), which is in a range comparable to that of the parental antibody ALZ-201 and similar to that of a chimera of variable regions of ALZ-201 (chALZ-201) with constant regions of human IgG1. Such affinity is appropriate for an effective therapeutic agent. The binding agents, as defined in the present invention, can therefore exhibit an affinity for the Aβ42CC antigen comparable to that of Ab11. The affinity can be conveniently measured by a surface plasmon resonance (SPR) assay, for example, by a Biacore assay, according to well-known methods in Petition 870250084592, dated 09 / 19 / 2025, pp. 130 / 194 37 / 75 state of the art. The Ae42CC peptide is described in Sandberg et al. 2010, PNAS, 107(35), 15595-15600 and in document WO2009 / 128772.

[146] As demonstrated in the examples below, other humanized antibodies tested in the development of the antibodies described in the present invention showed a slightly higher affinity. However, Ab11 was selected based on a combination of other characteristics. For example, as noted above, it exhibits a high percentage of humanity in the VH and VL sequences (88.7%). Since the VH and VL sequences of the Ab11 antibody are preserved in the antibodies described in the present invention, this beneficial property is preserved.

[147] Furthermore, as described in the Examples, Ab11 exhibits high thermal stability, characterized by a high denaturation temperature, also called melting temperature, (Tm) of 71 °C, as determined by differential scanning fluorimetry (DSF) analysis. The antibodies described in the present invention demonstrate similar thermal stability to that of Ab11. Consequently, an antibody described in the present invention may have a Tm of at least 69, 70, or 71 °C, as determined by differential scanning fluorimetry (DSF) analysis.

[148] Furthermore, as demonstrated in the Examples below, Ab11 can be obtained in high yield from a mammalian cell expression system. Consequently, in certain embodiments, the antibody described in the present invention can be expressed at a level of at least 4, 4.5 or 5 mg / mL from mammalian cells.

[149] As described above, the original antibody ALZ-201 has been shown to have a positive physiological and protective impact on the integrity and morphology of mouse neurons. The antibodies Petition 870250084592, dated 09 / 19 / 2025, pp. 131 / 194 38 / 75 described in the present invention may exhibit a similar protective effect. This can be determined in a neurotoxicity assay using brain extracts from AD patients to test their toxicity in neuronal cultures of mouse embryo neurons and examine the effect on antibody toxicity. Such an assay is described in the publication by Sandberg et al. 2022 cited above.

[150] Further details about the antibodies described in the present invention and their development are provided in the following non-limiting examples, with reference to the following figures. Brief Description of the Figures

[151] Figure 1: shows the protein purification profiles of recombinant antibodies after non-reduced SDS-PAGE analysis of the chromatographic fractions in Protein A. (A): pilot batch of chALZ-201 (Ab15). (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. Entrance. FT. Flow through. W. Washes. E. Eluted fractions.

[152] Figure 2: shows the results of reduced and non-reduced SDS-PAGE analysis of the combined final samples of each purified antibody (for quality verification purposes). (A): pilot batch of chALZ-201 (Ab15). (B): chALZ-201 (Ab15). (C): Ab1, Ab2 and Ab3. (D): Ab10 and Ab11. (E): Ab17. Coomassie blue staining was used. MW. Molecular weight marker.

[153] Figure 3: shows the average molecular weight of Ae42CC oligomers determined by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The results presented are the 280 nm normalized absorbance (ua) and molar mass (kDa) for the Ae42CC oligomers as they elute from the column. Figure adapted from Sandberg et al. 2022, Alz Res and Therapy 14:196. Petition 870250084592, dated 09 / 19 / 2025, pp. 132 / 194 39 / 75

[154] Figure 4: shows the results of the ELISA assay for the affinity of chALZ-201 or lecanemab for unstructured Aβ42, fibrillar Aβ42 and oligomeric Ae42CC. Figure adapted from Sandberg et al. 2022, Alz Res and Therapy 14:196.

[155] Figure 5: shows the results of the ELISA assay of the affinity of Ab1, Ab2, Ab3 and chALZ-201 for the Aβ42CC oligomers.

[156] Figure 6: shows the results of the ELISA assay of the affinity of Ab10, Ab11, Ab17 and chALZ-201 for the Aβ42CC oligomers.

[157] Figure 7: shows the thermal stability data Differential Scanning Fluorimetry (DSF) of antibodies, presented 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).

[158] Figure 8: shows the results of the expression comparison test for Ab10, Ab11 and chALZ-201 as expression yield in mg / L of culture. (A): Ab10 and Ab11. (B): Ab10 and chALZ-201. (C): Ab11 and chALZ201.

[159] Figure 9: shows the reduced and non-reduced SDS-PAGE analysis of the final purified Ab11 from each minipool (for quality verification purposes). 2 pg of Ab11 were applied to each strip. Coomassie blue staining was used. MW. Molecular weight marker. 1. Pool 1.2. Pool 2.

[160] Figure 10: shows the reduced and non-reduced SDS-PAGE analysis of the final purified Ab11 from each monoclone (for quality verification purposes). 2 pg of Ab11 were applied to each band. 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. Petition 870250084592, dated 09 / 19 / 2025, pp. 133 / 194 40 / 75

[161] Figure 11: shows an assessment of cell viability and stability of the three main monoclones 4H9, 1A3 and 2A6. Cells were cultured for 15 passages (30 generations), and cell density and viability were monitored at each passage.

[162] Figure 12: shows the results of agarose gel electrophoresis of PCR amplification of the VH and VL regions of Ab11 in the genome extracted from each of the three main monoclones 4H9, 1A3 and 2A6. Ethidium bromide staining was used. MW. Molecular weight marker.

[163] Figure 13: shows the reduced and non-reduced SDS-PAGE analysis of the final purified Ab11 from each monoclone submitted to cell viability and stability assessment (for quality verification purposes). 2 pg of Ab11 from cycles 5, 10 and 15 were applied to each band. Coomassie blue staining was used. MW. Molecular weight marker. 5, 10 or 15 indicate samples from passage number 5, 10 or 15.

[164] Figure 14: shows the results of agarose gel electrophoresis of the PCR-based mycoplasma test. Ethidium bromide staining was used. 1. 4H9. 2. 1A3. 3. 2A6. +. Positive control (290 bp). -. Negative control. MW. Molecular weight marker. Examples Example 1 Humanization of the ALZ-201 Antibody: Construction of an ALZ-201 Model

[165] A molecular model of the murine antibody clone ALZ201 was constructed according to established protocols. Variable heavy chain (VH) and variable light chain (VL) sequences were numbered / annotated according to Kabat and IMGT conventions to identify structural region (FW) and complementarity-determining residue (CDR) sequences.

[166] The sequences were used to search for structures of Petition 870250084592, dated 09 / 19 / 2025, pp. 134 / 194 41 / 75 murine antibodies were resolved to identify structures with the highest sequence identity to select templates for the VL and VH force field and CDRs, and these were used to construct partial models for the VH and VL. The best tertiary arrangement of the partial models was selected to construct the final model, using the PAPS (Packing Angle Prediction) server: http: / / www.bioinf.org.uk / abs / paps / . The PAPS server predicted a resolved murine antibody structure to provide the best tertiary arrangement of VH and VL, and the final model was assembled by fitting the coordinates of the main structure of the conserved anchor segments of the partial models of VH and VL to the selected murine antibody structure. Finally, the coordinates of the final model were subjected to a round of energy minimization employing GROMACS with the GROMOS96 force field.

[167] The model was used to assist the subsequent humanization project. In particular, by inspecting the model in light of the sequences of the human germline candidates for grafting, potential design errors that can arise from a design based purely on sequence considerations can be avoided. In particular, the model was used to select residues in the human FWQ that should be “backmutated” (replaced with the parental murine amino acid) in order to avoid loss of antibody stability or affinity due to structural incompatibility of FW with the grafted murine CDR sequences. In addition, the model was used to select residues from the grafted murine CDRs for replacement with human residues from the target human germline sequence (“germ-hnmg” - a process known as germline sequence-based humanization).This allows the “humanity” (or degree of humanization (human-ness)) of the resulting antibody to be increased without loss of affinity.

[168] A final challenge in humanization design is the reduction of Petition 870250084592, dated 09 / 19 / 2025, pp. 135 / 194 42 / 75 potential sequence-based liabilities, characteristic of parental murine CDRs; certain sequences are subject to chemical degradation over time and, in CDRs, this can result in loss of antibody affinity or stability. The model facilitated the selection of residue substitutions to reduce potential liabilities. Humanization project through CDR grafting of the mouse monoclonal antibody ALZ-201

[169] The murine antibody ALZ-201 was humanized by grafting the three CDRs, as defined by Kabat nomenclature, of the variable light chain (VL) region onto a human germline VL that was as homologous as possible to the murine antibody VL. Similarly, the three CDRs of the variable heavy chain (VH) region were grafted onto a human germline VH that was as homologous as possible to the murine antibody VH.

[170] The human germlines to be used as acceptor sequences for CDR grafting were selected based on sequence database searches and sequence identity comparisons. For VH, two human germlines were selected, IGHV2-5*09 and IGHV4-30-4*07. For VL, four human germlines were selected: IGKV3-11*01, IGKV320*01, IGKV1-39*01 and IGKV6-21*01.

[171] The ALZ-201 CDRs were analyzed for vulnerabilities in the following sequence (deamidation and isomerization motives).

[172] Murine VH / VL and corresponding human germline sequences were aligned and CDRs were grafted. In addition, some amino acid residues in the structural regions of the selected human germline variable regions were backmutated to the amino acid residues present Petition 870250084592, dated 09 / 19 / 2025, pp. 136 / 194 43 / 75 in the murine variable regions. Based on information about the structure of the immunoglobulin variable regions and with guidance from the previously mentioned murine monoclonal antibody ALZ-201 Fv homology molecular model, these few residues in the structural regions were identified as having key roles in maintaining the CDRs in the correct conformation or in VH / VL packaging, and therefore were retained in a humanized version (version A) or replaced by their human germline counterparts, if possible, in subsequent humanized versions.Under the guidance of the molecular homology model, in the subsequent version B, when deemed possible, CDR residues, as defined by Kabat, were also replaced by their human germline counterparts (germline) in order to increase the degree of humanity (i.e., percentage sequence identity for VH and VL between the humanized versions and the closest human germline used as the acceptor sequence for CDR grafting). The structural model, therefore, allowed the expansion of the limits of the humanization process, taking it beyond mere CDR grafting.

[173] The humanized A versions were designed as conservative versions that minimized / avoided CDR alternations. The subsequent versions B, C, and D were designed to increase the percentage of “humanity” (degree of humanization or humanness) (i.e., to achieve a higher percentage of sequence identity with the closest human germline (at least 85% or more) through germ-lining.

[174] For VH, 6 different humanized VH sequences were designed (based on two different human germlines, with 2 or 4 versions each, respectively). For VL, 8 different humanized VL sequences were designed (based on four human germlines with 2 versions each). This results in 48 Petition 870250084592, dated 09 / 19 / 2025, pp. 137 / 194 44 / 75 possible combinations between the humanized VH and VL sequences. The best combinations of VH and VL chains among the humanized VH and VL sequences were selected.

[175] The 48 combinations were then reduced to 18 based on several considerations, including % humanity and the sequence modifications introduced in the different versions B, C and D.

[176] Table 1 below shows the 18 combinations identified based on the selected humanized VH and VL sequences. Table 1 Humanized versions Heavy chain Light chain 1 ALZ201-25VHA-311VLA ALZ201-2-5-VHA ALZ201-3-11-VLA 2 ALZ201-25VHA-139VLA ALZ201-1-39-VLA 3 ALZ201-25VHA-621 VLA ALZ201-6-21-VLA 4 ALZ201-4304VHA-311 VLA ALZ201-4-30-4-VHA ALZ201-3-11-VLA 5 ALZ201-4304VHA-139 VLA ALZ201-1-39-VLA 6 ALZ201-4304VHA-621 VLA ALZ201-6-21-VLA 7 ALZ201-4304VHB-311 VLA ALZ201-4-30-4-VHC ALZ201-3-11-VLA 8 ALZ201-4304VHB-139VLA ALZ201-1-39-VLA 9 ALZ201-4304VHB-621 VLA ALZ201-6-21-VLA 10 ALZ201-25VHA-311VLBA ALZ201-2-5-VHA ALZ201-3-11-VLB 11 ALZ201-25VHA-139VLBA ALZ201-1-39-VLB 12 ALZ201-25VHA-621 VLBA ALZ201-6-21-VLB 13 ALZ201-4304VHA-311 VLA ALZ201-3-11-VLB 14 ALZ201 -4304VH A-139 VLA ALZ201-4-30-4-VHA ALZ201-1-39-VLB 15 ALZ201-4304VHA-621 VLA ALZ201-6-21-VLB 16 ALZ201-4304VHB-311 VLA ALZ201-4-30-4-VHC ALZ201-3-11-VLB 17 ALZ201-4304VHB-139VLA ALZ201-1-39-VLB 18 ALZ201-4304VHB-621 VLA ALZ201-6-21-VLB

[177] Of the 18 combinations, 16 were selected for further study (omitting humanized antibody variants 15 and 18)

[178] The various combinations were then subjected to evaluation and experimental testing, as described in the Examples below, based on the following criteria: Petition 870250084592, dated 09 / 19 / 2025, pp. 138 / 194 45 / 75 - Transient expression level in mammalian cells as human IgG1 / kappa antibodies; - Connection capacity (EC50 by ELISA, or Kd by Biacore); - Determination of biophysical properties, particularly differential scanning fluorimetry (DSF) analysis to determine the Tm of FAB, CH2 and CH3. Example 2 Vector Design and Recombinant Production: Chimeric vector design ALZ-201 (chALZ-201)

[179] From sequences of the variable regions of heavy chain (HC) and light chain (LC) of the murine antibody mAb20 (WO2009128772A1), a chimeric full-length human IgG1 antibody with kappa light chain (chALZ-201) was developed and used as a control reference for humanized variants of the parental murine antibody. The cDNA encoding HC and LC was chemically synthesized with optimization for expression in CHO cells. The coding sequences of the signaling peptides were added at the 5' / N-ter position. The two cDNA sequences constructed for recombinant expression of chALZ201 in CHO cells are shown in SEQ ID NOs: 9 and 10.

[180] chALZ-201 HC cDNA (SEQ ID NO: 9): GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTG GTGGCCGCCCCTCGGTGGGTTCTGAGCCAGGTGACCCTGAAGGAGTCCG GCCCTGGCATCAGCCAGCCTAGCCAGACCCTGAGCCTGACCTTGTTCCTTT AGCGGCTTCTCCCTGAGCACCTTTGGCTCGCCGGCCG GCCTAGCGGCAAGGGCCTGGAGTGGCTGGCTCACATCTACTGGGACGAC GATAAGCACTACATCCCAGCCTGAAGTCCCGGCTGACCATCAAGCAAGGA TACCAGCAACAATCAGGTGTTTCTGAAGATCACCACCGTGGACACCGCGC Petition 870250084592, of 19 / 09 / 2025, p. 139 / 194 46 / 75 ATACCGCCACCTATTTCTGCGCCCGGCGGGAGAGCCACTACTATGGCTCC GGCTACTACTTCGATTACTGGGGCCAGGGCACCACCCTGACCGTGTCCTC CGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTC CACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCC CAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTG CATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCC GTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAAC GTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAA GAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGC TGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGA TGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCAT GAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCA TAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGG TGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAG TACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAAC CATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGC CCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTG GTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGG CCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGC AGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCAC TATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGAGCGGCCGC.

[181] cDNA from LC of chALZ-201 (SEQ ID NO: 10): GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAG CCTGCTGCTGTGGATCTCCGGCGCCTATGGCCAGATCGTGCTGACCCAGT CCCCTGCTATCCTGTCCCAGCCCCGGCGAGAAGGTGACCATGACCATGC AGGGCCAGCAGCAGGTGAGCT GCAGCAGCCCCAAGCCCTGGATCTACGCTACCAGCAACCTGGCTTCCGG Petition 870250084592, of 19 / 09 / 2025, p. 140 / 194 47 / 75 CGTGCCTGCCAGGTTTAGCGGCTCCGGCTCCGGCACCTCCTATTCCCTGA CCATCTCCCGGGTGGAGGCCGAGGATGCCGCTACCTACTATTGTCAGCAG TGGAGGTCCGACCCCCTGACCTTCGGCGCTGGCACCAAGCTGGAGCTGA AGCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGC AGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACC CCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGG TAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACT CCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAA GTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAA AAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC.

[182] The tiny nucleic acid sequence, “gccgccacc”, present in SEQ ID NOs: 9 and 10, is the Kozak sequence, which functions as a protein translation initiation site, and the sequence “GAATTC” immediately preceding it is the Eco R1 restriction enzyme site. The two cDNA sequences were subcloned, using the Eco R1 enzyme, into the pXten1 mammalian cell expression vector (ProteoGenix, Schiltigheim, France). The expected protein sequence for the chALZ201 HC antibody is therefore shown in SEQ ID NO: 11.

[183] ​​HC of chALZ-201 (SEQ ID NO: 11): MKHLWFFLLLVAAPRWVLSQVTLKESGPGISQPSQTLSLTCSFS GFSLSTFGSGVSWIRQPSGKGLEWLAHIYWDDDKHYNPSLKSRLTISKDTSN NQVFLKITTVDTADTATYFCARRESHYYGSGYYFDYWGQGTTLTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY Petition 870250084592, dated 09 / 19 / 2025, pp. 141 / 194 48 / 75 TQKSLSLSPGK.

[184] In SEQ ID NO: 11, amino acids 1 to 19 are a secretion signal that is cleaved during protein translation, amino acids 20 to 143 comprise the VH domain and amino acids 144 to 473 comprise the constant heavy chain domain of IgG1.

[185] On the other hand, the expected protein sequence for the chALZ-201 LC antibody is presented in SEQ ID NO: 12.

[186] chALZ-201 LC (SEQ ID NO: 12): MVLQTQVFISLLLWISGAYGQIVLTQSPAILSSSPGEKVTMTCRA SSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRV EAEDAATYYCQQWRSDPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[187] In SEQ ID NO: 12, amino acids 1 to 20 are a secretion signal that is cleaved during protein translation, amino acids 21 to 126 comprise the VH domain and amino acids 127 to 233 comprise the constant light chain domain of IgG1. Vector design, and antibody variants 10 (Ab10) and 11 (Ab11)

[188] For Ab10 and Ab11, designed using in silico CDR grafting, as described above, the cDNA encoding the VH and VL chains of the antibodies was chemically synthesized with optimization for expression in CHO cells. The coding sequences of the signaling peptides were added at the 5' / N-ter position.

[189] The VH sequence is identical for both antibodies. The two cDNA sequences constructed for the expression of recombinant VH antibodies from Ab10 and VH from Ab11 in CHO cells are shown in SEQ ID NO: 13.

[190] Ab10 VH cDNA and Ab11 VH cDNA (SEQ ID NO: Petition 870250084592, dated 09 / 19 / 2025, pp. 142 / 194 49 / 75 13): GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTG GTGGCTGCTCCTCGGTGGGTGCTGTCCCAGGTGACCCTGAAGGAGTCCG GCCCCACCCTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTC AGCGGCTTTAGCCTGAGCACCTTTGGCAGCGGCGTGAGCTGGATCAGGC AGCCTCCCGGCAAGGCCCTGGAGTGGCTGGCTCACATCTATTGGGACGA CGACAAGCACTATAACCCTAGCCTGAAGAGCCGGCTGACCATCACCAAGG ACACCAGCAAGAACCAGGTGGTGCTGACCATCACAAACATGGACCCTGTG GATACCGCCACCTATTTTTGCGCCCGGAGGGAGAGCCACTACTATGGCAG CGGCTACTATTTCGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCA GC.

[191] As above, the lowercase nucleic acid sequence “gccgccacc” in SEQ ID NO: 13 is the Kozak sequence that functions as the protein translation initiation site, and the immediately preceding “GAATTC” sequence is the EcoR1 restriction enzyme site. The two cDNA sequences were subcloned, using the Eco R1 enzyme, into the pXten1 mammalian cell expression vector (ProteoGenix, Schiltigheim, France) containing the main structure sequence for the human IgG1 heavy chain constant region described above. The expected complete protein sequence for HC of the humanized antibodies Ab10 and Ab11 is therefore shown in SEQ ID NO: 7.

[192] Ab10 HC and Ab11 HC (SEQ ID NO: 7): MKHLWFFLLLVAAPRWVLSQVTLKESGPTLVKPTQTLTLTCTFS GFSLSTFGSGVSWIRQPPGKALEWLAHIYWDDDKHYNPSLKSRLTITKDTSKN QVVLTITNMDPVDTATYFCARRESHYYGSGYYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD Petition 870250084592, dated 09 / 19 / 2025, pp. 143 / 194 50 / 75 GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK.

[193] In SEQ ID NO: 7, amino acids 1 to 19 are a secretory signal that is cleaved during protein translation, amino acids 21 to 143 comprise the VH domain and amino acids 144 to 473 comprise the constant heavy chain domain of IgG1.

[194] The cDNA sequence constructed for recombinant VL antibody expression of Ab10 is presented in SEQ ID NO: 14.

[195] cDNA to VL of Ab10 (SEQ ID NO: 14): GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCCTC CCTGCTGCTGTGGATCAGGCGCGCTTATGGCGAGATCGTGCTGACCCAGA GCCCCGCCACCCTGTCCCTGAGCCCAGGAGAGCGGGCTACCCTGAGCTG TCGGGCCTCCTCCAGCGTGTCCTACATGCAGCCTAGCCTT GCCAGGCTCCTCGGCCCTGGATCTATGCCACCAGCAACCTGGCCACCGG CATCCCCGCCAGGTTCTCCGGAAGCGGCTCCGGAACCGATTTTACCCTGA CCATCTCCAGCCTGGAGCCTGAGGACTTTGCCGTGTACTACTGCCAGCAG TGGAGGAGCGATCCTCTGACCTTTAGCCAG GGCGAGCGAGGGATC.

[196] The cDNA sequence constructed for recombinant antibody expression of the VL of Ab11 is presented in SEQ ID NO: 15.

[197] cDNA and VL of Ab11 (SEQ ID NO: 15): GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAG CCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGT CCCCTTCCAGCCTGAGCCAGCGTGGGCGACAGGGTGACCATCAGCCTG GCAAGGCTCCCAAGCCTTGGATCTACGCTACCAGCAATCTGGCTAGCGGC Petition 870250084592, of 19 / 09 / 2025, p. 144 / 194 51 / 75 GTGCCTAGCCGGTTCTCCGGCTCCGGATCCGGCACCGACTTCACCCTGAC CATCTCCTCCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTG GCGGTCCGATCCCCTGACCTTCGGCGGCGGAACCAAGGTGGAGATCAAG.

[198] As mentioned above, the lowercase nucleic acid sequence “gccgccacc” in SEQ ID NOs: 14 and 15 is the Kozak sequence that functions as the protein translation initiation site, and the immediately preceding “GAATTC” sequence is the Eco R1 restriction enzyme site. The two cDNA sequences were subcloned, using the Eco R1 enzyme, into the pXten1 mammalian cell expression vector (ProteoGenix, Schiltigheim, France) containing the main structure sequence for the human IgG1 light chain constant region described above. The expected complete protein sequences for LC of the humanized antibodies Ab10 and Ab11 are therefore shown in SEQ ID NOs: 16 and 8.

[199] Ab10 LC (SEQ ID NO: 16): MVLQTQVFISLLLWISGAYGEIVLTQSPATLSLSPGERATLSCRA SSSVSYMHWYQQKPGQAPRPWIYATSNLATGIPARFSGSGSGTDFTLTISSLE PEDFAVYYCQQWRSDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[200] Ab11 LC (SEQ ID NO: 8): MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCRA SSSISYMHWYQQKPGKAPKPWIYATSNLASGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQWRSDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[201] In SEQ ID NOs: 16 and 8, amino acids 1 to 20 are a secretory signal that is cleaved during protein translation, amino acids 21 to 126 comprise two VL domains, and amino acids 127 Petition 870250084592, dated 09 / 19 / 2025, pages 145 / 194 52 / 75 to 233 comprise the constant light chain domain of IgG1.

[202] The humanized variants Ab10 and Ab11 of ALZ-201 exhibit 88.7% sequence identity with a homologous human IgG. All other humanized ALZ-201 candidates described in the present invention have been similarly subcloned for expression in CHO cells. These are designated Ab1 (87.6% human residue), Ab2 (87.6% human residue), Ab3 (86.1% human residue), and Ab17 (85.6% human residue). Transient expression in Chinese hamster ovary cells

[203] XtenCHO cells were used for transient expression of the antibodies described herein (PX-XTE-004; ProteoGenix, Schiltigheim, France). This cell line is derived from the CHO-K1 Chinese hamster ovary cell line. Vials containing 1 mL of cell suspensions frozen at 1 χ¹⁰⁷ cells / mL in XtenCHO expression medium (PX-XTE-002; ProteoGenix, Schiltigheim, France) with 8 mM L-glutamine, 0.5% anti-caking agent (0010057AE; Gibco / Thermo Fisher Scientific, USA) and 10% DMSO were thawed for a maximum of 90 seconds by submerging and gently swirling the vial in XtenCHO expression medium at 37 °C until only a small amount of ice remained. The vials were decontaminated with 70% ethanol and transferred to a laminar flow hood. The contents were transferred to 8 mL of pre-warmed XtenCHO Expression Medium, supplemented with 8 mM L-glutamine, and centrifuged at 300 xg for 5 minutes.The supernatant solution was discarded and the cells resuspended in 2 mL of XtenCHO Expression medium with 8 mM L-glutamine, and the number of viable cells and viability were determined using an automated cell counter (Countstar Biotech, Model IC 1000), using the default cell counting mode settings. Briefly, a sample of the cell suspension was removed from... Petition 870250084592, dated 09 / 19 / 2025, pp. 146 / 194 53 / 75 vial and diluted 1:1 with 0.4% trypan blue stain. After gently mixing, 20 μL of the sample mixture were added to the chamber port on a cell counting chamber slide and inserted into the automated cell counter. Three horizons (upper, middle, and lower) were chosen for counting, and the system then calculated the average cell density and viability.

[204] A sterile, vented, disposable Erlenmeyer shaker flask (TAB-012-125; Guangzhou Jet Bio-Filtration Co., Guangzhou, China) containing 30 mL of XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C was seeded at a density of 0.2 to 0.3 x 106 cells / mL and an anti-caking agent was added to a final concentration of 0.5%. The cells were incubated at 37 °C with relative humidity > 80% and 5% CO2 on an orbital shaker platform (IS-RDS6C5 Incubator, Crystal Technology & Industries, USA).

[205] When the cells reached 1.5 to 2.5 χ 106 viable cells / mL, typically 2-3 days after thawing, the cells were subcultured. The culture was first centrifuged at 300 χ g for 5 minutes and the supernatant solution discarded. The cells were then transferred to 30 mL of XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C to a density of 0.2 - 0.3 χ 106 cells / mL and an anti-caking agent was added to a final concentration of 0.5%. The culture was returned to incubation at 37 °C with > 80% relative humidity and 5% CO2 on an orbital shaker platform. When the cells again reached 1.5 to 2.5 χ¹⁰⁶ viable cells / mL, the process was repeated at least once before transfection. Transfection was never performed on cells that had undergone more than 20 passages.

[206] The day before transfection, cells were subcultured in XtenCHO Expression Medium with 8 mM L-glutamine, but Petition 870250084592, dated 09 / 19 / 2025, pp. 147 / 194 54 / 75 without the anti-caking agent, using the subculture procedure described above. After 24 h, the cells typically reached a density of 2 to 3 χ¹⁰⁶ viable cells / mL, with a viability of > 90%. The culture was centrifuged at 300 χ¹⁰ g for 5 minutes and the supernatant solution discarded. The cells were then co-transfected with pXtenl plasmids carrying the respective cDNA sequences for the HC and LC of each antibody using the following protocol. The cells were first transferred to 15 mL of XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C to a density of 5 χ¹⁰⁶ viable cells / mL. 48 pg of each expression plasmid, pXten1, containing the cDNA sequences for the HC and LC ALZ-201 antibody derivative to be expressed, were added to the cells, and 144 pL of XtenFect reagent Working Solution (PX-XTE-003; ProteoGenix, Schiltigheim, France) were added dropwise, under gentle agitation of the cells.After incubating the cells at 37 °C with relative humidity >80% and 5% CO2 on an orbital shaker platform for 2 h, an additional 15 mL of XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C was added to the cells, along with 96 pL of XtenCHO Enhancer Solution (PX-XTE-003; ProteoGenix, Schiltigheim, France). The cells were then returned to 37 °C with >80% relative humidity and 5% CO2 on an orbital shaker platform.

[207] 24 hours after transfection, the anti-caking agent was added to a final concentration of 0.5% and the temperature was reduced to 33 °C. Cell density and cell viability were checked regularly during this expression phase.

[208] For initial expression analyses of all humanized ALZ-201 antibodies and the reference chALZ-201 described herein, cells from 30 mL cultures were collected when viability fell below 50% on day 14 post-transfection by centrifugation at 300 xg for 5 Petition 870250084592, dated 09 / 19 / 2025, pp. 148 / 194 55 / 75 minutes. The supernatant solution was removed and clarified by centrifugation at 5,000 x g for 30 minutes and passed through a sterile Millex-GP 0.22 μm filter (SLMPL25SS; Merck KGaA, Darmstadt, Germany).

[209] The antibodies were then purified using Protein A. In summary, Protein A chromatography resin (Mabselect SuRe LX #17547402; Cytiva, USA) was soaked in 0.5 M NaOH for 30 minutes, then washed and equilibrated with phosphate-buffered saline at pH 7.5 (PBS: 10 mM Na2HPO4, 2.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl). Supernatant solutions from the cell cultures were added and binding was allowed for 4 hours at room temperature. After washing the beads with PBS pH 7.5 at 50 times the bead volume, the bound antibodies were eluted with 20 mM 2-hydroxypropane-1,2,3tricarboxylic acid (citric acid) buffer solution at pH 2.7. The eluted fractions were neutralized with 1M tris-(hydroxymethyl)aminomethane with HCl (Tris-HCl) at pH 9.0.

[210] Antibody-containing fractions were confirmed by polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS) using the Mini-PROTEAN Tetra vertical electrophoresis system (1658000FC; Bio-Rad, USA) and Tris-HCl gels (8% and 12%) with Tris-Glycine as running buffer (25 mM Tris-HCl, 200 mM Glycine, 0.1% w / v SDS, pH 8.3). 10 μL samples were mixed in a 4:1 ratio with loading buffer (10% w / v SDS, 20% v / v glycerol, 0.2 M Tris-HCl pH 6.8, 0.05% w / v Bromophenolblue) with or without a reducing agent (10 mM beta-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 gels were subjected to electrophoresis at 120 V and stained with Coomassie brilliant blue G-250.

[211] The antibody-containing fractions were pooled and the buffer was changed to PBS pH 7.5 by dialysis against 200x the sample volume. Petition 870250084592, dated 09 / 19 / 2025, pp. 149 / 194 56 / 75 using 3.5K MWCO snakeskin tubes (88244; Thermo Scientific, USA). Dialysis was performed overnight (for approximately 16 h) at 4 °C under continuous agitation, after which the dialysis buffer was changed and dialysis continued for a further 3 h at 4 °C. Samples were then filtered through a 0.22 µm Millex syringe filter (SLGV013SL or SLGV004SL depending on the final sample volume; Merck KGaA, Germany) and analyzed for endotoxin levels using an endotoxin assay kit (ToxinSensor® Chromogenic LAL Endotoxin Assay Kit; No. L00350; Genscript, USA). The pooled final sample of each purified antibody was quality checked by SDS-PAGE as described above.

[212] A pilot batch of 30 mL of chALZ-201 (also named Ab15) indicated that the antibody was well expressed in XtenCHO cells, and Ab10 and Ab11 were subsequently expressed in parallel with a second batch of chALZ-201, in addition to the humanized variants Ab1, Ab2, Ab3, and Ab17. Figure 1 shows the SDS-PAGE analyses of the Protein A chromatographic fractions, and the SDS-PAGE analyses of the pooled samples are shown in Figure 2. All antibodies were found to conform to expected sizes, where IgG antibodies with reduced disulfide bonds typically migrate as approximately 50 kDa and 25 kDa species in SDS-PAGE gels, corresponding to the heavy and light chains, respectively. Under non-reducing conditions, on the other hand, IgG antibodies give rise to a single band in SDSPAGE gels, with a size of at least 150 kDa.

[213] Based on the SDS-PAGE results, it was estimated that all antibodies expressed here had a purity >90%. Antibody concentration was determined by UV spectrophotometry at 280 nm, using an extinction coefficient of 210,000 M-1cm-1. The expression results of the 30 mL cell culture batches are presented in Table 2. It was verified- Petition 870250084592, dated 09 / 19 / 2025, pp. 150 / 194 57 / 75 shows that Ab11 and Ab17 were very well expressed, while Ab10 was poorly expressed compared to all derivatives. Table 2 CHO Cell Culture Expression Test in 30 mL Antibody Yield (mg / 30mL) Concentration (mg / mL) Estimated Purity chALZ-201 Pilot 4.05 0.90 >90% chALZ-201 3.33 1.34 >90% Ab1 2.22 0.60 >90% Antibody Yield (mg / 30mL) Concentration (mg / mL) Estimated Purity Ab2 2.78 0.78 >90% Ab3 2.33 0.76 >90% Ab10 1.09 0.87 >90% Ab11 5.37 1.85 >90% Ab17 5.15 1.65 >90% Example 3 Binding Affinities Amyloid-beta (Ab) peptide preparations

[214] To evaluate reactivity towards the unstructured monomeric antigen, synthetic Αβ42 peptides (H-1368; Bachem, Switzerland) were reconstituted at 1 mg / mL in 0.1 M aqueous ammonia solution (pH 9) and used within 2 hours of preparation. The fibrillar form of the antigen was obtained by reconstituting the lyophilized peptide (H-1368; Bachem, Switzerland) at 1.0 mg / mL in PBS with 0.02% azide and then shaking the solution for 55 hours at 700 rpm and 37 °C. The fibrils were then incubated for 90 hours without agitation at room temperature before being frozen at -20 °C. Frozen vials of fibrillar Αβ42 were thawed immediately before use.

[215] The Aβ42CC peptide is a non-fibrillogenic derivative of the Aβ42 peptide with two alanine-cysteine ​​substitutions at amino acid positions 21 and 30, and an intramolecular disulfide bond connecting the two (WO2009128772A1; Sandberg, A. et al., 2010, Proc. Natl. Acad. Sci. Petition 870250084592, dated 09 / 19 / 2025, pp. 151 / 194 58 / 75 (USA, 107:15595-600). An oligomeric form of this peptide was used in the development of murine ALZ-201 (WO2012120035A1; Sandberg, A. et al., 2022, Alz. Res. Therapy 14:196). The synthetic Ae42CC used in this study was custom-made by solid-state peptide synthesis and purified to 95% by reverse-phase HPLC using standard methods and practices (AmbioPharm Inc., North Augusta, SC, USA). Oligomeric peptide preparations were obtained by initially dissolving the peptides at pH 10.0 to 10.4 and then allowing them to oligomerize in PBS. The oligomers were then frozen to prevent further oligomerization. Frozen vials of oligomeric Aβ42CC were thawed immediately before use.

[216] The size of the Aβ42CC oligomers used in the present invention was determined by size exclusion HPLC (1100 Series; Agilent Technologies, Santa Clara, CA, USA), where a 100 pL sample was injected into a 7.8 x 300 mm TSK-GEL® G4000SWxl column (Tosoh, Tokyo, Japan) with 20 mM sodium phosphate buffer, 150 mM NaCl, pH 7.4, as running buffer. The flow rate was 0.6 mL / min. 280 nm UV and multi-angle light scattering (MALS; MiniDAWN Treos from Wyatt Technology Corporation, Santa Barbara, CA, USA) were used to determine the weight-average molecular weight using ASTRA 6.1 software (Wyatt Technology). The oligomers used had an average molecular weight of 702 ± 3.5 kDa and an oligomer content >94% (Figure 3).

[217] Monomeric and fibrillar Aβ42 concentrations were inferred from the net peptide content determined by the peptide manufacturer (Bachem, Bubendorf, Switzerland), while the Aβ42CC concentration was determined by ultraviolet (UV) spectroscopy using an extinction coefficient of 1401 cm-1M-1 for the difference in absorbance at 280 Petition 870250084592, dated 09 / 19 / 2025, pp. 152 / 194 59 / 75 nm and 300 nm. Antibody specificity using ELISA

[218] The antigenic specificities of chimeric and humanized derivatives of ALZ-201 were evaluated using ELISA to ensure that the unique binding characteristics of the original antibody were preserved.

[219] 96-well Maxisorp™ Nunc ELISA plates (44-240421; Thermo Fisher Scientific, Waltham, MA, USA) were coated with 5 μg / mL of antigen (100 μL / well) for 2 ha at 37 °C. Monomeric Αβ42 was coated at high pH to maintain its random spiral conformation. Plates were blocked with 1% BSA in PBS (150 μL / well) for 40 min at 37 °C. Washing between steps was performed by washing three times with PBS supplemented with 0.05% Tween-20 (300 μL / well). Primary antibodies were diluted from 1000 ng / mL to 0.46 ng / mL with 0.1% BSA in PBS, added at 100 μL / well, and incubated for 1.5 h at room temperature. After washing, a secondary monoclonal antibody conjugated with HRP was added at 1000 ng / mL (100 μL / well), and the plates were incubated for 45 min at 37 °C. The plates were washed again, and then the substrate 3,3',5,5'-tetramethylbenzidine was added (100 μL / well), and the plates were incubated for 5 to 10 min at 37 °C.The reaction was stopped with 2 M HCl (50 μL per well). The difference in absorbance at 450 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 ​​and extracting half the maximum effective antibody concentration (EC50).

[220] The ELISA results for chALZ-201 against unstructured Aβ42, fibrillar Aβ42 and oligomeric Aβ42CC are shown in Figure 4. There was no binding to unstructured or fibrillar Aβ42, but there was strong Petition 870250084592, dated 09 / 19 / 2025, pp. 153 / 194 60 / 75 binding with oligomeric Ae42CC, indicating that the chALZ-201 antibody maintained the specificity of the original antibody for the oligomeric antigen, as expected. In contrast, the lecanemab antibody (an exact sequence copy of the BAN2401 / lecanemab antibody expressed in XtenCHO cells; PX-TA1746; ProteoGenix) was used here as a positive control and demonstrated nearly equal binding affinity to all three different forms of the Aβ peptide in the forward ELISA configuration used in this example.

[221] The EC50 values ​​obtained from the best-fit equation are shown in Table 3. Here, the EC50 value is the average of two measurements. The standard deviation (SD) shown in the table is the average of the two standard errors used to determine the individual EC50 values ​​(calculated as SD = √((sd1² + sd2²) / 2), where sd1 and sd2 are the two standard errors). Table 3 Concentration Required to Induce 50% of the Maximum Binding (EC50) of chALZ-201 and Lecanemab Bound to Different Ab42 Peptide Derivatives in an ELISA Assay Anticopo EC50 ± DP (ng / mL) Ae42CC Oligomers Aβ42 Monomers Aβ42 Fibrils chALZ-201 28±1 No binding No binding Lecanemab copy 59±17 89±11 114±26 Antibody affinities using ELISA and surface plasmon resonance (SPR)

[222] The fact that humanized variants retain their affinities with the target antigen was evaluated with ELISA analyses, as well as with SPR techniques using Biacore™.

[223] The ELISA protocol here was similar to that described above, although only Ae42CC oligomers were used as antigen in this example. The antibodies Ab1, Ab2, Ab3, Ab10, Ab11, Ab17 and chALZ-201 were evaluated. The results are shown in Figures 5 and 6, with the values ​​of Petition 870250084592, dated 09 / 19 / 2025, pp. 154 / 194 61 / 75 EC50 values ​​obtained are shown in Table 4. One of the experiments with the Ab3 antibody did not yield data that could be easily fitted to the Hill equation, leading to substantial errors in the EC50 determination. The affinities of chALZ-201 and the humanized derivatives for the oligomeric antigen Ae42CC are within the same range, indicating that the affinity for the antigen is maintained after humanization. Table 4 Concentration that induces 50% of the maximum effect (EC50) of antibodies that bind to β42OO oligomers by ELISA assay. Plate Antibody EC50 ± SD (ng / mL) Ab1 76.6 ± 22.3 Plate 1 Ab2 54.2 ± 20.2 Ab3 74.7 ± 121.8 chALZ-201 66.4 ± 11.0 Ab10 72.3 ± 8.7 Plate 2 Ab11 92.3 ± 9.1 chALZ-201 62.6 ± 11.4 Plate 3 Ab17 41.6 ± 7.5 chALZ-201 82.1 ± 27.2

[224] SPR affinity determination was performed using a Biacore™8k instrument. SPR analyzes biomolecular interactions in real time, providing quantitative measurements of reaction kinetics and affinity constants. SPR has proven to be the preferred method for studies requiring sensitive and reliable detection of antibody binding rate constants (Yang, D. et al., 2016, Anal. Biochem. 508:78-96). A human IgG anti-Fc antibody (BR-1008-39; GE Healthcare, USA) was coupled 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). Healthcare, USA). The running buffer was HBS-EP+ (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.01% Surfactant P20), and the regeneration buffer was Glycine pH 1.5. The chALZ-201 antibody and the antibodies Petition 870250084592, dated 09 / 19 / 2025, pages 155 / 194 62 / 75 humanized Ab1, Ab2, Ab10, Ab11, and Ab17 antigens were diluted in running buffer and captured by human anti-Fc antibody. The oligomer antigen in running buffer was carried over the sensor chip at defined oligomer concentrations (calculated using the 702 kDa Mw data obtained by SEC-MALS in Figure 3) ranging from 1.25 nM to 80 nM, and the response was captured over time and the data from a reference channel subtracted. The sensor chip was washed with regeneration buffer between each concentration. Association and dissociation rates were obtained from the sensorgrams, and the kinetic parameters of association (association constant, ka) and dissociation (dissociation constant, kd) were calculated, along with the dissociation equilibrium constant (binding constant, Kd), using the BIAevaluation software (Biacore™). The results are presented in Table 5, demonstrating that all antibodies derived from ALZ-201 are high-affinity ligands. Table 5 Kinetic Binding Parameters Determined by SPR Antibody ka (M-1 s-1) kd (s-1) kd (M) Ab1 4.87 x 10⁵ 6.92 x 10⁻⁴ 1.42 x 10⁻⁹ Ab2 4.89 x 10⁵ 4.30 x 10⁻⁴ 8.80 x 10⁻¹⁰ Ab10 5.37 x 10⁵ 3.06 x 10⁻⁴ 5.70 x 10⁻¹⁰ Ab11 2.99 x 10⁵ 4.67 x 10⁻⁴ 1.56 x 10⁻⁹ Ab17 6.49 x 10⁵ 1.85 x 10⁻⁴ 2.85 x 10⁻¹⁰ chALZ-201 4.49 x 10⁵ 6.79 x 10⁻⁴ 1.51 x 10-9 Example 4 Thermal Stability

[225] The tendency of biological macromolecules to resist thermal denaturation is commonly used as a rough measure of their inherent stability to 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 changes in the intrinsic fluorescence of Petition 870250084592, dated 09 / 19 / 2025, pp. 156 / 194 63 / 75 protein as it unfolds. Protein unfolding typically exposes fluorescent groups, such as the side chains of the amino acids tryptophan and tyrosine, to water molecules that inhibit fluorescence.

[226] Differential scanning fluorimetry (DSF) was used here to measure the temperature at which each antibody unfolds, Tm, taken as the inflection point of the change in the fluorescence ratio at 350 nm and 330 nm (F350 / F330) as a function of temperature. 50 μL samples were analyzed undiluted in PBS pH 7.5 at the concentrations indicated in Table 2. The instrument used was a Prometheus NT.48 nanoDSF (NanoTemper Technologies GmbH, Germany) operated at a scan rate of 1 °C / min from 40 °C to 90 °C, and fluorescence at 350 nm and 330 nm collected at a rate of 10 data points per minute. The data are shown as the first derivative of the F350 / F330 ratio (dF / dT) in Figures 7A and 7B, and the thermal denaturation points (Tm) extracted in Table 6. All antibodies exhibit a transition, Tm1, within a temperature range of 67 to 71 °C. Antibodies Ab2, Ab11, and Ab17 also exhibit a second transition, Tm2, ​​around 76 to 78 °C.The two distinct unfolding events of antibodies Ab2, Ab11, and Ab17 are likely a reflection of different thermal stabilities of the Fab and Fc domains. Antibodies Ab2, Ab11, and Ab17 exhibited the highest thermostabilities, where the pretransitional baselines for Ab11 and Ab17 indicated a slightly later onset of denaturation compared to Ab2. Table 6 Thermal Denaturation Points Determined by Differential Scanning Fluorimetry Antibody Tm1 (°C) Tm2 (°C) Ab1 67 — Ab2 71 78 Petition 870250084592, dated 09 / 19 / 2025, pp. 157 / 194 64 / 75 Antibody Tm 1 (°C) Tm 2 (°C) Ab3 69 — Ab10 69 — Ab11 71 78 Ab17 71 76 chALZ-201 67 — Example 5 Comparison Test of the Expression of Ab10, Ab11 and chALZ-201

[227] It was unexpected that Ab10 had a much lower expression level than Ab11, and this was confirmed here by transient expression in CHO cells with quantification using a Sartorius Octet RED96 device (FortéBio / Sartorius, USA).

[228] Gene synthesis, cloning, transfection, and recombinant production of Ab10, Ab11, and chALZ-201 were performed as described in Example 2, with the following modifications made during transfection and expression. Before transfection, cells were transferred to 7.5 mL of XtenCHO Expression Medium with 8 mM L-glutamine at 37 °C at a density of 5 χ 106 viable cells / mL. 24 pg of each expression plasmid (pXten1), containing the respective HC and LC domains of the antibodies Ab10, Ab11, or chALZ-201, were added to the cells, and 106 pL of XtenFect Working Solution (PX-XTE-003; ProteoGenix, Schiltigheim, France) were added dropwise under gentle agitation of the cells.After incubating the cells at 37°C with >80% relative humidity and 5% CO2 on an orbital shaker platform for 2 hours, an additional 7.5 mL of XtenCHO Expression Medium with 8 mM L-glutamine at 37°C was added to the cells, along with 48 µL of XtenCHO Enhancer Solution (PX-XTE-003; ProteoGenix, Schiltigheim, France). The cells were then returned to 37°C with >80% relative humidity and 5% CO2 on an orbital shaker platform. 24 hours after transfection, the anti-caking agent was added to a final concentration of 0.5%, and the temperature was reduced to 33°C. Petition 870250084592, dated 09 / 19 / 2025, pages 158 / 194 Cell density and viability were regularly checked during this expression phase (65 / 75).

[229] Three separate transfections were performed in parallel for each antibody construct. 200 μL of culture medium were collected on days 4, 7, 10, and 14, and the IgG1 concentration was determined using an Octet RED96 device equipped with a G Protein Biosensor (18-5082; FortéBio / 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. Using the quantification experiment module of the Octet system's data acquisition software, the binding rates of Ab10, Ab11, and chALZ-201 to the G Protein Biosensor were measured. Higher antibody concentrations result in faster binding rates. The data were analyzed using Octet Data Analysis HT software, and the calculated concentrations for each rate were based on values ​​from a standard human IgG1 curve (FHJ92850; Antibody Systems).The reference standard was prepared in PBS and analyzed in parallel at 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 1 / 20 in PBS before analysis. The sample agitation speed was 1,000 rpm, the quantification time was set to 300 seconds, the temperature to 30 °C, and the data acquisition time to 5 Hz.

[230] The results are presented as expression yield in mg / L of culture in Figure 8, where antibodies are compared in pairs in different panels for clarity. Pairwise statistical comparisons for each data point are presented in Table 7. Here, the difference between the means observed in two independent samples is reported with significance values ​​(P-values) and 95% confidence intervals (CI) of the difference. The P-value is the Petition 870250084592, dated 09 / 19 / 2025, pp. 159 / 194 66 / 75 probability of obtaining the observed difference between the samples if the null hypothesis were true. The null hypothesis is the hypothesis that the difference is 0. Table 7 Statistical Comparison of Differences in Expression Levels of Antibodies in CHO Cells Comparison Day P Diff ± SE 95% CI 4 0.0004 42 ± 3.9 32.2469 to 53.7531 Ab10 - Ab11 7 <0.0001 77 ± 3.7 66.7359 to 87.2641 10 0.0045 109 ± 18.9 56.4917 to 161.5083 14 0.0069 130 ± 25.4 59.3960 to 200.6040 Comparison Day P Diff ± SE 95% CI 4 0.0444 27 ± 9.3 1.1030 to 52.8970 Ab10 - chALZ-2017 0.0032 79 ± 12.5 44.3964 to 113.6039 10 0.049 89 ± 31.9 -0.5212 to 177.4788 14 0.0022 121 ± 17.4 72.7772 to 169.2228 4 0.1443 -16 ± 8.8 -40.5209 to 8.5209 Ab11 - chALZ-201 7 0.8791 2 ± 12.3 -32.2678 to 36.2678 10 0.5337 -20 ± 29.4 -101.6262 to 61.6262 14 0.6852 -9 ± 20.6 -66.2828 to 48.2828 Example 6 Generation of Stable Monoclonal CHO Cell Lines Expressing Ab11 Vector design used for stable transfection.

[231] For the development of a stable CHO cell line expressing the target antibody Ab11, the cDNA sequence from VH with SEQ ID NO: 13 and the cDNA sequence from VL with SEQ ID NO: 15 were combined with the respective cDNA sequences for the constant HC and LC regions of IgG1 described in Example 2. The full-length cDNA sequences used for the stable expression of recombinant Ab11 were:

[232] Ab11 HC cDNA (SEQ ID NO: 19): GAATTCgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGC TGGTGGCTGCTCCTCGGTGGGTGCTGTCCCAGGTGACCCTGAAGGAGTC CGGCCCCACCCTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACC Petition 870250084592, dated 09 / 19 / 2025, pp. 160 / 194 67 / 75 TTCAGCGGCTTTAGCCTGAGCACCTTTGGCAGCGGCGTGAGCTGGATCAG GCAGCCTCCCGGCAAGGCCCTGGAGTGGCTGGCTCACATCTATTGGGAC GACGACAAGCACTATAACCCTAGCCTGAAGAGCCGGCTGACCATCACCAA GGACACCAGCAAGAACCAGGTGGTGCTGACCATCACAAACATGGACCCTG TGGATACCGCCACCTATTTTTGCGCCCGGAGGGAGAGCCACTACTATGGC AGCGGCTACTATTTCGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGCGCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAA GTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACT TCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGA GTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCT TCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGC AACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCC AAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAAC TGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACC CTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTC TCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAG TGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTAC AGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAA GGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACA CTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATG CCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCA GACGGCAGCTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGG CAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAA TCACTATACTCAGAAAAGCCCTGAGCCTGTCCCCCGGGAAATGAGCGGCCG C.

[233] cDNA and LC of Ab11 (SEQ ID NO: 20): Petition 870250084592, of 19 / 09 / 2025, p. 161 / 194 68 / 75 GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAG CCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGCTGACCCAGT CCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTG TCGGGCTTCCTCCAGCATCTCCTATATGCACTGGTATCAGCAGAAGCCCG GCAAGGCTCCCAAGCCTTGGATCTACGCTACCAGCAATCTGGCTAGCGGC GTGCCTAGCCGGTTCTCCGGCTCCGGATCCGGCACCGACTTCACCCTGAC CATCTCCTCCCTGCAGCCTGAGGATTTTGCTACCTACTACTGCCAGCAGTG GCGGTCCGATCCCCTGACCTTCGGCGGCGGAACCAAGGTGGAGATCAAG CGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAG CTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCC AGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTA ATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCC CTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGT GTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAA GCTTTAATAGGGGGGAGTGCTGAGCGGCCGC.

[234] The lowercase nucleic acid sequence, “gccgccacc” in SEQ ID NOs: 19 and 20 is the Kozak sequence, which functions as a protein translation initiation site, and the sequence “GAATTC” immediately preceding it is the EcoR1 restriction enzyme site. The genes were chemically synthesized and subcloned into the pTXs7-GSbis expression vector for the development of stable mammalian cell lines (ProteoGenix, Schiltigheim, France). Note that HC and LC are subcloned into a single plasmid. Creation of stable transfected pools

[235] The pTXs7-GSbis construct obtained as described above was used to develop stable pools of the CHO-K1 cell line expressing Ab11. This vector also contains a gene for glutamine synthetase (GS) that confers resistance to methionine sulfoxamine toxicity. Petition 870250084592, dated 09 / 19 / 2025, pp. 162 / 194 69 / 75 (MSX). MSX is a glutamate-like drug that binds to GS, thereby inhibiting the production of glutamine, which is necessary for cell growth. This ensures that CHO cells containing one or multiple copies of the vector and therefore the gene for Ab11 are selected as the concentration of MSX increases in the cell culture medium.

[236] After determining the natural resistance to MSX of the initial CHO-K1 suspension culture, cells were transfected with the pTXs7-GS construct linearized with the Pvu I enzyme. After 48 h of post-transfection incubation, cells were seeded into six 96-well plates and stable clones were selected by culturing in the presence of 30 μM MSX for four weeks. After four weeks of screening, all wells were examined under a microscope and by ELISA using the 702 ± 3.5 kDa Ae42CC oligomer antigen described in Example 3.

[237] In parallel, three cell pools were also generated in 15 mL T75 flasks cultured similarly in selective medium containing 30 μM MSX.

[238] For the ELISA assay, 96-well Maxisorp™ Nunc ELISA plates (44-2404-21; Thermo Fisher Scientific, Waltham, MA, USA) were coated with 5 μg / mL of antigen (100 μL / well) for 2 h at 37 °C. The plates were blocked with 3% BSA in PBS (300 μL / well) at 37 °C for 1.5 h. 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 were added per well. The plates were incubated at 37 °C for 1 h, washed, and a secondary mAb conjugated with HRP was added at 1000 ng / mL (100 μL / well), and the plates were incubated again for 1 h at 37 °C. After washing, the substrate 3,3',5,5'-tetramethylbenzidine was added (100 μL / well), and the plates were incubated for 7 min at 37 °C. The reaction was stopped with 2 M HCl (50 μL per well). The difference in Petition 870250084592, dated 09 / 19 / 2025, pp. 163 / 194 Absorbance at 450 and 630 nm was measured using a spectrophotometer. The ELISA screening results identified 74 positive minipools, the results of which are shown in Table 8. Table 8 ELISA Results for the 74 Positive Minipools of Transfected CHO Cells Sample OD450 Sample OD450 Sample OD450 Sample OD450 1A4 0.23 2E8 0.21 4A7 0.23 5E4 1.41 1B1 0.21 2E11 0.21 4A11 0.26 5E7 0.21 1B6 0.26 2G3 0.22 4B9 0.24 5E10 0.71 1C1 2.15 2G4 0.22 4B12 0.23 5E11 0.23 1C9 0.22 2G7 0.24 4C3 0.22 5E12 0.24 1D2 0.22 2H5 0.23 4D2 0.22 5F1 0.24 1E4 1.75 3A5 0.22 4D11 0.22 5F6 0.26 1F3 0.20 3B7 0.20 4E2 0.23 6C1 0.24 1F4 0.22 3B9 0.21 4E10 0.23 6C4 0.71 1F5 0.21 3C3 0.21 4F5 0.25 6D3 0.20 1F6 0.21 3C5 0.22 4F11 0.24 6D6 0.36 1F12 0.21 3D8 0.22 4G6 0.94 6E2 0.24 1H2 0.20 3E9 0.24 4G9 0.21 6E6 0.23 1H5 0.21 3E12 0.22 4H6 0.22 6E9 0.23 1H9 1.88 3F3 0.23 4H10 0.23 6E11 0.53 2B2 0.24 3F5 0.20 5A4 0.24 6F12 0.63 2B9 0.24 3F10 0.20 5A12 0.24 6H3 0.22 2C3 0.21 3G7 0.20 5C1 0.25 - - 2C9 0.20 4A2 0.24 5C3 0.24 - - Small-scale production and purification tests

[239] Five minipools (1E4, 5E10, 6C4, 1C1 and 4G6) and the 3 generated pools were used for expression assessments in fed-batch culture. Cells were cultured in 30 mL of selective medium (30 μM MSX) in 125 mL shake flasks and incubated for at least 3 generations (37°C, 5% CO2, 130 rpm). When viability reached > 95%, expression tests were performed. Petition 870250084592, dated 09 / 19 / 2025, pages 164 / 194 71 / 75 cells were cultured in 30 mL fed-batch cultures. Cells were seeded at 5 x 10⁶ cells per mL in 30 mL of expression medium in 125 mL shaker flasks and incubated as previously described. 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.

[240] Cultures were stopped when viability fell < 50% (11 days), and culture medium samples were purified and analyzed by SDS-PAGE and UV absorbance using general protocols and procedures such as those used for transient CHO expression analyses described in Example 2. Purity assessment after purification is shown in Figure 9. The yield and purity obtained are summarized in Table 9. A stable minipool (5E10) and Pool 1 showed high expression and were selected for further development. Table 9 Yield and Purity Obtained for Small-Scale Expression Tests Antibody Quantity* (mg) Yield** (mg / L) Purity*** Pool 1 32.5 1083 >90% Pool 2 0.93 31 >90% 1E4 17.02 566 >90% 5E10 20.59 687 >90% 6C4 7.68 256 >90% 1C1 9.77 325 >90% 4G6 6.48 216 >90% * Obtained after purification of a 30 mL culture test. **Extrapolated from the 30 mL test. ***Estimated from the analysis of full-length (complete) antibodies observed in unreduced SDS-PAGE analyses. Petition 870250084592, dated 09 / 19 / 2025, pages 165 / 194 72 / 75 Isolation and screening of stable monoclones

[241] From the stable pools of 5E10 and Pool 1, isolation and screening of monoclones were performed using standard methods. Briefly, monoclones were isolated using the limiting dilution method with a seeding density of 0.2 cells / well in 96-well plates. Monoclones were confirmed by microscopic observation and expression screening was performed by ELISA with anti-Fc antibodies. After two rounds of limiting dilution and expression screening by ELISA, the 10 monoclones with the best expression were amplified by culture in 6-well plates and used to perform small-scale production assessments with 30 mL cultures in 125 mL shaker flasks using the same protocol described above.The antibodies were then purified and analyzed as previously described, and the final samples obtained were buffered with PBS pH 7.5 and then analyzed qualitatively and quantitatively by SDS-PAGE and UV analysis methods, respectively, as described above. The purity assessment after purification is shown in Figure 10. The concentrations obtained are shown in Table 10. All 10 isolated monoclones produced antibodies in high purity and exhibited good expression levels in flask cultures (~1 g / L), with 3 of them showing a high expression level (>1.5 g / L) and one of them showing a very high expression level (>2 g / L). TABLE 10 Production Yields in Fed-Batch Culture for the 10 Best Monoclones Clone Quantity* (mg) Yield** (g / L) 2D7 31.5 1.050 4H9 61.2 2.040 13F11 34.2 1.140 8B6 35.00 1.166 Petition 870250084592, dated 09 / 19 / 2025, pages 166 / 194 73 / 75 Clone Quantity* (mg) Yield** (g / L) 8F9 25.5 0.850 15H5 26.25 0.875 1H10 44.20 1.473 1A3 48.60 1.618 2A6 47.52 1.584 5A9 46.20 1.540 * Obtained after purification of the 30 mL culture test. **Extrapolated from the 30 mL test. RCB stability study Cell growth stability analysis

[242] Monoclones 4H9, 1A3, and 2A6 were seeded at a density of 0.5 x 10⁶ cells / mL in 30 mL of selective medium in 125 mL shake flasks and incubated at 37 °C, 5% CO₂, and 130 rpm for 15 passages (30 generations). Cell density and viability were monitored for each passage, and the stability of the entire growth cycle was analyzed. The results are shown in Figure 11, for which the coefficient of variation (CV; in %) is shown in Table 11. Table 11 Analysis of Cell Growth Stability Across 15 Passages Clone CV% of VCD* CV% of viability 2A6 7.21% 0.28% 1A3 6.82% 0.26% 4H9 5.91% 0.25% *VCD: Viable cell density Transgene DNA sequence

[243] Genomic DNA was extracted for PCR amplification and genetic sequencing for each of the three monoclones from cell passages 5, 10, and 15. The DNA was amplified by PCR using the following primers:

[244] HC-direct (Forward): Petition 870250084592, dated 09 / 19 / 2025, pp. 167 / 194 74 / 75 GCAGTCACCGTCCTTGACACGGGATCCGCCGCCACCATGAA GCACCTGTGG (SEQ ID NO: 21).

[245] HC-reverse (Reverse): ATGGCTGATTATGATCAATCTCGAGTCATTTCCCGGGGGACA GGCTCAG (SEQ ID NO: 22).

[246] LC-direct (Forward): CAGTCACCGTCCTTGACACGAAGCTTGCCGCCACCATGGTG CTGCAGAC (SEQ ID NO: 23).

[247] LC-reverse (Reverse): GTATGGCTGATTATGATCAATGAATTCTCAGCACTCCCCCCTA TTAAAGC (SEQ ID NO: 24).

[248] The PCR amplification results are illustrated in Figure 12, confirming the presence of the Ab11 gene. The PCR products were then sequenced and an alignment was performed, which indicated that no point mutation occurred in the Ab11 gene in any clone during 15 passages. Production yield analysis

[249] Fed-batch expression tests of 30 mL were performed for each monoclone from passages 5, 10, and 15, and the antibodies were purified as described above. Purity assessments by SDS-PAGE are shown in Figure 13, and yields are shown in Table 12. No stability problems were observed during the study for monoclones 1A3 and 4H9. Monoclone 2A6 showed lower production yields during the stability analysis, with CV% > 20%. Table 12 Yield Obtained Over 15 Cycles During Stability Analysis Clo Initial Yield CV Yield Yield ne (g / L)P5* (g / L)P10* (g / L)P15* (g / L)% 2A6 1.58 0.87 0.72 0.83 39.6 Petition 870250084592, dated 09 / 19 / 2025, pp. 168 / 194 75 / 75 Clo ne Initial Yield Yield Yield P10*(g / L) Yield P15*(g / L) CV % (g / L) P5* (g / L) 1A3 1.62 1.88 1.61 1.35 13.4 8 4H9 2.04 2.06 2.22 1.73 10.2 Initial yield taken from Table 10 for comparison. *P5 / P10 / P15: Passage 5 / 10 / 15 Mycoplasma test

[250] 2 to 3 χ 105 cells from each of the clones 2A6, 1A3 and 4H9 were collected for mycoplasma testing using a mycoplasma PCR test kit. The results are shown in Figure 14, which shows that the monoclonal cell cultures were free of mycoplasma infection. Conclusion

[251] In conclusion, the Ab11 antibody was successfully introduced into the genome of the CHO-K1 cell line and demonstrated robust and stable high expression, producing up to 2 g of antibody per liter of culture with an estimated purity of > 90%. Viable monoclonal cell lines were isolated and confirmed to be free of mycoplasma bacteria. Three research monoclonal cell banks were isolated in this study, named 2A6, 1A3, and 4H9. Of these, clone 4H9 showed the highest expression level and also proved to be more stable over 15 passages than the other two clones.

Claims

1. ANTIBODY, characterized by comprising an antigen-binding domain capable of specifically binding to prefibrillar β42 oligomers with a β structure, wherein said antigen-binding domain comprises: (i) a variable heavy chain (VH) region comprising the sequence SEQ ID NO: 1; or (ii) a variable light chain (VL) region comprising the sequence SEQ ID NO: 2; or a combination thereof.

2. ANTIBODY, according to claim 1, characterized in that the antigen-binding domain comprises both the VH of (i) and the VL of (ii) in combination.

3. ANTIBODY, according to either claim 1 or 2, characterized in that the antibody comprises two antigen-binding domains.

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

5. ANTIBODY, according to claim 4, characterized in that the antibody is an IgG antibody, or an antigen-binding fragment thereof.

6. ANTIBODY, according to any one of claims 4 or 5, characterized in that the antibody is an IgG1Kappa antibody, or a fragment thereof.

7. ANTIBODY, according to any one of claims 1 to 6, characterized by the antigen-binding domain comprising: Petition 870250084592, dated 09 / 19 / 2025, p. 170 / 194 2 / 4 (i) a heavy chain sequence comprising 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 having; (ii) at least 90% sequence identity with SEQ ID NO: 3; and / or (iii) a light chain sequence comprising the VL sequence of SEQ ID NO: 2 linked to the light chain constant region sequence of SEQ ID NO: 4; or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

4.

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

9. ANTIBODY, as defined in any one of claims 1 to 7, characterized by being for use in therapy.

10. ANTIBODY, as defined in any one of claims 1 to 7, characterized by being for use in the treatment of an amyloid disease.

11. ANTIBODY for use, according to claim 10, characterized by amyloid disease being a neurodegenerative condition associated with Aβ.

12. ANTIBODY for use, according to any one of claims 10 or 11, characterized by the amyloid disease being a disease associated with soluble β-structured Αβ42 oligomers.

13. ANTIBODY for use, according to any one of claims 10 to 12, characterized by the amyloid disease being Alzheimer's disease (AD), Down syndrome or inclusion body myositis (IBM).

14. PHARMACEUTICAL COMPOSITION, characterized by Petition 870250084592, dated 09 / 19 / 2025, page 171 / 194 3 / 4 comprising an antibody, as defined in any of claims 1 to 7, in a mixture with at least one pharmaceutically acceptable vehicle or excipient.

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

16. ANTIBODY or pharmaceutical composition for use, according to claim 15, characterized in that SOC therapy is with an agent selected from among an anti-plaque agent, lecanemab and donanemab.

17. ANTIBODY, as defined in any one of claims 1 to 7, or a conjugate, as defined in claim 8, characterized in that it is for use in the in vivo diagnosis of an amyloid disease in a subject.

18. ANTIBODY for use in in vivo diagnosis, according to claim 17, characterized by amyloid disease being a disease associated with soluble β-structured Αβ42 oligomers.

19. NUCLEIC ACID MOLECULE, characterized by comprising a nucleotide sequence encoding an antibody, as defined in any one of claims 1 to 7, or a polypeptide comprising a VH and / or VL region thereof.

20. VECTOR, characterized by comprising a nucleic acid molecule as defined in claim 19.

21. VECTOR, according to claim 20, characterized in that the vector is an expression vector.

22. HOST CELL, characterized by comprising the vector as defined in either of claims 20 or 21.

23. HOST CELL, according to claim 22, Petition 870250084592, dated 09 / 19 / 2025, pp. 172 / 194 4 / 4 characterized as being a mammalian host cell.

24. METHOD FOR PRODUCING AN ANTIBODY, as defined in any one of claims 1 to 7, characterized in that said method comprises cultivating a host cell, as defined in any one of claims 22 or 23, under conditions suitable for expression of the antibody, as defined in any one of claims 1 to 7.

25. ANTIBODY, as defined in any one of claims 1 to 7, characterized by being for combined use with a second therapeutic agent for use in the therapy of an amyloid disease.

26. ANTIBODY for use according to claim 25, characterized in that the second agent is selected from an anti-plaque agent, lecanemab and donanemab.

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