ANTICORPO BIESPECÍFICO QUE SE LIGA AO DOMÍNIO DO TIPO PROTEASE DO RECEPTOR DE TRANSFERRINA HUMANA HTFR1

Abstract

The present disclosure relates to a binding protein, comprising a first transferrin receptor 1 binding moiety M1 comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) and having the capacity to bind selectively to an epitope located in the protease-like domain of hTfR1, and a second moiety M2 comprising an antibody Fc domain. In the binding protein, M1 and M2 are connected to each other by at least one peptide linker between M1 and M2.

Description

1 / 102 Bispecific antibody that binds to the protease-like domain of the human transferrin receptor hTfRl. Field

[0001] The present disclosure relates to a binding protein comprising a first transferrin receptor 1 binding portion M1 comprising a variable immunoglobulin heavy chain (VH) region and a variable immunoglobulin light chain (VL) region and having the ability to selectively bind to an epitope located in the protease-like domain of hTfR1, and a second portion M2 comprising an antibody Fc domain. In the binding protein, M1 and M2 are connected to each other by at least one M1-M2 linker peptide. Background

[0002] Treatment options for brain and neurological diseases are limited due to the impermeability of brain blood vessels to most substances transported in the bloodstream (Freskgard and Urich (2017), Neuropharmacology 120:38-55; Stanimirovic et al. (2018), BioDrugs 32:547-559). The small blood vessels (capillaries) of the brain, collectively called the blood-brain barrier (BBB), are unique when compared to blood vessels found in the periphery of the body. The tight apposition of endothelial cells (ECs) of the BBB to neural cells, such as astrocytes, pericytes, and neurons, induces phenotypic characteristics that contribute to the observed impermeability. Tight junctions between ECs in the BBB limit paracellular transport, while the absence of passive pinocytotic vesicles and fenestrae limits nonspecific transcellular transport.These factors combine to restrict molecular flow from the blood to the brain in general to molecules less than 500 Da in size and lipophilic. Thus, the promising prospect of using the large mass transfer surface area (more than 20 m² of 600 km of capillaries in a human brain) of the bloodstream as a... Petition 870250082258, dated 12 / 09 / 2025, page 21 / 171 2 / 102 delivery vehicles become largely impractical, except in circumstances where a drug with the desired pharmacological properties fortuitously possesses size and lipophilicity attributes that allow it to cross the BBB. Due to these restrictions, it is estimated that more than 98% of all small molecule drugs and almost 100% of the emerging class of protein and gene therapies do not cross the BBB.

[0003] Document WO91 / 03259 proposes a principle for the transport of a neuropharmaceutical agent across the BBB, which involves conjugating the agent to an antibody that is reactive with the transferrin receptor. According to this disclosure, the binding of the conjugate to the transferrin receptor leads to the active transport of the conjugate across the BBB. Subsequent work has further developed this concept, for example, as described in WO2012 / 075037, WO2014 / 033074, WO2018 / 011353 and WO2022 / 258841, all describing different formats for achieving the transport of a biopharmaceutical agent across the BBB using the transferrin receptor.

[0004] There are two forms of the human transferrin receptor. Transferrin receptor 1 (TfR1) is the target of the binding protein of the present disclosure. TfR1 is an iron transporter protein that maintains cellular iron levels by recognizing and internalizing iron through specific binding of the iron transporter proteins transferrin (Tf) and ferritin (Ft) into cells via clathrin-coated vesicle-mediated endocytosis. TfR1 is expressed in numerous cells and organs, but expression levels vary, and most importantly, TfR1 is expressed to a greater extent in BBB endothelial cells than in other endothelial cells, making the receptor a target for neuropharmaceutical delivery. Structurally, TfR1 is a dimeric transmembrane glycoprotein comprising the amino acid sequence SEQ ID NO: 66, which has a large ectodomain (residues 89 to 760), a region Petition 870250082258, dated 12 / 09 / 2025, page 22 / 171 3 / 102 intramembranous (residues 62 to 88) and a cytoplasmic domain (residues 1 to 61). The ectodomain, in turn, has three distinct domains kept separate from the cell surface by a rod region (residues 89 to 120). These three parts of the ectodomain are the helical domain (residues 606 to 760), the protease-like domain (residues 121 to 183, 384 to 605), and the apical domain (residues 184 to 383) (Lawrence et al. (1999), Science 286:779-782).

[0005] By using TfR1 to transport a molecule comprising a TfR1 ligand across the blood-brain barrier in this way, binding to TfR1 will first occur in the circulation, i.e., before the molecule is transported to the brain compartment. While in circulation, the complex between the molecule and TfR1 will be exposed to blood components and cells in the peripheral system where the molecule is located and presented on cells expressing TfR1. The complex, when displayed on cell surfaces, will then be exposed to various endogenous blood components or other factors present in the local environment around the TfR1-expressing cells. This potential interaction between the displayed TfR1 / ligand complex and the environment can induce or trigger various pathways with various modes of action. If and when this happens, undesirable side effects may occur in the periphery before the molecule is transported to the brain.This is especially important if the molecule comprising the TfR1 ligand comprises an antibody or a part thereof known to mediate an immune response.

[0006] In fact, disadvantages of targeting TfR1 with antibodies have already been described, such as acute clinical signs and decreased circulating reticulocytes. This was previously resolved by eliminating the Fc effector function of the antibodies. This has been shown to improve acute clinical signs and partially recover the reduction in reticulocytes. Both Petition 870250082258, dated 12 / 09 / 2025, page 23 / 171 4 / 102 Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been described as being involved in these processes. Mutation of the Fc portion of antibodies to reduce or eliminate ADCC and / or CDC has been suggested as a potential strategy to mitigate the development of TfR1-based therapies designed to cross the blood-brain barrier (Couch et al. (2013), Sci Transl Med 5:183ra57). However, this strategy modifies the antibody structurally and removes the antibody's effector function when it is bound to its target. Thus, Fc mutations risk reducing or even preventing the therapeutic function of a therapeutic antibody.For example, it has been postulated that the cerebral clearance mechanism of β-amyloid is promoted by the Fc effector function of an antibody, including a role for microglia and expressed Fc gamma receptors, the resident immune and phagocytic cells in the central nervous system (Condello et al. (2015), Nat Commun 6:6176). Thus, for example, in certain uses of antibodies for the treatment of brain diseases, an Fc-mediated function is necessary to achieve the desired clinical effect.

[0007] The administration of therapeutic monoclonal antibodies is frequently accompanied by severe first-infusion reactions (FIOs). This has been described as being induced by the effector function of the antibodies and can be a complicating factor in the clinical use of therapeutic antibodies, particularly if they have TfR1 as one of their targets (Weber et al. (2018), Pharm Res 35(9):169). Another observation is that, in certain cases, when an antibody binds to TfR1 via a TfR1-binding scFab module connected to the C-terminal end of the antibody's Fc portion, the antibody's Fab arms can, to some extent, block the ADCC and / or CDC pathways mediated by FcyR binding to the antibody's Fc portion (Weber et al. (2018), Cell Rep 22:149-162). Weber et al. described the effect as being due to Petition 870250082258, dated 12 / 09 / 2025, p. 24 / 171 5 / 102 reverse orientation of the bound antibody compared to a standard antibody.

[0008] There is still a need for biopharmaceutical agents, for example, for the treatment of brain diseases, that can be transported across the blood-brain barrier and have a beneficial profile with regard to reducing the risk of side effects, for example, side effects mediated by the Fc effector function of antibodies. Revelation of the invention

[0009] One goal of the breakthrough is to meet this need by providing a binding protein that can cross the blood-brain barrier.

[0010] Another goal of the discovery is to provide a binding protein with therapeutic function to the brain, while reducing or eliminating the risk of Fc-mediated adverse effects, such as ADCC, ADCP, or CDC.

[0011] Another goal of the discovery is to improve existing binding proteins based on bi- or multi-specific formats for the purpose of treating neurological diseases.

[0012] Another goal of the discovery is to provide a binding protein with a new binding orientation in the interaction with hTfR1.

[0013] One or more of these objects, and / or any other object that is evident to a person skilled in the art in the present disclosure, is / are satisfied by the various aspects described herein.

[0014] Thus, in a first aspect, the present disclosure provides a binding protein comprising: - a first M1 portion, which is a human transferrin receptor 1 (hTfR1) binding portion comprising a variable immunoglobulin heavy chain (VH) region and a variable immunoglobulin light chain (VL) region, said VH and VL regions forming a VH / VL pair comprising a surface Petition 870250082258, dated 12 / 09 / 2025, page 25 / 171 6 / 102 antigen binding, in which said antigen-binding surface provides the binding protein with the ability to selectively bind to an epitope located in the protease-like domain of hTfR1 defined by amino acid residues 121 to 183 and 384 to 605 in SEQ ID NO: 66, and - a second M2 portion comprising an Fc domain of the antibody, wherein M1 and M2 are connected to each other by at least one linker peptide between M1 and M2, said linker being arranged in such a way that M2 induces an Fc-mediated reduction when administered to a human and when M1 binds to hTfR1 present on a cell.

[0015] In a first alternative aspect, the revelation provides a binding protein, comprising: - a first M1 portion, which is a human transferrin receptor 1 (hTfR1) binding portion comprising a variable immunoglobulin heavy chain (VH) region and a variable immunoglobulin light chain (VL) region, said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, wherein said antigen-binding surface provides the binding protein with the ability to selectively bind to an epitope located in the protease-like domain of hTfR1 defined by amino acid residues 121 to 183 and 384 to 605 in SEQ ID NO: 66, and - a second M2 portion comprising an Fc domain of the antibody, wherein M1 and M2 are connected to each other by at least one linker peptide between M1 and M2, said linker being arranged in such a way that M2 is oriented towards the cell surface when M1 binds to hTfR1 present on a cell. Petition 870250082258, dated 12 / 09 / 2025, p. 26 / 171 7 / 102

[0016] In one embodiment, M2 is selected from the group consisting of an antibody and an Fc fusion protein. In one specific embodiment, M2 is an antibody. In another specific embodiment, M2 is an Fc fusion protein.

[0017] In one embodiment, the aforementioned Fc domain of M2 is capable of inducing an Fc-mediated response when administered to a human, for example, an Fc-mediated cytotoxic response. The binding protein of the first aspect is designed to reduce this Fc-mediated response. Without wanting to get bogged down in theory, the binding of M1 to an epitope in the protease-like domain of hTfR1 is contemplated to orient the entire binding protein comprising M1 and M2 in such a way that, upon binding to hTfR1, the Fc domain is oriented away from the environment and towards a space created below and / or beside the hTfR1 protein when anchored to the surface of a cell. In this way, the interaction between the Fc domain and Fc receptors, necessary for the Fc-mediated response to occur, is prevented or diminished. This effect is believed to occur only when the binding protein is bound to hTfR1.As a result, once the binding protein has crossed the BBB and entered an environment where the Fc domain of the binding protein has an intended function and a desired effect, that Fc-mediated effect will be functional when needed. M1 portion of the hTfRl binding

[0018] As described above, in the first aspect, the present disclosure provides a binding protein in which the M1 portion is a human transferrin receptor 1 (hTfR1) binding portion, capable of selectively binding to an epitope located in the protease-like domain of hTfR1 defined by amino acid residues 121 to 183 and 384 to 605 in SEQ ID NO: 66. Without intending to be limited by theory, the binding of hTfR1 to an epitope, or binding site, within the protease-like domain is contemplated to offer advantages in terms of Petition 870250082258, dated 12 / 09 / 2025, page 27 / 171 8 / 102 avoid the disadvantages associated with known hTfR1 ligands, in particular, those known ligands that have affinity for epitopes or binding sites located in the apical domain of TfR1.

[0019] The binding of M1 to hTfR1 at an epitope in the protease-like domain is illustrated in Figure 1, which is a surface density model of the structure between a VH / VL pair in an exemplary M1 module described herein, determined by X-ray crystallography, as described in Example 5. As shown in the M1 portion bound to the left side of the hTfR1 homodimer, the pairing of the VH and VL regions and the binding orientation of the M1 portion in hTfR1 provide four peptide chain ends that are available for binding of the second M2 portion via a linker between M1 and M2.

[0020] In one specific embodiment, the epitope or binding site for the binding portion of hTfR1 comprises amino acid residues 150, 151, 154, 158, 159, 161, 163, and 385 in SEQ ID NO: 66. In another embodiment, the epitope or binding site for the hTfR1 binding protein of the development consists of amino acid residues 150, 151, 154, 158, 159, 161, 163, and 385 in SEQ ID NO: 66. In an alternative specific embodiment, the epitope or binding site for the hTfR1 binding proteins comprises or consists of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO: 66.As shown in the following examples, for instance with reference to Figure 16, this epitope modality for the binding proteins identified and revealed here ensures a binding that does not interfere with the natural ligands of hTfR1, transferrin and ferritin.

[0021] As is known to a person skilled in the art, an epitope (or “antigenic determinant”) is a group of amino acids or other chemical groups exposed on the surface of Petition 870250082258, dated 12 / 09 / 2025, p. 28 / 171 9 / 102 a molecule, often a protein, in this case hTfRl, that can generate an antigenic response and bind to the antibody. An epitope is a region located on the surface of an antigen that is recognized by the immune system, specifically by antibodies. A conformational epitope is composed of neighboring amino acid residues located on the surface structure of an antigenic protein. Conformational epitopes bind their complementary paratopes on B cell receptors and / or antibodies. In one embodiment of the revelation, the epitope bound by the binding molecule is a conformational epitope.

[0022] In one embodiment, the linkage to hTfRl by the M1 linking moiety is monovalent.

[0023] As described above, the first M1 portion comprises a VH / VL pair with an antigen-binding surface. For clarity, the VH / VL designation as used in relation to a VH / VL pair does not limit the construct to any specific order of the VH and VL regions in the polypeptide chain, but is used only to convey that both VH and VL regions are present and are capable of pairing to form an Ig domain with an antigen-binding surface. Thus, the term VH / VL pair encompasses, for example, constructs in which the VL region precedes the VH region in a single Fv chain, constructs in which the VH region precedes the VL region in a single Fv chain, and constructs in which the VH and VL regions are non-covalently associated with each other. In a specific embodiment of the binding protein, the VH / VL pair in M1 is arranged so that the VL region precedes the VH region in a single-chain Fv construct.

[0024] The VH / VL pair comprised in Ml comprises an antigen-binding surface. In one embodiment, said antigen-binding surface is composed of three complementarity-determining regions (CDRs) of each of the Petition 870250082258, dated 12 / 09 / 2025, page 29 / 171 10 / 102 VH and VL regions. In one modality, the aforementioned CDRs comprise the following amino acid sequences: VHCDR1: VHCDR2: X1X2NMX3 (SEQ ID NO: 1), where: X1 is selected from D and A; X2 is selected from Y and A; and X3 is selected from D and A; X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO: 2), VHCDR3: where: X4 is selected from D and A; X5 is selected from D and A; X6 is selected from Y and A; X7 is selected from D and A; and X8 is selected from Y and A; GGX9SGSSX10X11HPMX12X13 (SEQ ID NO: 3) VLCDR1: where: X9 is selected from Y and A; X10 is selected from Y and A; X11 is selected from Y and A; X12 is selected from D and A; and X13 is selected from Y and A; KSSQSLLX14STNQKNX15LA (SEQ ID NO: 4), VLCDR2: VLCDR3: where: X14 is selected from Y and A; and X15 is selected from Y and A; X16ASTRES (SEQ ID NO: 5) where: X16 is selected from W and A; and QQX17FIX18PRT (SEQ ID NO: 6) where X17 is selected from Y and A; and X18 is selected from Y and A. Petition 870250082258, dated 12 / 09 / 2025, p. 30 / 171 11 / 102

[0025] In one embodiment, the amino acid sequence of said VHCDR1 is selected from the group consisting of SEQ ID NO: 7 and 13 to 15.

[0026] In one embodiment, the amino acid sequence of said VHCDR2 is selected from the group consisting of SEQ ID NO: 8 and 16 to 20.

[0027] In one embodiment, the amino acid sequence of said VHCDR3 is selected from the group consisting of SEQ ID NO: 9 and 21 to 25.

[0028] In one embodiment, the amino acid sequence of said VLCDR1 is selected from the group consisting of SEQ ID NO: 10, 26 and 27.

[0029] In one embodiment, the amino acid sequence of said VLCDR2 is selected from the group consisting of SEQ ID NO: 11 and 28.

[0030] In one embodiment, the amino acid sequence of said VLCDR3 is selected from the group consisting of SEQ ID NO: 12, 29 and 30.

[0031] In some embodiments, CDR sequences can be freely combined among the options listed above. Such embodiments include, for example, but are not limited to, those combinations exemplified in Example 9 for variants. replaced by alanine of the representative M1 portion of the h26D3 binding protein.

[0032] In a specific embodiment of the revelation, the antigen-binding surface CDR sequences of the M1 binding portion are as follows: VHCDR1: DYNMD (SEQ ID NO: 7), VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO: 8), VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO: 9), VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO: 10), VLCDR2: WASTRES (SEQ ID NO: 11), VLCDR3: QQYFIYPRT (SEQ ID NO: 12). Petition 870250082258, dated 12 / 09 / 2025, p. 31 / 171 12 / 102

[0033] In another specific embodiment of the revelation binding protein, the CDR sequences of the antigen-binding surface of the M1 binding portion are as follows: VHCDR1: DYNMD (SEQ ID NO: 7), VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO: 18), VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO: 9), VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO: 10), VLCDR2: WASTRES (SEQ ID NO: 11), VLCDR3: QQYFIYPRT (SEQ ID NO: 12).

[0034] In one embodiment, the CDR sequences in a Antigen-binding interfaces comprised in a revelation-binding protein are as defined using the Kabat convention, which is well known to those skilled in the art of antibody technology (see, for example, Kabat (1991), Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242 of the U.S. Department of Health and Human Services).

[0035] In one embodiment, the said VH region of the VH / VL pair in M1 comprises or consists of an amino acid sequence selected from: (i) the group consisting of SEQ ID NO: 31 to 44, for example, the group consisting of SEQ ID NO: 31 and 37; and (ii) a sequence with at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of a sequence defined in (i).

[0036] In one embodiment, the said VL region of the VH / VL pair in M1 comprises or consists of an amino acid sequence selected from: (i) the group consisting of SEQ ID NO: 45 to 51; and Petition 870250082258, dated 12 / 09 / 2025, p. 32 / 171 13 / 102 (ii) a sequence having at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of a sequence defined in (i).

[0037] In a particular embodiment, the VH region and the VL region are both as defined immediately above, that is, a VH comprising or consisting of a sequence selected from SEQ ID NO: 31 to 44 and sequences with at least 80% sequence identity with the same, and a VL comprising or consisting of a sequence selected from SEQ ID NO: 45 to 51 and sequences with at least 80% sequence identity with the same.

[0038] In one embodiment, said region VH comprises SEQ ID NO: 31 and said region VL comprises a sequence selected from SEQ ID NO: 45 to 51.

[0039] In one embodiment, said VH region comprises a selected sequence of SEQ ID NO: 31 to 44 and said VL region comprises SEQ ID NO: 45.

[0040] In one embodiment, said region VH comprises SEQ ID NO: 31 and said region VL comprises SEQ ID NO: 45.

[0041] In one embodiment, said region VH comprises SEQ ID NO: 37 and said region VL comprises SEQ ID NO: 45.

[0042] In certain embodiments, the VH and VL sequences in the linking molecule are selected from any of the listed sequences and sequences that have at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with each other. Connection between M1 and M2 Petition 870250082258, dated 12 / 09 / 2025, p. 33 / 171 14 / 102

[0043] In an important embodiment of the development-binding protein, the M1 portion comprises an scFv. In other words, the VH / VL pair in M1 constitutes part of an scFv, in which the VH and VL regions are coupled by an scFv linker peptide. In one of these embodiments, the scFv linker can be linked to the C-terminal amino acid residue of the VH region and the N-terminal amino acid residue of the VL region, or to the C-terminal amino acid residue of the VL region and the N-terminal amino acid residue of the VH region. In the first configuration, the VH region precedes the VL region in the polypeptide chain that constitutes the scFv, while in the second configuration, the VL region precedes the VH region. The two different configurations are denoted “VH first” and “VL first” and are illustrated in Figures 2A and 2B, respectively, by the exemplary h26D3 M1-binding moiety structure in complex with hTfR1.As shown in Figure 2A, in the “VH first” configuration, the scFv linker between VH and VL is located under the M1 portion, close to the cell membrane surface. This allows M2 to bind to M1 using the N-terminal amino acid residue of VH (VH-N) or the C-terminal amino acid residue of VL (VL-C), or both. As shown in Figure 2B, in the “VL first” configuration, the scFv linker between VH and VL is located on top of the M1 portion, further from the cell membrane surface. This allows M2 to bind to M1 using the C-terminal amino acid residue of VH (VH-C) or the N-terminal amino acid residue of VL (VL-N), or both.When the M1 binding portion is used as a scFv in the “first VL” configuration, or even in any configuration where VH-C and / or VL-N binding sites are available, the Fc domain containing the second M2 portion can, by binding to one or both binding sites, be oriented close to the cell membrane in the space below most of the hTfR1 homodimer (schematically illustrated in Figure 3, again with reference to the X-ray structure of h26D3 in complex with hTfR1). Unintentionally. Petition 870250082258, dated 12 / 09 / 2025, page 34 / 171 15 / 102 being limited by theory, this is contemplated to reduce the likelihood that the antibody will be available for harmful interaction with other components in circulation when bound to hTfR1.

[0044] The design and selection of suitable linker peptides for use within and between domains and portions of fusion proteins, antibody constructs, and other engineered polypeptides are within the capabilities of a person skilled in the art. In some embodiments where M1 comprises or consists of an scFv, the scFv linker is a flexible linker peptide, consisting of 5 to 40 amino acid residues, for example, 10 to 30 amino acid residues, for example, 15 to 25 amino acid residues, for example, about 15 amino acid residues, for example, 15 amino acid residues, for example, comprising or consisting of the sequence (G4S)3 (SEQ ID NO: 88).

[0045] The same design considerations or similar considerations apply to the linkers used to attach the hTfR1-binding M1 portion to the second M2 portion. In one embodiment, said at least one linker peptide between M1 and M2 is linked, on the M1 side, to the C-terminal amino acid residue of the VH region of M1 or to the N-terminal amino acid residue of the VL region of M1.

[0046] In one of these embodiments, at least one linking peptide between M1 and M2 is linked, on the M2 side, to the C-terminal residue of a CH3 region of said Fc domain and, on the M1 side, to the N-terminal amino acid residue of the VL region of M1. An example of this embodiment in this document has a full-length antibody as M2 and is called “Gen 2D”. The “Gen 2D” design is illustrated in the right panel of Figure 4. As shown in this design, the hTfR1-binding portion of M1 is C-terminally fused to an antibody heavy chain of M2, which pairs with another antibody heavy chain without an hTfR1 linker. Along with two copies of the M2 antibody light chain, the construct Petition 870250082258, dated 12 / 09 / 2025, p. 35 / 171 The complete 16 / 102 is formed with an hTfR1 ligand attached to a standard Y-shaped antibody structure. In an example of testing an scFv in the “VL-first” configuration as M1, i.e., where the hTfR1-binding M1 portion is an scFv fused to the antibody via the VL-N site, there was no detectable antibody-dependent cellular cytotoxicity (ADCC) in the assay described in Example 18 (Figure 32B).

[0047] In another such embodiment, M2 comprises an antibody with two antibody light chains, and M1 and M2 are connected to each other by means of two linker peptides, the first linker being attached, on the M2 side, to the C-terminal amino acid residue of the first light chain of M2 and, on the M1 side, to the N-terminal amino acid residue of the VL region of M1, and the second linker being attached, on the M2 side, to the N-terminal amino acid residue of the second light chain of M2 and, on the M1 side, to the C-terminal amino acid residue of the VH region of M1. An example of this embodiment in this document has a full-length antibody as M2 and is designated “Gen 2A”. The “Gen 2A” design is illustrated in the left panel of Figure 4 and described in detail in WO2022 / 258841 (incorporated by reference).By linking a single-stranded hTfR1-binding M1 portion to the C-terminal of one of the M2 light chains and to the N-terminal of the other M2 light chain, a symmetrical construct composed of only two different polypeptide chains is created. In an example of testing an scFv in the “VL-first” configuration as M1 in this “Gen 2A” format, i.e., in which the hTfR1-binding M1 portion is an scFv fused to the antibody via the VL-N and VH-C sites, there was no detectable complement-dependent cytotoxicity (CDC) in the assay described in Example 14 (Figure 29).

[0048] As described above, the design and selection of suitable linker peptides for use within and between domains and portions of fusion proteins, antibody constructs, and other engineered polypeptides are within the capabilities of a Petition 870250082258, dated 12 / 09 / 2025, page 36 / 171 17 / 102 technical subject matter. In one embodiment of the revealing linker protein, M1 and M2 are linked by at least one flexible linker peptide. In one embodiment, the at least one flexible linker peptide comprises glycine, serine, alanine, and / or threonine residues. In a more specific embodiment, said linker(s) has / have a general formula selected from (GnSm)pe (SnGm)p, wherein independently, n = 1 to 7, m = 0 to 7, n + m < 8 and p = 1 to 10. In some embodiments, at least one linker has between 10 and 50 amino acid residues in length, as between 10 and 30 amino acid residues in length, as between 15 and 25 amino acid residues in length, or between 10 and 20 amino acids in length. If M1 and M2 are linked by two or more connectors, all optional connector designs revealed apply individually to each connector present, regardless of the other connectors.Thus, for example, if there are two linkers, they may have the same or different lengths and have the same amino acid sequence or different amino acid sequences. Second portion M2

[0049] With respect to the second M2 portion in the revelation-binding protein, it is selected between an antibody and an Fc fusion protein. Through the presence of its Fc domain, M2 may be able to induce an Fc-mediated response, such as an Fc-mediated cytotoxic response. In one embodiment, such a cytotoxic response is selected from the group consisting of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and combinations thereof. In another embodiment, the response is selected from ADCC, CDC, and combinations thereof. In one specific embodiment, the response is ADCC. In another specific embodiment, the response is CDC. Petition 870250082258, dated 12 / 09 / 2025, p. 37 / 171 18 / 102

[0050] In a developmental embodiment of the binding protein, M2 is an antibody capable of selectively binding to a target present in the brain of a mammal. In some modalities, the aforementioned target is selected from the group consisting of β-amyloid peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, TAR 43 DNA-binding protein (TDP-43) or derivatives or fragments thereof, myeloid cell-expressed triggered receptor 2 (TREM2), beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, Tau, phosphorylated Tau or fragments thereof, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, β-amyloid precursor protein, p75 neurotrophin receptor, neuregulin, and caspase 6.In a more specific embodiment, the target is selected from the group consisting of β-amyloid peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, TAR 43 DNA-binding protein (TDP43) or derivatives or fragments thereof, receptor-triggered myeloid cell-expressed 2 (TREM2), Tau, phosphorylated Tau or fragments thereof, and apolipoprotein E4. In an even more specific embodiment, the target is selected from the group consisting of β-amyloid peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, and TAR 43 DNA-binding protein (TDP-43) or derivatives or fragments thereof.

[0051] In one embodiment of the developmental binding protein, where M2 is an antibody capable of selectively binding to a target present in the brain of a mammal, said antibody is an anti-Αβ antibody, for example, an antibody selected from the group consisting of Petition 870250082258, dated 12 / 09 / 2025, p. 38 / 171 19 / 102 lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06 and KHK6640.

[0052] In another embodiment of the revelation binding protein, where M2 is an antibody capable of selectively binding to a target present in the brain of a mammal, the said antibody is an anti-alpha-synuclein antibody, for example, an antibody selected from the group consisting of prasinezumab, UCB7853, Lu AF82422, TAK-341 and BAN0805. Affinity for a target

[0053] As used herein, the terms specific X binding, selective X binding, and X affinity, where X is a target (e.g., an antigen or an epitope, such as TfR1 bound by the VH / VL pair in the M1 portion of the binding protein as defined above), refer to a property of a binding protein, such as a property of an antibody or antigen-binding fragment thereof, or of a bi- or multispecific construct incorporating such antibody or antigen-binding fragment thereof, which can be tested, for example, by ELISA, surface plasmon resonance (SPR), or biolayer interferometry (BLI). A person skilled in the art is familiar with these and other methods.

[0054] For example, the binding affinity for a target, antigen, or epitope X can be tested in an experiment in which a binding protein to be tested is captured on ELISA plates coated with X or a molecule comprising the epitope X, and a biotinylated detector antibody is added, followed by streptavidin-conjugated horseradish peroxidase (HRP). Alternatively, said detector antibody can be directly conjugated with HRP. Tetramethylbenzidine (TMB) substrate is added and the absorbance at 450 nm is measured using a multi-well plate reader for ELISA. The person skilled in the art can then interpret the results obtained from such experiments to establish at least one measure. Petition 870250082258, dated 12 / 09 / 2025, page 39 / 171 20 / 102 qualitative measurement of the binding affinity for X of the binding protein. If a quantitative measurement is desired, for example, to determine the EC50 value (half the maximum effective concentration) for the interaction, ELISA can also be used. The response of the binding protein against a series of dilutions of X can be measured using ELISA, as described above. The person skilled in the art can then interpret the results obtained from such experiments and the EC50 values ​​can be calculated from the results, using, for example, GraphPad Prism v.9 and nonlinear regression.

[0055] As used herein, the term EC50se refers to half the maximum effective concentration of the binding protein that induces a response midway between baseline and maximum after a specified exposure time.

[0056] Alternatively, or in addition, the inhibition ELISA can be used to obtain a quantitative measure of interaction by determining the IC50” (half the maximum inhibitory concentration). In an inhibition ELISA, the concentration of target X in a fluid sample is measured by detecting interference in an expected signal output. In principle, a known substance containing a target or epitope is used to coat a multi-well plate. Simultaneously, a binding protein with supposed putative affinity for X is added and incubated with a solution containing the target at varying concentrations. Following standard blocking and washing steps, samples containing the mixture of said binding protein and target are added to the well. The detection antibody labeled with affinity for the binding protein is then applied for detection using relevant substrates (e.g., TMB).In principle, if there is a high concentration of the target in the fluid sample, a significant reduction in the signal output will be observed. Conversely, if there is very little target in the fluid sample, there will be very little reduction in the expected signal output. The expert understands. Petition 870250082258, dated 12 / 09 / 2025, page 40 / 171 21 / 102 that the signal output also depends on the affinity of the binding protein to the target in question.

[0057] As used herein, the term IC50 refers to half the maximum inhibitory concentration of a binding protein that induces a response midway between baseline and maximum inhibition after a specified exposure time. Here, a lower IC50 value indicates that a lower target concentration is required to interfere with the binding of the detection antibody to the known target coated on the plate, compared to a higher IC50 value. Thus, a lower IC50 value typically corresponds to higher affinity.

[0058] The binding affinity of a binding protein can also be tested by surface plasmon resonance (SPR). For example, affinity can be tested in an experiment in which the target or epitope X is immobilized on a sensor chip of the instrument and the sample containing the binding protein to be tested is passed over the chip. Alternatively, the binding protein to be tested can be immobilized on a sensor chip of the instrument, and a sample containing X is passed over the chip. The person skilled in the art can then interpret the results obtained from such experiments to establish at least a qualitative measure of the binding affinity for X of the binding protein. If a quantitative measure is desired, for example, to determine a KD value for the interaction, SPR can also be used. Binding values ​​can, for example, be set on a Biacore (Cytiva) or ProteOn XPR 36 (Bio-Rad) instrument.The target or epitope is suitably immobilized on a sensor chip of the instrument, and samples of the binding protein whose affinity is to be determined are prepared by serial dilution and injected. KD values ​​can then be calculated from the results using, for example, the 1:1 Langmuir binding model of Biacore Insight Evaluation Software 2.0 or... Petition 870250082258, dated 12 / 09 / 2025, page 41 / 171 22 / 102 other suitable software, usually provided by the instrument manufacturer.

[0059] Binding affinity can also be measured by biolayer interferometry (BLI), a label-free technology for measuring biomolecular interactions within the interactome. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a protein layer immobilized at the tip of the biosensor and an internal reference layer. Binding between a ligand (target or epitope X) immobilized on the surface of the biosensor tip and an analyte (such as a binding protein with a presumed affinity for X) in solution produces an increase in optical thickness at the biosensor tip, resulting in a change in wavelength, Δλ, which is a direct measure of the change in the thickness of the biological layer. Interactions are measured in real time, providing the ability to monitor binding specificity, association and dissociation rates, or concentration, with precision and accuracy.

[0060] A person skilled in the art is aware of the methods mentioned above and of other methods for measuring the affinity of a binding protein for a target or epitope X, qualitatively or quantitatively, or both. Pharmaceutical compositions

[0061] In a second aspect, the disclosure provides a pharmaceutical composition comprising a binding protein as described herein and at least one pharmaceutically acceptable excipient or carrier.

[0062] Techniques for formulating polypeptides, such as antibodies and their derivatives for human therapeutic use, are well known in the field and are reviewed, for example, in Wang et al. (2007), J Pharm Sci, 96:1-26, the content of which is incorporated here in full. Petition 870250082258, dated 12 / 09 / 2025, page 42 / 171 23 / 102

[0063] Pharmaceutically acceptable excipients that may be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial mixtures of glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool grease.

[0064] In certain embodiments, pharmaceutical compositions are formulated for administration to a subject by any suitable route of administration, including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual) and transdermal injection. In preferred embodiments, the composition is formulated for intravenous or subcutaneous administration. Methods of prevention, treatment, diagnosis, prognosis and detection

[0065] The binding protein according to the present disclosure may be useful as a therapeutic, prophylactic, diagnostic and / or prognostic agent.

[0066] Therefore, in a further aspect of the disclosure, a binding protein is provided according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a medicament.

[0067] In another aspect of the revelation, a binding protein is provided according to the first aspect, or a Petition 870250082258, dated 12 / 09 / 2025, p. 43 / 171 24 / 102 pharmaceutical composition according to the second aspect, for use as a diagnostic agent.

[0068] In another aspect of the disclosure, a binding protein is provided according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a prognostic agent.

[0069] Methods are also provided for the prevention, treatment or diagnosis of diseases or for the assessment of disease prognosis, wherein a binding protein, as disclosed herein, is administered to an individual in need thereof, normally a human individual.

[0070] The use of the disclosed binding protein for the manufacture of compositions (such as medicines) for use in the prevention, treatment, diagnosis and / or prognosis of any of the listed diseases is also provided.

[0071] Thus, in one embodiment, the binding protein, or pharmaceutical composition comprising it, is useful in the treatment, prevention, diagnosis and / or prognosis of a neurodegenerative disorder, for example, a disorder selected from Alzheimer's disease and other disorders associated with Aβ protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease and familial amyloid neuropathy.

[0072] In a more specific embodiment, the aforementioned disorder is selected from Alzheimer's disease and other disorders associated with Aβ protein aggregation, Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, disease of Petition 870250082258, dated 12 / 09 / 2025, page 44 / 171 25 / 102 Parkinson's disease (PD), Parkinson's disease dementia (PDD), and the Lewy body variant of Alzheimer's disease.

[0073] In an even more specific embodiment, the aforementioned disorder is selected from Alzheimer's disease and other disorders associated with Aβ protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), in particular Alzheimer's disease.

[0074] In an alternative embodiment, the binding protein, or pharmaceutical composition comprising it, is useful in the treatment, prevention, diagnosis and / or prognosis of another disorder, for example, a disorder selected from brain cancer, multiple sclerosis and lysosomal storage diseases.

[0075] In another aspect, a method is provided for the treatment, prevention, diagnosis and / or prognosis of a disorder as listed above, said method comprising administering to said mammal an amount, such as a therapeutically effective amount, of a binding protein, or pharmaceutical composition comprising it. Incorporation by reference

[0076] Several publications are cited in this application, each of which is incorporated herein by reference in its entirety. Brief description of the figures

[0077] Figure 1 shows the X-ray structure, determined as described in Example 5, of the complex between the ectodomain of the human transferrin receptor (hTfR1) and the VH / VL pair of the h26D3 ligand of hTfR1, here representing M1. As shown, h26D3, or another ligand with the same epitope specificity, binds to the hTfR1 side in the protease-like domain, placing the ligand in a unique position in the hTfR1 structure. Because hTfR1 is a homodimer, each hTfR1 can bind to two h26D3 ligands, one on each side. When used as a moiety in a protein of Petition 870250082258, dated 12 / 09 / 2025, page 45 / 171 26 / 102 According to the revelation, the VH / VL pair provides four different possible binding points indicated in the structure image: (VL-C) C-terminal amino acid residue of the variable region of the light chain; (VH-N) N-terminal amino acid residue of the variable region of the heavy chain; (VH-C) C-terminal amino acid residue of the variable region of the heavy chain; and (VL-N) N-terminal amino acid residue of the variable region of the light chain.

[0078] Figure 2 illustrates two different design options for creating an hTfR1-binding scFv for use, in this document, as M1. Depending on the order in which the VH and VL variable domains are linked in the scFv, different locations in the scFv can be used as binding sites for the second M2 portion, for example, in the form of an antibody or other Fc-containing protein. (A) illustrates the “first VH” configuration of the hTfR1-binding scFv and shows that the scFv linker between the variable domains is located below the scFv relative to the orientation of hTfR1 when anchored to a cell membrane. In this configuration, binding of the M2 portion is possible to one or both VL-C and VH-N binding sites. (B) illustrates the “first VL” configuration of the hTfR1-binding scFv and shows that the scFv linker between the variable domains is located above the scFv relative to the orientation of hTfR1 when anchored to a cell membrane.In this configuration, the M2 portion can be fixed at one or both of the VH-C and VL-N connection points.

[0079] Figure 3 is a schematic illustration of hTfR1 anchored to the cell surface and shows how the stem region of hTfR1 provides a space (“Hidden Space”) below the interaction site between portion M1 and hTfR1. Also shown in the figure are the attachment points for M2 in a development embodiment in which M1 is a scFv with “VL first” orientation. In this preferred embodiment, M2 is suitably attached to M1 via one or both VH-C and VL-N. Petition 870250082258, dated 12 / 09 / 2025, page 46 / 171 27 / 102

[0080] Figure 4 shows schematic illustrations of two embodiments of the revealing binding protein. In the embodiment called “Gen 2A”, M1 is provided as a scFv fused between two antibody light chains, which couple to two identical heavy chains to form an intact antibody as M2. This M2 antibody is bound to two binding sites on the scFv M1 via two linkers at the C-terminal of the first light chain and the N-terminal of the second light chain of the M2 antibody. This is a symmetrical IgG structure built from two different polypeptide chains and contains an hTfR1 linker (M1 portion) in each binding protein. More details of the Gen 2A form are revealed in WO2022 / 258841. In the so-called “Gen 2D” modality, M1 is provided as a scFv fused to the C-terminal amino acid residue of a heavy chain, HC (knob), comprising the knob portion of an asymmetric IgG knob-into-hole construct.Together with a light chain (LC) and a heavy chain (HC) with a corresponding knob-into-hole portion, but without scFv, this forms the complete IgG structure as the M2 portion of the development, having attached to it an M1 portion in the form of an scFv at the C-terminal of the Fc portion, using one of the available binding sites on the VH / VL pair of M1.

[0081] Figure 5 shows the results of a binding screening of the indicated IgG antibodies from the immunization described in Example 1 in relation to human (hTfR1), cyno (cTfR1) and mouse (mTfR1) TfR1 in crude hybridoma supernatants by biolayer interferometry (BLI).

[0082] Figure 6 shows the result of the BLI binding analysis described in Example 2 for the indicated Fab fragments of the mouse antibodies 24B4, 26D3 and 37D10, as well as for a Fab fragment of the control antibody 8D3.

[0083] Figure 7 shows the mapping of antibody-binding epitopes to the protease-like domain of hTfR1, as described in Example 2, by selective antibody binding to Petition 870250082258, dated 12 / 09 / 2025, p. 47 / 171 28 / 102 ELISA plates coated with human TfR1 receptors, mouse TfR1 receptors, or one of three different chimeric human / mouse TfR1 receptors. Antibodies 24B4, 26D3, and 37D10 bind to hTfR1 (A), but not to mTfR1 (B). In addition, 24B4, 26D3, and 37D10 also bind to the chimeric h / m protease-like domain (D), but not to any of the plates coated with the other chimeric receptors (C and E).

[0084] Figure 8 illustrates the epitope clustering assay described in Example 2, with the following four main steps: Step 1 - immobilization of bio-TfR1 on the sensor chip; Step 2 - washing away non-binding material; Step 3 - binding of the competing ligand to TfR1; Step 4 - association of the ligands to the TfR1:ligand complex formed in Step 3. The data from Step 4 determine whether the two ligands investigated compete for binding to hTfR1.

[0085] Figure 9 shows the result of performing the epitope clustering assay, as described in Example 2, showing the degree of competition between antibodies for simultaneous binding to hTfR1. Binding of (A) antibody 26D3, (B) antibody 24B4 and (C) control antibody 15G11-1 to pre-formed hTfR1 complexes and any of the indicated antibodies. The binding responses for all antibodies are normalized to the binding response measured to free hTfR1 (no competing antibody).

[0086] Figure 10 shows the binding of the indicated ligands to hTfR1 on the surface of the cells studied as described in Example 2. The Y-axes of both diagrams show the mean fluorescence intensity when labeling cells with (A) whole antibodies and (B) Fab fragments of the indicated ligands. No background labeling is detected with the IgG negative isotype control (A) or the unrelated Fab fragment, Ly128 (B).

[0087] Figure 11 shows the result of the competition analysis of the indicated ligands with ferritin and transferrin, Petition 870250082258, dated 12 / 09 / 2025, page 48 / 171 29 / 102 as described in Example 3. The diagrams show (A) MFI of the indicated ligands binding to TfR1 expressed on the surfaces of THP-1 cells, (B) MFI of ferritin on the cell surface when exposed to the indicated ligands, with the positive control antibody MA-712 competing with ferritin, and (C) MFI of transferrin on the cell surfaces when exposed to the indicated ligands.

[0088] Figure 12 is a collection of sensorgrams showing the result of the SPR analysis of the original 26D3 and the humanized 26D3 as described in Example 4 (h26D3) in Fab formats when connected to hTfR1 and cTfR1 as indicated.

[0089] Figure 13 shows the results of BLI and ELISA binding studies performed on mouse and humanized versions of 26D3 in an scFv format, as described in Example 4. (A) Sensorgrams obtained by BLI measurement of the binding of the indicated constructs to hTfR1. (B) Binding responses from ELISA measurement of the binding of the indicated constructs to coated TfR1.

[0090] Figure 14 presents X-ray structure representations of the 26D3-Fab and hTfR1 complex, determined as described in Example 5. The chain names used in the coordinate files are indicated. (A) Refined structure showing the overall folding of three independent complexes in the asymmetric unit. (B) Example of electron density (2m|Fo|-D|Fc|) contoured at the 1σ level. Protein chains are drawn in ribbon representation, while sugars are shown in stick representation.

[0091] Figure 15 is a ribbon representation of the human TfR1 h26D3-Fab complex determined by X-ray crystallography, as described in Example 5. h26D3-Fab is shown in dark gray and hTfR1 in white. The binding interface (epitope / paratope) is encircled. Petition 870250082258, dated 12 / 09 / 2025, page 49 / 171 30 / 102

[0092] Figure 16 is a representation of the surface area of ​​hTfR1 with the binding sites for the indicated natural ligands ferritin and transferrin, as well as the epitope for the 26D3 ligand of the present disclosure. The different binding sites and epitopes are represented with a circle around each specific site.

[0093] Figure 17 illustrates the work of generating and characterizing an hTfR1-KI mouse model, as described in Example 6. (A) Schematic illustration of the hTfR1-KI mouse transgenic construct. The extracellular domain of human TFRC was inserted into the murine Tfrc gene by homologous recombination. (B) Quantitative analysis by reverse transcription PCR (RT-qPCR) of the expression of mouse Tfrc and human TFRC genes in the brain (N = 3 / genotype). hTfR1-KI mice (gray circles) express human TFRC and mouse Tfrc in the total brain homogenate, WT littermate siblings express only mouse Tfrc (white). (C) Western blot analysis for hTfR1, total TfR1, and hTfR1-KI in the brain. hTfR1-KI animals at 6 to 8 months (N = 5) and 15 months (N = 4) express comparable levels of the hTfR1 protein. Total TfR1 levels are comparable between hTfR1-KI and WT transgenic siblings (N=3).

[0094] Figure 18 shows the results of in vivo analysis of brain and plasma exposure to various indicated hTfR1 binding molecules in hTfR1KI transgenic mice, as described in Example 7. (A) Brain exposure 24 h after intravenous administration of the indicated hTfR1 ligands. (B) Plasma exposure 24 h after intravenous administration of the indicated hTfR1 ligands. (C) Brain:plasma ratio 24 h after intravenous administration of the indicated hTfR1 ligands. The negative control is denoted 158 and the positive control 15G11-1. Error bars represent mean ± SD. (n=4 per construct tested). Petition 870250082258, dated 12 / 09 / 2025, page 50 / 171 31 / 102

[0095] Figure 19 shows the results of in vivo analysis of brain exposure to various hTfR1-binding molecules indicated in hTfR1-KI mice by immunohistochemistry, as described in Example 8. Cortical cerebral capillary staining observed for various binding molecules, including h26D3. The hTfR1 reference ligand 15G11-1” and the TfR1 non-binding Rec158” were used as positive and negative controls, respectively.

[0096] Figure 20 shows the BLI sensorgrams for the indicated alanine variants of h26D3, as described in Example 9. Each variant exhibited a different kinetic profile, illustrating the possibility of generating variants with different affinities against human TfR1 with specific mutations in the CDR regions of the heavy or light chain.

[0097] Figure 21 shows representative SPR sensorgrams of the interaction between the indicated alanine variants of h26D3 with hTfR1 and cTfR1, measured as described in Example 9.

[0098] Figure 22 shows the results of the indirect ELISA analysis of the binding of the indicated alanine variants of h26D3 with hTfR1 and cTfR1, measured as described in Example 9.

[0099] Figure 23 shows the SPR sensorgrams of the interaction between the indicated alanine variants of h26D3, studied as building blocks of scFv within a bispecific protein format, as described in Example 9.

[0100] Figure 24 is an illustration of two different Gen 2A constructs designed and produced as described in Example 10.

[0101] Figure 25 shows the result of the purification of different Gen 2A constructs, produced and purified as described in Example 11. The monomeric content of the bispecific binding proteins was high (generally >98%) and they were produced at low mg / l levels. Purity was analyzed using Coomassie blue staining for SDS-PAGE. Petition 870250082258, dated 12 / 09 / 2025, page 51 / 171 32 / 102

[0102] Figure 26 shows sensorgrams from the SPR binding analysis of the fourteen Gen 2A binding protein constructs and control, as described in Example 12. One sensorgram for each indicated variant is shown, illustrating that all constructs were functional and bound to hTfR1.

[0103] Figure 27 shows cell binding data for the indicated Gen 2A constructs, measured as described in Example 13. All constructs tested bound similarly to cells expressing hTfR1.

[0104] Figure 28 is a collection of diagrams showing the results of CDC measurements in Branch cells, as described in Example 14, with the indicated test constructs #1-7 (setting “VH first”).

[0105] Figure 29 is a collection of diagrams showing the results of CDC measurements in Branch cells, as described in Example 14, with the indicated test constructs #8-14 (setting “VL first”).

[0106] Figure 30 is a collection of diagrams showing the results of the in vivo pharmacokinetic study described in Example 15. Plasma and brain exposure is shown after iv administration of the indicated test constructs, from terminal samples collected at 4, 24, 72, 168, and 240 h, with n = 3 mice per time point and test construct. Data are presented as mean ± SD.

[0107] Figure 31 is a diagram showing the plasma concentration profiles versus continuous sampling time with n = 3 mice per test construct after iv administration, as described in Example 15. Data are presented as mean ± SD.

[0108] Figure 32 shows 40x z-stack images of the cerebral cortex of hTfR-KI mice stained for hIgG as described in Example 16, 24 h post-dose. Three replicates (n = 3) per indicated group (LC1, HC6, and LC5) are shown. A Petition 870250082258, dated 12 / 09 / 2025, page 52 / 171 33 / 102 perfusion score (0 to 3) of each brain is given within the white squares in the lower left corner. Blood vessels are indicated by arrows.

[0109] Figure 33 shows representative 63x z-stack images of each group with hIgG vs collagen IV staining, as described in Example 16. The perfusion score (0 to 3) of each brain is given within the white squares in the lower left corner.

[0110] Figure 34 is a pair of diagrams showing the results of the in vivo study described in Example 17. Plasma (A) and brain (B) concentrations at 24 h for the indicated test constructs at the three indicated doses: 11 nmol / kg (circles), 40 nmol / kg (triangles), and 60 nmol / kg (inverted triangles). The 2A2#2-8D3 level is based on the calibrator prepared from 2A3#2-WT.

[0111] Figure 35 shows the result of the cytokine response analysis described in Example 17, displaying individual and median plasma levels of the indicated cytokines (A: TNF and KC / GRO; B: IL-6 and IL-10) for the indicated test constructs at the three indicated doses 11 nmol / kg (diamonds), 40 nmol / kg (triangles) and 60 nmol / kg (inverted triangles), pre-dose and 2 h post-dose.

[0112] Figure 36 shows the results of the production and purification of a Gen 2D construct, as described in Example 18, after gel analysis of the purified construct by Coomassie blue gel detection staining. The bands are as follows: (1) Marker; (2) mAb158-2D-h26D3-HC6, unreduced; (3) mAb158-2D-h26D3-HC6, reduced; (4) mAb158-2Dh26D3, unreduced; (5) mAb158-2D-h26D3, reduced.

[0113] Figure 37 shows sensorgrams from the SPR binding analysis of Gen 2D binding protein constructs and controls, as described in Example 19. Sensorgrams for the binding of each indicated variant to hTfR1 and cTfR1 are shown, Petition 870250082258, dated 12 / 09 / 2025, page 53 / 171 Figure 34 / 102 illustrates that all constructs are functional and link to hTfR1 and cTfR1. The Fab fragment of h2 6D3 was included as a positive control.

[0114] Figure 38 shows the results of the ADCC assay described in Example 20, using rituximab as a positive control. (A) Validation of the assay configuration with rituximab, which induces a strong induction (in times) in the presence of target cells (diagram labeled Rituximab). Without target cells, there is no ADCC induction by rituximab (diagram labeled Rituximab (without target cells)”). Similarly tested with and without target cells was the mAb158 antibody, i.e., antibody without the hTfR1-binding M1 portion. (B) Analysis of the 2D Gen format with scFvde h26D3 in the VL configuration first” as M1 and mAb158 as M2, showing no ADCC induction. In the same assay, rituximab exhibits a strong induction, while the mAb158 antibody alone shows no indication of ADCC activity.

[0115] Figure 39 shows cell linkage data for the indicated Gen 2D construct in K562 cells, measured as described in Example 20.

[0116] Figure 40 shows the results of the in vivo pharmacokinetic study described in Example 21. A: Mean (±SD) plasma (filled) and brain (open) concentration profiles at time of mAb000-Gen2D-h26D3-HC6 (diamonds) and mAb000 (squares) in hTfR-KI mice after a single intravenous injection of 40 nmol / kg (n = 3 to 5 per sampling point). B: Mean (±SD) plasma concentration profiles at time of mAb000-Gen2D-h26D3-HC6 (diamonds) and mAb000 (squares) from continuous sampling in hTfR-KI mice after a single intravenous injection of 40 nmol / kg (n = 5).

[0117] Figure 41 shows 40x Z-stack images of the frontal cortex of 5XFAD x hTfR-KI mouse brains 72 h after injection with mAb000-Gen2D-h26D3-HC6 (A) or mAb000 (B), as Petition 870250082258, dated 12 / 09 / 2025, p. 54 / 171 35 / 102 described in Example 22. Representative image of total β-amyloid and hIgG from two replicates (n = 2) per group.

[0118] Figure 42 is a diagram showing the competition between the indicated test constructs for hTfR1 binding by the M-A712 antibody on cell surfaces, measured by flow cytometry, as described in Example 23.

[0119] Figure 43 is a diagram showing the competition of human ferritin for binding to hTfR1 by antibody M-A712 on cell surfaces, measured by flow cytometry, as described in Example 23. Examples

[0120] Although the invention has been described with reference to several exemplary aspects and embodiments, it will be understood by those skilled in the art that various alterations may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. Furthermore, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from its essential scope. Therefore, it is intended that the invention is not limited to any specific embodiment but includes all embodiments that fall within the scope of the appended claims.

[0121] The invention will be further illustrated by the following non-limiting Examples. They are offered for illustrative purposes only and are not intended to limit the invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that can be altered or modified to produce essentially the same results. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations may be present. Unless otherwise indicated, the practice of the invention employs conventional methods of protein chemistry, biochemistry, Petition 870250082258, dated 12 / 09 / 2025, page 55 / 171 36 / 102 recombinant DNA and pharmacology techniques, within the scope of the art. Such techniques are fully explained in the existing literature. Furthermore, it will be evident to a person skilled in the art that the protein modification methods applied herein can also be applied to other constructs described herein and contemplated by the present inventors to fall within the scope of the disclosure. Example 1 Identification of human TfR1 ligands by immunization and screening. Immunization and screening of hybridomas.

[0122] To identify monoclonal antibodies that bind to human transferrin receptor 1 (hTfR1), four 6–10 week-old Balb / c or C57BL / 6 mice were subcutaneously immunized with the adjuvant-associated immunogen. The hTfR1 immunogen was designed to contain the ectodomain of the human TfR1 protein, N-terminally fused to a tetanus toxin T-cell epitope, P2 (Kovacs-Nolan and Mine (2006), Biochim Biophys Acta 1760:1884–1893) via a GSS linker and an N-terminal 1Qx histidine tag (His10-P2-hTfR1; SEQ ID NO: 52). Following gene constructing, recombinant His10-P2-hTfR1 protein was generated by transient transfection into Hek293 cells using the Expi293™ expression system (Gibco), purified on a nickel column (HisTrap FF, cat. no. 17-525501, GE Healthcare), exchanged for PBS buffer, and concentrated to 1 mg / ml. The expressed TfR1 immunogen was aliquoted and stored at -80 °C until use.The adjuvant Quil-A (vac-quil, InvivoGen) was used in all immunizations except for the final booster injection, in which no adjuvant was included. For use, Quil-A was resuspended in ddH2O at a concentration of 1 mg / ml, sterile filtered, and aliquoted into 0.1 ml aliquots stored at -80 °C. Quil-A was administered at a dose of 10 g / mouse.

[0123] The animals were immunized monthly with the recombinant immunogen, His10-P2-hTfR1, mixed and Petition 870250082258, dated 12 / 09 / 2025, p. 56 / 171 37 / 102 co-administered with Quil-A. Three weeks after each immunization, blood samples were collected and plasma was analyzed for the presence of antibodies reactive to human TfR1 and recombinantly produced murine TfR1. Titers were considered high enough when the ELISA response at a 1 / 100,000 dilution exceeded the mean of the whites (i.e., background signal) plus 3 standard deviations from the whites. The four mice used in this study received between 4 and 6 immunizations each.

[0124] Three days prior to fusion, the final intraperitoneal booster injection was administered to the mice in the absence of adjuvant. At sacrifice, the mice were anesthetized with isoflurane. Intact spleens were collected through an opening in the abdominal cavity and dissected. Briefly, a single cell suspension from the spleen of an immunized mouse was prepared and mixed with Sp2 / 0 cells in a 3:1 ratio. The cells were fused using PEG and added to a vial of ClonaCell™-HY Medium D (STEMCELL Technologies). 60 to 70 µl per well were then dispensed into 96-well plates. After 6 to 7 days, 150 µl of HAT medium were added to each well in the 96-well semi-solid plates. The following day, 120 µl of supernatant were discarded from each well and 100 µl of fresh HAT medium were added.The following day, 100 µl of supernatant from each well were collected and transferred to a storage plate and tested for the presence of antibodies against mouse TfR1 using indirect ELISA on nickel-coated plates, according to the protocol below. A repeat screening of the hybridoma plates was performed by adding 120 µl of HAT medium on day 12 and 3 days later transferring 25 µl of supernatant to ELISA plates for screening for reactivity against mouse TfR1 (both screenings are referred to as “primary screening”). Clones that were positive for mouse TfR1 with OD>0.2 were transferred to 24-well plates and cultured. Petition 870250082258, dated 12 / 09 / 2025, page 57 / 171 38 / 102 for at least 3 days and subjected to a secondary screening for reactivity toward murine, human, and cynomolgus TfR1 in solution using biolayer interferometry (BLI) (referred to as “secondary screening”). Although binding of hTfR1 and cynomolgus TfR1 was indicated, only very weak binding or no binding was detected for mTfR1 in the secondary screening. The supernatants from the 24-well plates were also screened for binding to His-labeled hTfR1, as well as lack of binding to His-labeled β-amyloid precursor protein (APP; negative control) using direct-coated TfR1 plates and nickel-coated plates, as described below. Binding to cynomolgus TfR1 (cTfR1) was also analyzed using direct-coated TfR1.Notably, the ELISA responses (OD450 values) were very low for mTfR1 compared to hTfR1 and cTfR1, indicating weaker binding to mTfR1 compared to binding to hTfR1 and cTfR1 for all positive clones.

[0125] The selected clones were diluted using limiting dilution assays (LDA) to achieve monoclonality. Reactivity against mouse TfR1 and human TfR1 was retested by ELISA in monoclonal cultures after LDA and expansion. Indirect ELISA screening

[0126] ELISA assays were performed according to standard ELISA protocols to screen plasma samples for reactivity to target antigens after immunizations, or to identify hybridoma clones producing antibodies reactive to the target protein TfR1. Briefly, 96-well half-area plates (Corning) were coated with 1 pg / ml of His10-mTfR1 (SEQ ID NO: 53) or His10hTfR1 (SEQ ID NO: 54). His10-mTfR1 and His10-hTfR1 were recombinantly produced and purified using the procedure described above for the immunogen His10-P2-hTfR1. The plates were blocked with 150 pg / well of blocking solution free of Petition 870250082258, dated 12 / 09 / 2025, page 58 / 171 39 / 102 protein (Pierce) for 1 h at room temperature with shaking (600 to 900 rpm). The plates were washed four times with PBS containing 0.1% TWEEN®-20 and Kathon™. Serially diluted plasma samples from an initial dilution of 1 / 450 or 1 / 2 diluted hybridoma supernatants were added to the plates (50 pl / well; dilution buffer: PBS with 0.1% BSA and 0.05% TWEEN®-20) and incubated for 2 h at room temperature, after which the plates were washed four times. The detection antibody (HRP-conjugated with anti-mouse IgG, Southern Biotech, cat. no. 1030-05, diluted 1 / 5000 in dilution buffer) was added at 50 µl / well and the plates were incubated for 1 h at room temperature. After another wash (as above), 50 µl / well of TMB substrate (K-Blue® Aqueous, Neogen) was added and the reaction was stopped after 10–15 min with 50 µl / well of 0.5 M H2SO4. The optical density at 450 nm was read using a plate reader (Tecan).Final yields were defined as the above-average dilution of the blank wells (bottom signal) plus 3 standard deviations from the blank wells.

[0127] Primary screening of hybridoma clones producing antibodies reactive against the target protein was performed using nickel-coated ELISA plates. Briefly, 96-well Ni-coated plates (PIERCE), pre-blocked with BSA, were incubated with 3 pg / ml (100 µl) of His10-mTfR1, without agitation, overnight at 4 °C. The plates were washed four times with PBS containing 0.1% TWEEN®-20 and Kathon™. Hybridoma supernatants diluted 1 / 4 were added to the plates (dilution buffer: PBS with 0.1% BSA and 0.05% TWEEN®-20) and incubated for 2 h at room temperature, after which the plates were washed four times. The detection antibody (HRP-conjugated anti-mouse IgG, Southern Biotech, cat. no. 1030-05, diluted 1 / 5000 in dilution buffer) was added at 100 µl / well, and the plates were incubated for 1 h at room temperature. After another wash (as above), 100 µl / well of Petition 870250082258, dated 12 / 09 / 2025, page 59 / 171 40 / 102 aqueous K-Blue® substrate (Neogen) was added and the reaction was stopped after 10 to 15 min with 100 μl / well of 0.5 M H2SO4. The optical density at 450 nm was read using an ELISA plate reader (Tecan).

[0128] Examples of clones considered positive for mouse TfR1 and human TfR1 binding are shown in Table 1. It was also confirmed that these clones bind to both His-tagged hTfR and cTfR by ELISA, and do not bind to His-tagged APP (negative control). The selected clones were subsequently characterized in several assays. Petition 870250082258, dated 12 / 09 / 2025, page 60 / 171 Table 1: Examples of clones identified in hybridoma screening Clone Specificity for mTfRl by indirect ELISA (Ni Capture) Binding to hTfR by BLI Binding to cynoTfR by BLI Binding to mTfRl by BLI Isotype (from sequencing) 24B4 OD>0.2 Yes Yes No / weak IgG2a / κ 26D3 OD>0.2 Yes Yes No / weak IgG2a / κ 37D10 OD>0.2 Yes Yes No / weak IgG2a / κ 41 / 102 Petition 870250082258, dated 12 / 09 / 2025, page 61 / 171 42 / 102 Measurements by biolayer interferometry

[0129] The selected clones were investigated using biolayer interferometry (BLI) on an Octet instrument (Octet Red384, ForteBio). In the configuration used, the method adopted involves capturing IgG from the respective clone at the individual sensor tips to allow the detection of antibodies that bind to the target in solution. In addition to providing a binding measure, BLI measurements provide more details about the overall binding properties, as they include estimates of the activation and deactivation rate.

[0130] Figure 5 shows the results of BLI measurements for three selected clones provided as examples, with binding measured directly in the crude hybridoma supernatant. Briefly, mouse IgG antibody clones in hybridoma supernatants, diluted 1:1 in running buffer (PBS, 0.02% TWEEN®-20 and 0.01% BSA), were captured on anti-mouse capture biosensors (anti-mouse capture, AMC, Molecular Devices, Cat. 18-5580). Subsequently, the sensors with immobilized IgGs were briefly washed for 10 s before incubation in running buffer to establish a reference signal. The association with the target antigens was measured by incubating the sensors for 120 s in assay plate wells containing the following concentrations of the respective target antigens: 500 nM mTfR1, 250 nM hTfR1, and 250 nM cTfR1. All proteins were diluted in running buffer.Target dissociation was measured by incubating the biosensors in running buffer for 90 s. All clones tested, namely 24B4, 26D3, and 37D10, bind to human and cynomolgus TfR1, but very weakly to mouse TfR1. Overall, most clones showed greater cross-reactivity towards human and cynomolgus TfR1 than towards mouse TfR1. Petition 870250082258, dated 12 / 09 / 2025, page 62 / 171 43 / 102 Sequencing of selected clones

[0131] The clones of interest were cryopreserved and sequenced by shotgun sequencing of the whole transcriptome. Among the sequenced hybridoma clones were those designated 26D3, 24B4, and 37D10. Amino acid sequences were obtained for the respective variable heavy chain (VH) and variable light chain (VL) regions of these antibodies, and the complementarity-determining regions (CDRs) of these antibodies were identified using Kabat's definition. The amino acid sequences of the CDRs of the selected mouse antibody 26D3 are provided in Table 2 below. Table 2: CDR sequences of the primary antibody 26D3 Antibody VHCDR1 VHCDR2 VHCDR3 2 6D3 DYNMD (SEQ ID NO: 7) DINPDYDTTSYNEKFKG (SEQ ID NO: 8) GGYSGSSYYHPMDY (SEQ ID NO: 9) VLCDR1 VLCDR2 VLCDR3 2 6D3 KSSQSLLYSTNQKNY LA (SEQ ID NO: 10) WASTRES (SEQ ID NO: 11) QQYFIYPRT (SEQ ID NO: 12) Example 2 In vitro binding to human and cynomologus TfR1 and epitope screening

[0132] A more detailed BLI binding analysis was performed on purified and selected antibodies. The binding of Fab fragments from murine antibodies 26D3, 24B4, and 37D10 to human TfR1 and cynomolgus TfR1 was investigated. For example, the Octet Red384 BLI instrument was used to measure the binding between immobilized TfR1 and the tested Fab fragments in solution. Antibody binding to TfR1 was measured with TfR1 complexed to human transferrin (Tf) ligand. Tf / TfR1 complexes were formed on streptavidin biosensors, first loading the sensors with biotinylated human holotransferrin, followed by a complex formation step capturing hTfR1 or cTfR1 on the sensors. The final complex density on the sensors was similar for hTfR1 and cTfR1. The binding Petition 870250082258, dated 12 / 09 / 2025, page 63 / 171 44 / 102 of the antibody to TfR1 was measured during a 120 s association phase and a 300 s dissociation phase. Figure 6 shows sensorgrams for 15 nM of each of 24B4-Fab, 26D3-Fab, and 37D10-Fab, as well as for a Fab derived from the known antibody 8D3 that binds to TfR1 (Boado et al. (2009), Biotechnol Bioeng 102:1251-1258). The data indicate a similar binding profile against human and cTfR1 for 24B4-Fab and 26D3-Fab, and cross-reactive binding to both species was also detected for 37D10-Fab, while no significant binding of 8D3-Fab against human or cynomologus TfR1 was detected. It is important to highlight that the experiment shows that 24B4-Fab, 26D3-Fab, and 37D10-Fab bind to TfR1 when the natural ligand transferrin is in complex with TfR1.

[0133] Next, an ELISA experiment showed that antibodies 26D3, 24B4, and 37D10 bind to the protease-like domain of TfR1. In the ELISA experiment, human, mouse, or three different chimeric TfR1 receptors were used to coat ELISA plates (Figure 7). The ELISA protocol was slightly modified as follows from the indirect ELISA described in Example 1.In summary, ELISA plates were coated with the following His-labeled antigens at 1 μg / ml: human TfR1 ectodomain (His10hTfR1; SEQ ID NO: 55), mouse TfR1 ectodomain (His10mTfR1; SEQ ID NO: 56), chimeric TfR1 consisting of a human apical domain grafted onto the mouse TfR1 ectodomain (h / m apical domain chimera, mhHD_TFR1; SEQ ID NO: 57), chimeric TfR1 consisting of a human helical domain grafted onto the mouse TfR1 ectodomain (h / m helical domain chimera, mhHD_TfR1; SEQ ID NO: 58), or chimeric TfR1 consisting of a human protease-like domain grafted onto the mouse TfR1 ectodomain (h / m helical domain chimera, mhHD_TfR1; SEQ ID NO: 58). of the protease h / m type, mhPLD_TfR1; SEQ ID NO: 59). The coated plates were then blocked. Serial IgG dilutions of the antibodies were performed. Petition 870250082258, dated 12 / 09 / 2025, page 64 / 171 45 / 102 mice analyzed were prepared in PBS and incubated in ELISA plates. Unbound antibodies were then washed before incubating the wells with an HRP-conjugated anti-mouse IgG secondary antibody for 1 h. The plates were then washed again before adding the HRP TMB substrate for antibody development and detection of well binding. TMB development was stopped by adding 0.5 M H2SO4 to the wells, and ELISA responses were measured with OD at 450 nm in an ELISA plate reader. As illustrated in Figure 7, 26D3, 24B4, and 37D10 bind only to hTfR1 (A) and not to mTfR1 (B). There is no binding of 26D3, 24B4, or 37D10 to the construct with the human apical domain grafted onto the remainder of the mTfR1 ectodomain (C). The control antibody 15G11-1 (Yu et al. (2014), Sci Transl Med 6:261ra154) known to bind to the human apical domain shows binding to the h / m apical domain chimera as expected (C).Furthermore, 26D3, 24B4, and 37D10 bind to the chimera of the h / m protease-like domain (D), but not to any of the plaques coated with the other chimeric receptors (C and E). Additionally, the control antibody 8D3, with an epitope in the apical domain of mTfR1, binds to all plaques coated with TfR1 antigens, including this domain, i.e., mTfR1 (B), chimera of the h / m protease-like domain (D), and chimera of the h / m helical domain (E). In summary, the experiment demonstrates that the epitope or epitopes for 26D3, 24B4, and 37D10 are predominantly within the protease-like domain of hTfR1, and that this contrasts with the control antibodies 15G11-1 and 8D3.

[0134] In another BLI experiment conducted with the aim of epitope binning, it was demonstrated that the binding of 26D3 and 24B4 is directed to the same or overlapping regions of hTfR1, with one epitope located outside the apical domain (Figure 8). The BLI epitope binning experiment was performed on an Octet Red384 instrument (ForteBio) initially (Step 1) immobilizing Petition 870250082258, dated 12 / 09 / 2025, page 65 / 171 46 / 102 biotinylated hTfR1 in streptavidin biosensors (high-precision biosensors, ForteBio). Next (Step 2), a washing step was performed. Then (Step 3), the sensors loaded with hTfR1 were incubated in buffer (non-competitive reference) or 200 nM of the respective antibody (Ab) to form hTfR1:Ab complexes on the sensors. Finally (Step 4), the sensors with free hTfR1 (reference) or the respective pre-formed hTfR1:Ab complex were incubated in 200 nM of the respective antibody to measure the binding to hTfR1 in complex with the competing antibody. Figure 8 shows the representative BLI sensorgrams obtained during the main steps of the assay indicated. The signal in Step 4 is indicative of the degree of competition between the two antibodies analyzed. If antibodies compete for binding to the same epitope or to overlapping epitopes, there will be no increase in the sensorgram signal in Step 4.On the other hand, if the two antibodies tested bind to distinct and different epitopes, there will be an increase in signal in Step 4.

[0135] The results of competitive screening of antibody binding to epitopes in hTfR1 by epitope clustering, as described above, are illustrated in Figure 9. It was demonstrated that antibodies 26D3 (dark gray bars) and 24B4 (light gray bars) bind to an overlapping epitope, which is different from the apical domain epitope of hTfR1 of the control antibody 15G11-1 (black bars). Figure 9A shows that the binding response for 26D3 is reduced by more than 70% when hTfR1 is in complex with 24B4. As expected, the binding of 26D3 to the pre-formed hTfR1:26D3 complex is almost completely inhibited, illustrating that it is self-blocking. Similarly, Figure 9B shows that the binding response to 24B4 is 70% lower when hTfR1 is in complex with 26D3 and almost completely inhibited on its own. Both 24B4 and 26D3 retain the full binding response to hTfR1 when hTfR1 is in complex with the antibody. Petition 870250082258, dated 12 / 09 / 2025, page 66 / 171 47 / 102 of the control antibody 15G11-1, which has its binding epitope within the apical domain of hTfR1 (Figures 9A and 9B, black bars). As shown in Figure 9C, the control antibody 15G11-1 has similar binding responses to the apical domain of hTfR1, regardless of whether it is tested against hTfR1 without competing antibody or when the receptor is in complex with 24B4 or 26D3. In Figure 9, all responses have been normalized to the maximum binding response of the respective antibody to free hTfR1.

[0136] In addition, antibody binding to endogenous hTfR1 in brain endothelial cells was studied. Endogenous hTfR1 binding on cell surfaces was monitored using flow cytometry and human hCMEC / D3 cells (Weksler et al. (2013), Fluids Barriers CNS 10:16), which are known to express significant levels of hTfR1 on their surface. Cells that showed positive staining were plotted and the mean fluorescence intensity (MFI) is shown in Figure 10. Both Figure 10A (IgG1 antibodies) and 10B (Fab fragments) show that the cells were positively stained for hTfR1 with 24B4 and 26D3 to a similar degree (MFI) compared to the positive control antibody 15G11-1, which has a high affinity for hTfR1, and to a greater degree than the low-affinity control antibody 15G11-2 (Yu et al. (2014), supra).No background staining was detected with the negative isotype control (Figure 10A) or the unrelated Fab fragment Ly128 (Figure 10B). These data illustrate that both 24B4 and 26D3 bind to hTfR1 expressed on the cell surface. Example 3 Competition for hTfR1 binding with ferritin and transferrin

[0137] The unique binding to hTfR1 of the ligands according to the revelation, binding to the protease-like domain of hTfR1 and identified as described in Example 1, was evaluated for competition with the natural TfR1 ligands, ferritin (Ft) and Petition 870250082258, dated 12 / 09 / 2025, page 67 / 171 48 / 102 transferrin (Tf). To test ferritin competition with the antibody, the human monocytic cell line THP-1 (Sigma / ECACC) was used. Binding of scFv-Fc formate (see Example 4 below) and control antibody (M-A712) to hTfR1 on the surface of the THP-1 cell was confirmed, as shown in Figure 11A. To assess competition between ferritin and the described ligands, cells were incubated with serially diluted test ligands along with human liver ferritin (BioRad, 4420-4804) for 1 h at 4 °C. After incubation, ferritin bound to hTfR1 on the cell surface was captured using a primary sheep antibody against human liver ferritin (BioRad, AHP2179G) and analyzed by flow cytometry.The results are shown in Figure 11B and demonstrate that 26D3 scFvFc does not compete with ferritin on the cell surface, while the anti-CD71 control antibody, clone M-A712, known to bind to the same epitope on hTfR1 as Ft (Maier et al. (2016), Mol Ther Nucleic Acids 5: e321), clearly competes with Ft binding. Furthermore, for the identified 26D3 hTfR1 ligand, the impact on Ft binding is much smaller, illustrating that 26D3 has a different epitope on hTfR1 than the binding site for Ft (Figure 11B).

[0138] For transferrin competition, K562 lymphoblastic cells (Sigma / ECACC) were used. Cells were incubated with serially diluted test constructs along with Alexa Fluor 488-conjugated human holotransferrin (Thermo Fisher; T13342) and incubated for 1 h at 4 °C. hTfR1-bound transferrin on the cell surface was captured by flow cytometry, and the mean fluorescence intensity was plotted. Figure 11C shows that there is no competition between the 26D3 ligand and transferrin. When unlabeled (unconjugated) Tf was used as a positive control for competition, the binding of the labeled Tf signal (AF488) was reduced in a concentration-dependent manner. The experiment illustrates that a ligand Petition 870250082258, dated 12 / 09 / 2025, page 68 / 171 49 / 102 targeting the protease-like domain of TfR1 does not directly compete for the same epitope as transferrin.

[0139] Overall, this example shows that the binding of 26D3 to hTfR1 does not negatively affect the ability of the two endogenous ligands, ferritin and transferrin, to bind to the receptor. Example 4 Humanization of the 26D3 ligand of hTfR1

[0140] The mouse antibody Fab sequence 26D3, identified and characterized as described in Examples 1 to 3, was analyzed and an in silico model of the 3D structure of Fab 26D3 was generated using Bioilluminate Software (Schrodinger). This murine Fab model was used as input for humanization. In this process, the CDRs of the VH and VL regions of 26D3 (see Table 2; SEQ ID NO: 10 to 15) were grafted in silico into several human variable domains and some residues were mutated back to the murine structure at certain positions. Three variants with the fewest reverse mutations and other desirable characteristics were generated and extracted from the software. One of these humanized variants was selected for expression and named h26D3. h26D3 has the VH region sequence defined at SEQ ID NO: 31 and the VL region sequence defined at SEQ ID NO: 45.The humanized h26D3 version and the original murine 26D3 sequence were expressed as His-tagged Fabs by transient transfection of Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. The harvested supernatant was purified using HiTrap IMAC Sepharose FF (Cytiva) followed by size exclusion chromatography on HiLoad Superdex 200pg 26 / 600 (Cytiva). The following buffers were used: Ni-NTA wash buffer: 20 mM Tris pH 8.0, 10 mM imidazole, and 200 mM NaCl; Ni-NTA elution buffer: 20 mM Tris pH 8.0, 200 mM NaCl, and 500 mM imidazole; size exclusion buffer (SEC): 1xdPBS (Thermo Fisher). Petition 870250082258, dated 12 / 09 / 2025, page 69 / 171 50 / 102

[0141] The binding of purified Fabs to human and cynomolgus TfR1 was evaluated using surface plasmon resonance (SPR) on a Biacore 8K instrument (Cytiva), and the results are shown in Figure 12. 1 pg / ml of human TfR1 (truncated hTfR1 from SEQ ID NO: 89) or cynomolgus TfR1 (truncated cTfR1 from SEQ ID NO: 90) was immobilized on a CM5 sensor chip (Cytiva, #BR100399) using the type 2 amine coupling kit (Cytiva, #BR100633) according to the manufacturer's instructions. The h26D3 and 26D3 Fabs were injected onto the chip using serial 1:2 dilutions in five steps, starting with 25 nM. The interaction was measured using the single-cycle kinetic method with a contact time of 120 s at a flow rate of 30 µl / ml, followed by a dissociation time of 600 s. Surface regeneration between cycles was performed by injecting 3M MgCl2. Binding data were fitted to a 1:1 interaction model. The Fabs were diluted in HBS-EP+ (Cytiva, #BR100669).The experiments were performed at 25 °C. The data confirm that the humanized variant of 26D3, i.e., h26D3, maintained the ability to bind to human and cynomologus TfR1 (Figure 12). The kinetic parameters obtained in the experiment are presented in Table 3 below. Table 3: SPR analysis of murine and humanized Fabs 26D3 vs. hTfR1 and cTfR1 Ligand tested Target ka (1 / MS) kd (1 / s) Kd (M) 26D3 hTfr1 2.8 x 106 1.0 x 10-3 3.6 x 10-9 26D3 cTfR1 5.7 x 106 6.2 x 10-3 1.0 x 10-8 h26D3 hTfr1 1.2 x 106 4.5 x 10-3 3.2 x 10-9 h26D3 cTfR1 2.7 x 106 1.5 x 10-3 5.8 x 10-9

[0142] Both murine 26D3 and the humanized h26D3 variant were converted to the scFv format and confirmed to maintain target binding as scFv (Figure 13). Murine and humanized 26D3 were reformatted to scFv (SEQ ID NO: 60 and SEQ ID NO: 61, respectively) and produced as Fc-fused monovalent scFv antibody fragments using the Petition 870250082258, dated 12 / 09 / 2025, pp. 70 / 171 51 / 102 knob-into-hole (KiH) technology. In this format, an scFv fragment is fused only to half of the Fc knob (SEQ ID NO: 62), while half of the Fc hole (SEQ ID NO: 63) is left unfused. The resulting antibody format is a single-arm scFv-Fc. The 26D3 scFv fused to half of the Fc knob has the complete amino acid sequence SEQ ID NO: 64, while the h26D3 scFv fused to half of the Fc knob has the complete amino acid sequence SEQ ID NO: 65. The binding profiles for murine and humanized 26D3 in this scFv format are similar and confirm binding activity in the scFv format. The binding responses are consistent with those of the antibody in Fab format. This was confirmed by several methods, including a kinetic experiment using BLI (results shown in Figure 13A) and an ELISA (results shown in Figure 13B).The binding kinetics for murine and humanized 26D3-scFvFc were measured by BLI, first immobilizing biotinylated hTfR1 on streptavidin biosensors (Fortebio). The sensors were then washed in buffer (Kinetics buffer, Fortebio) before measuring the association of 26D3-scFv-Fc (murine) and h26D3-scFv-Fc (humanized) at concentrations of 25 nM, followed by a 500 s dissociation phase. In the ELISA experiment, hTfR1 was used to coat the plates for standard binding ELISA experiments using the protocol for indirect ELISA described in Example 1. Example 5 Crystallization and structure determination of h26D3-Fab in complex with hTfR1

[0143] This example describes the crystallization of a complex between h26D3-Fab and hTfR1 and the determination of the binding interface. The ectodomain of human TfR1 (SEQ ID NO: 55) was expressed by transient transfection of human embryonic kidney cells (Expi297; Thermo Fisher Scientific) according to the manufacturer's instructions. The harvested supernatant was purified using Petition 870250082258, dated 12 / 09 / 2025, pp. 71 / 171 52 / 102 HiTrap IMAC Sepharose FF (Cytiva) followed by size exclusion chromatography on HiLoad Superdex 200pg 26 / 600 (Cytiva). The buffers used and the purification of the humanized Fab were as described in Example 4.

[0144] The formation of a complex between humanized h26D3-Fab and hTfR1 was achieved by mixing the two components in a 1:1 molar ratio in 1x dPBS and incubating at room temperature for 1 h. Subsequently, the complex was purified using size exclusion chromatography on a HiLoad Superdex 200pg 26 / 600 (Cytiva), as described in Example 4.

[0145] Crystallization was performed using a 15 mg / ml hTfR1-h26D3 stock solution in PBS, which was diluted to 4 mg / ml in PBS supplemented with 4 mM β-mercaptoethanol. A 100+100 nl drop was prepared using the reservoir additive screen: 0.1 M sodium potassium phosphate pH 6.5, 10% PEG 3000, 0.05% dichloromethane, and 2 mM β-mercaptoethanol. The crystal was flash-frozen in reservoir solution supplemented with 8% glycerol and 16% PEG 400.

[0146] X-ray data collection and refinement were performed as follows. Data were collected at 3.87 Å on beamline I04 of the Diamond Light Source. The beamline was equipped with a DECTRIS Eiger2 XE 16M detector. The dataset was integrated using XDS (Kabsch (2010), Acta Crystallogr D Biol Crystallogr 66:125-132) with anisotropic scaling STARANISO (Tickle et al. (2018), Global Phasing (Ltd) and diffracted to 3.87 Å along the c* direction of the reciprocal lattice and to 4.82 Å in the a* / b* plane. Three complexes were found in the asymmetric unit. The structure was refined using the Buster refinement software, and model building was performed in Coot. The parameters and statistics of data collection and refinement are presented in Table 4 below. Table 4: Statistics of X-ray diffraction data collection and refinement Petition 870250082258, dated 12 / 09 / 2025, page 72 / 171 53 / 102 Resolution (λ) 110.81 at 3.87 (4.27 at 3.87) Wavelength (λ) 0.97950 Space group P31 2 1 Unit cell (λ) a = b = 127.95, c = 472.91 Spherical completeness (%) 63.9 (12.7) Ellipsoidal completeness (%) 93.8 (74.0) No. of observations / unique reflections 546922 / 27520 (27874 / 1378) Redundancy 19.9 (20.2) <I / o(I) >9.8 (1.7) CC(l / 2) (%) 99.9 (81.5) Rmerge (I) (%) 20.6 (200.9) Rpim (I) (%) 6.6 (62.8) Rmodel (F) (%) 26.0 (31.6) Rfree (F) (%) 31.4 (34.3) No. of non-hydrogen atoms 24858 No. of water molecules 0 rms deviations from ideal geometry Bond lengths (Â) 0.009 Bond angles (°) 1.1 Average factor B protein chain A / B / C / D / E / F / G / H / L (Â2) 76.9 / 110.0 / 103.0 / 132.2 / 134.4 / 87.2 / 101.0 / 79.9 / 83.4 Average factor B glycosylations (Â2) 70.7 Ramachandran plot quality Favored regions (%) 93.4 Allowed regions (%) 4.3 Outliers (%) 2.3 ​

[0147] The final, refined structure of the complexes showing the overall folds is shown in Figure 14. As shown in Figure 14A, there were three independent complexes in the asymmetric unit. The chain names used in the coordinate files are indicated. Figure 14B shows an example of the electron density contoured at the interface between human TfRl and the heavy / light chain of h26D3-Fab. Protein chains are drawn in ribbon representation, while sugars are shown in stick representation. The interaction of the binding interface between h26D3 and human TfRl was extracted from the structure Petition 870250082258, dated 12 / 09 / 2025, page 73 / 171 54 / 102 X-rays and described below to provide information on the precise binding of h26D3 to human TfRl.

[0148] The binding interface between human TfRl and h26D3Fab is shown in Figures 14 and 15, and the interaction was observed between the amino acid residues indicated in Table 5. Table 5: Amino acid residues involved in the interaction between h26D3 and hTfRl Human h26D3 heavy chain TfRl: 50ASP interacts with 150Asn; 57Thr interacts with 150Asn; 59Ser interacts with 150Asn; 105Ser interacts with 154Pro; 105Ser interacts with 159Ser; 105Ser interacts with 161Lys; 106Tyr interacts with 161Lys; 106Tyr interacts with 158Gly; 107Tyr interacts with 151Ser. h26D3 light chain: 32Ser interacts with 163G1U; 32Ser interacts with 385Lys; 33Thr interacts with 160Gln.

[0149] Table 5 describes the main residues on both sides involved in the epitope / paratope interface, as determined by the crystal structure. Additional residues in the vicinity are also likely important for the binding between h26D3 and human TfRl. Furthermore, as described in Example 9 below, several positions outside the observed binding interaction show important participation in the binding of h26D3 to human TfRl.

[0150] Table 6 below lists the amino acids of human TfRl that are involved in the respective interactions with h26D3, Et, and Tf. Notably, no amino acid involved in the binding of h26D3 is part of any of the binding interfaces for the endogenous ligands. This illustrates that the ligands of the present disclosure, as exemplified by h26D3, bind to human TfRl outside of the binding sites used by Et and Tf. Petition 870250082258, dated 12 / 09 / 2025, pp. 74 / 171 55 / 102 Table 6: Amino acid residues in hTfRl that interact with the respective ligand. h26D3 Ferritin* Transferrin# 150Asn 195Ser 121Arg 151Ser 197Gln 123Tyr 154Pro 199Ser 125Asp 158Gly 201Ile 12 6Asp 159Ser 208Arg 622Val 161Lys 209Leu 623Arg 163G1U 210Val 62 6Asn 385Lys 212Leu 62 9Arg 215Asn 640Gln 343G1U 643Tyr 343Lys 651Arg 344Lys 6 61Gly 348Asn 6 62Asn 374Lys 6 63Wing 6 64Glu 6 67Asp 7 57Asp 7 58Asn * Montemiglio et al. (2019), Nat Commun 10:1121 # Eckenroth et al. (2011), Proc Natl Acad Sei USA 108:13089

[0151] The different epitopes in the hTfRl structure (pdb: 1SUV) are illustrated in more detail in Figure 16. As shown in Figure 16, the Ft binding site is located in the apical domain of hTfRl, the Tf binding site is located primarily in the helical domain of hTfRl, and the h26D3 epitope is located in the protease-like domain of hTfRl. The structure illustrates that the different ligands and ligands use distinct and specific surface areas in the hTfRl structure. hTfRl is a homodimer with two identical chains, and the epitopes are indicated only on one of these chains. Example 6: Generation and characterization of knock-in mice for hTFR1

[0152] Knock-in mice for human TfRl (hTfRl-KI; TFR1C-KI) were generated by homologous recombination (experimental work carried out at Cyagen US). A cDNA vector containing the TFR1C ectodomain (NCBI reference sequence: Petition 870250082258, dated 12 / 09 / 2025, page 75 / 171 56 / 102 The murine Tfrc transmembrane and intracellular domain (NM_001128148.3) and the transmembrane and intracellular domain were introduced by pronuclear microinjection into ES C57BL / 6N Tfrc cells. The coding region of exon 2 of Tfrc plus partial intron 2 were replaced by the chimeric cassette TFR1C (Figure 17A). Correct insertion of the hTfR1 cDNA was verified by Southern blot and PCR. Transgene expression in hTfR1-KI mice was confirmed in brain tissue by qRT-PCR (Figure 17B) and Western blot (Figure 17C), indicating levels of endogenous expression. hTfR1-KI mice were maintained on a C57BL / 6N genetic background, and only heterozygous hTfR1-KI mice were used for experiments. Example 7: Brain uptake of hTfRl binding constructs in vivo

[0153] To evaluate hTfR1-mediated brain uptake in vivo, Fc-scFv constructs (see Example 4) were produced for four different binding proteins. A known ligand for hTfR1, 15G11-1, was used as a control (Yu et al. (2014), supra). This hTfR1 ligand has been described as active in vivo and is used as a positive reference control for brain uptake. In addition, a construct containing a non-hTfR1 scFv ligand based on the anti-ε-amyloid antibody mAb158 was designed and included as a negative control in the form of an Fc fusion construct (Fc-scFv158, also referred to simply as 158 here and in the figures). The different Fc-scFv constructs were injected intravenously (iv) into hTfR1 knock-in mice (hTfR1-KI) produced as described in Example 6 (n=4 per construct) at equimolar doses of 30 nmol / kg (corresponding to approximately 2.3 mg / kg). Plasma and brain exposure was assessed 24 h post-dose.

[0154] The animals were anesthetized with isoflurane and terminal blood samples were collected from the orbital plexus in BD Microtainer K2EDTA tubes. The samples were inverted and centrifuged at 2,400 xg for 10 min at 4 °C. Plasma was extracted Petition 870250082258, dated 12 / 09 / 2025, pp. 76 / 171 57 / 102 and transferred to Eppendorf tubes and frozen at -80 °C. Immediately after blood collection, the animals' abdomens were opened and a cannula (21 G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium and transcardiac perfusion was performed with at least 50 ml of cold PBS. After perfusion, the brains were extracted and the olfactory bulbs removed. The brains were separated into left and right hemispheres, and the cerebellum was removed from the left hemisphere. The left hemisphere was then weighed and flash-frozen in dry ice and stored at -80 °C until further preparation and analysis of the concentrations of the injected constructs using a Meso Scale Discovery (MSD) based assay.The right hemispheres were placed in 4% formaldehyde and stored at 4 °C for 24 h; then they were washed in cold PBS, transferred to a cold 30% sucrose solution prepared in PBS, and stored at 4 °C for subsequent immunohistochemistry (IHC) processing (see Example 8 below).

[0155] For brain concentration measurements, frozen left hemispheres were thawed on ice and homogenized in TBS by automated bead homogenization. Triton was added to the homogenate to a final Triton concentration of 0.5% before centrifugation at 16,000 xg, after which the supernatants were collected.

[0156] Plasma and brain concentrations of antihTfR1 Fc-scFv were determined using a customized MSD assay to detect human Fc. A standard 96-well MSD plate (MSD, #L15XA-3) was coated with 0.5 pg / ml of goat anti-human IgG antibody, Fcy-specific fragment (Jackson Immuno Research Europe Ltd, #109-005-098) diluted in 1x PBS (Medicago AB, #09-9400-100). After overnight incubation at 4°C, the plate was washed 4x in 1x-TWEEN PBS (Fisher Scientific, #09-9410-100) and blocked with 150 µl of 1% BlockerA in TWEEN PBS (MSD, #R93BA-4) per well. Samples and standards Petition 870250082258, dated 12 / 09 / 2025, page 77 / 171 58 / 102 corresponding molecules, ranging from 400 pM to 0.1 pM in 1:4 dilution steps, were added and incubated for 2 hours at 900 rpm at room temperature. A 1-hour incubation step with anti-human mouse IgG antibody (Mabtech, 38501-1000, MT145) diluted to 0.5 pg / ml was included, followed by a 1-hour incubation of anti-mouse antibody conjugated with SULFO-TAG (MSD, R32AC-1) diluted to 0.5 pg / ml, after which the plate was incubated for a further hour at room temperature and 900 rpm. 150 µl of MSD reading buffer (MSD, #R92TC) were added per well before reading the plates on an MSD SECTOR Imager. Between each incubation step, four washes in 1x-TWEEN PBS were performed. All antibodies and samples, except the coating antibody, were diluted in 1% Blocker A in PBS-TWEEN and added at a volume of 50 µl / well.The concentration of analytes in the samples was evaluated using MSD Workbench software, employing a 4PL curve fitting algorithm and 1 / Y2 curve weighting for the standard curve. Statistical analysis was performed in GraphPad Prism (v. 9.0.0) using one-way ANOVA with Tukey's post-hoc test.

[0157] The results are shown in Figure 18. As shown in Figure 18A, substantially higher brain concentrations were observed for both test constructs and the positive control 15G11-1, compared to the negative control (158) 24 hours after dosing. As shown in Figure 18B, plasma concentrations of both test constructs and the positive control 15G11-1 were lower at 24 h compared to that of 158, indicating that hTfR1 involvement leads to faster plasma clearance. Concentration ratios between brain and plasma are shown in Figure 18C. Both test constructs and the positive control 15G11-1 showed significantly increased brain exposure relative to plasma compared to the negative control. Taken together, the data Petition 870250082258, dated 12 / 09 / 2025, pp. 78 / 171 59 / 102 support hTfR1-mediated transport across the BBB in this experiment for the novel hTfR1 ligands tested. Example 8 Immunohistochemistry data on brain exposure

[0158] The in vivo involvement of hTfR1 by the FcscFv construct was studied in more detail using a qualitative immunohistochemical (IHC) analysis. In summary, coronal brain sections with a thickness of 20 µm were obtained from hemispheres of PBS-perfused mouse brains described in Example 7 using a cryostat (Microm NX50 CryoStar, Epredia). The sections were collected on Superfrost plus slides (Menzel-Glâser, #J1800AMNZ) and air-dried before IHC. The brain sections were washed with PBS (pH 7.4) for 15 minutes and incubated in blocking buffer (5% BSA, 0.25% Triton-X in PBS) for 2 hours at room temperature. To visualize constructs measured intravenously, brain sections were incubated with a secondary goat anti-human IgG (specific for heavy and light chains) conjugated to Alexa Fluor 488 (Invitrogen, #A11013) for 120 min at room temperature, followed by 3 washes of 15 min in PBS.The slides were mounted with Fluoromount-G (Invitrogen, #00-4958-02) for image analysis. Confocal images of the cerebral cortex were captured using a Leica Stellaris 5 confocal system equipped with an HC PL APO 40x / 1.25 GLYC motCORR CS2 objective (Leica, #11506423).

[0159] Distinct immunofluorescence signals in IHC were observed in brain capillaries with the positive reference module 15G11-1, while a minimal IHC signal was detected in brain sections of mice injected with the negative control 158 (Figure 19). The IHC signal in the brain capillary was observed for both test constructs h26D3 and 37D10, of which h26D3 showed the strongest immunofluorescence signal, comparable to the positive control 15G11-1. Taken together, the Petition 870250082258, dated 12 / 09 / 2025, pp. 79 / 171 60 / 102 MSD (Example 7) and IHC (this Example) analyses demonstrate that hTfR1 ligands in an scFv format exhibit increased brain exposure in hTfR1-KI mice. Example 9: Affinity variant generation and affinity determinations

[0160] Several variants of the parental antibody h26D3 were generated by replacing tyrosine, tryptophan, and aspartic acid residues in the CDRs, one by one, with alanine residues. The resulting variant VH regions were named HC1-HC13, and their amino acid sequences are provided in the sequence listing as SEQ ID NO: 32 to 44, respectively. The variant CDR sequences comprised within these variant VH regions are listed as SEQ ID NO: 13 to 25, respectively. The resulting variant VL regions were named LC1-LC6, and their amino acid sequences are provided in the sequence listing as SEQ ID NO: 46 to 51, respectively. The variant CDR sequences comprised within these variant VL regions are listed as SEQ ID NO: 26 to 30, respectively. Table 7 below provides a summary of the specific mutations in each of the alanine variants. Table 7: Alanine substitution variants of VH and VL of h26D3 Variant Mutation Variant Mutation Variant Mutation LC1 Y31A HC1 D31A HC8 Y60A LC2 Y38A HC2 Y32A HC9 Y101A LC3 Y55A HC3 D35A HC10 Y10 6A LC4 W5 6A HC4 D50A HC11 Y107A LC5 Y97A HC5 D54A HC12 D111A LC6 Y100A HC6 Y55A HC13 Y112A HC7 D5 6A

[0161] The alanine variants generated were expressed as unique mutant Fabs labeled with His by transient transfection of Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. The clarified medium in which the Fabs were secreted, Petition 870250082258, dated 12 / 09 / 2025, page 80 / 171 61 / 102 was used to evaluate binding to hTfR1 by BLI (Octet RED384, ForteBio). Expressed Fabs were immobilized from cell supernatants on anti-Fab biosensors for 240 s. Subsequently, the association of the hTfR1 ectodomain (SEQ ID NO: 55), diluted to 3.75 pg / ml in Kinetics lx buffer (ForteBio), to the loaded sensors was measured for 300 s, followed by dissociation for 300 s. It was confirmed that all variants bind to hTfR1, but were affected to different extents (Figure 20).

[0162] Variants that showed impaired binding to hTfRl in screening were selected for further characterization. In addition, double mutants were generated by combining heavy and light chains with alanine substitutions. Table 8 below provides a summary of the specific mutations in each of the alanine variants that were selected. Table 8: Selected h26D3 VH and VL variants for further characterization. Variant Mutation VL Mutation VH LC1 Y31A None LC5 Y97A None LC6 Y100A None HC2 None Y32A HC3 None D35A HC4 None D50A HC5 None D54A HC6 None Y55A HC8 None Y60A HC10 None Y10 6A HC11 None Y107A HC3 / LC1 Y31A D35A HC3 / LC5 Y97A D35A HC6 / LC1 Y31A Y55A HC6 / LC5 Y97A Y55A HC8 / LC1 Y31A Y60A HC8 / LC5 Y97A Y60A HC10 / LC1 Y31A Y10 6A HC10 / LC5 Y97A Y10 6A

[0163] The selected variants were expressed as His-tagged Fabs by transient transfection of cells Petition 870250082258, dated 12 / 09 / 2025, page 81 / 171 62 / 102 Chinese hamster ovary (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. Fabs were purified on a small scale with HisPur™ Ni-NTA Magnetic Beads (Thermo Scientific) according to the manufacturer's instructions, followed by buffer change to DPBS pH 7.4. Selected variants were also purified on a larger scale by application on a HisTrap Excel column (Cytiva), which was washed with 20 mM Tris, 200 mM NaCl, and 5 mM imidazole. Proteins were eluted with 20 mM Tris, 200 mM NaCl, and 500 mM imidazole, followed by buffer change to DPBS pH 7.4 using a HiPrep 26 / 10 desalting column (Cytiva). Proteins were concentrated using an Amicon Ultra centrifugal concentrator (30 MWCO; Millipore). The selected variants were subsequently polished by size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex 200; Cytiva) on DPBS pH 7.4.The analytical characterization of the protein was performed by UV protein determination, SDS-PAGE, and HPLC-SEC.

[0164] The binding of purified Fabs to human and cynomolgus TfR1 was evaluated using SPR (Figure 21) or indirect ELISA (Figure 22). For SPR, a Biacore 8K instrument (Cytiva) was used. 1 pg / ml of hTfR1 (SEQ ID NO: 89) or cTfR1 (SEQ ID NO: 90) was immobilized on a Cm5 sensor chip (Cytiva, #BR100399) using the type 2 amine coupling kit (Cytiva, #BR100633) according to the manufacturer's instructions. Fabs were injected onto the chip using a series of 1:2 serial dilutions in four steps, starting with 100 nM. The interaction was measured using the single-cycle kinetic method with a contact time of 120 s at a flow rate of 30 µl / min, followed by a dissociation time of 1000 s. Surface regeneration between cycles was performed by injecting 3M MgCl2. Binding data were fitted to a 1:1 interaction model. The Fabs were diluted in HBS-EP+ (Cytiva, #BR100669). The experiments were Petition 870250082258, dated 12 / 09 / 2025, page 82 / 171 63 / 102 performed at 25 °C. The results are shown in Figure 21 and the calculated KD values ​​are presented in Table 9 below. Table 9: SPR analysis of Fabs from the h26D3 variant vs. hTfR1 and cTfR1 Fab of variant h26D3 hTfr1 Kd (nM) cTfR1 Kd (nM) HC2 8.7 12 HC3 16 168 HC4 5.1 8 HC5 20 39 HC6 156 164 HC8 10 19 HC10 9.2 4.6 HC11 388 2.7 LC1 14 70 LC5 240 ND LC6 8.5 9.4 h26D3 10 12

[0165] For the indirect ELISA, 96-well half-area plates (Corning, No. 3690) were coated with 1 g / ml of recombinant hTfR1 ectodomain (SEQ ID NO: 74) in PBS overnight at 4 °C. The coated plates were blocked with Pierce's protein-free blocking solution (Thermo Fisher Scientific, No. 37572) for 1 h at room temperature, under shaking, and washed four times in PBS containing 0.1% TWEEN20. Serial dilutions (1:3) of various constructs expressed in incubation buffer (1% BSA, 0.1% TWEEN-20 in PBS) were incubated for 1 h at room temperature. After the four washing steps, the bound test constructs were detected by adding the anti-human IgG antibody F(ab')2-HRP (Jackson Immuno Research, #109-036-003) at a 1:5000 dilution in incubation buffer (1 h, at room temperature).After four washing steps, the K-Blue® Aqueous TMB substrate (Neogen, #331177) was added to the wells for 15 minutes at room temperature before the reaction was stopped with a 1:1 dilution of 0.5 M H2SO4. The optical density at 450 nm was recorded (Spark, Tecan) and the background signal was subtracted before analysis. The results obtained are shown in Figure 22. Petition 870250082258, dated 12 / 09 / 2025, page 83 / 171 64 / 102

[0166] Based on Biacore and ELISA measurements, several variants were identified within a wide range of affinities for human TfR1. Many variants exhibited retained cross-reactivity to cynomolgus TfR1.

[0167] Finally, selected variants were reformatted to scFv and used in the context of the bispecific linking molecule format disclosed in WO2022 / 258841. Bispecific linking molecules comprising scFv modules constructed from h26D3 and selected alanine mutants were expressed in ExpiCHO cells as described above. The filtered supernatants were applied to a MabSelect SuRe column (Cytiva) which was subsequently washed with DPBS pH 7.4. The expressed linking molecules were eluted by applying 0.7% HAc pH 2.5, followed by immediate neutralization of the sample to pH 7.5. Purified samples were subsequently polished by subjecting them to size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex 200; Cytiva) on DPBS pH 7.4. The purified constructs were concentrated using Amicon Ultra centrifugal concentrators (30 MWCO, Millipore).Each purified expressed construct was characterized using SDS-PAGE, size exclusion chromatography (Superdex 200 Increase 3.2 / 300; Cytiva), and UV protein determination. Binding to hTfR1 was assessed using SPR as described above, with adjustments to the concentration range depending on the variant. As shown in Figure 23 and Table 10 below, the different variants tested exhibited a range of affinities for the hTfR1 target. Table 10: SPR analysis of the scFv of the h26D3 variant in bispecific format vs. hTfR1 Petition 870250082258, dated 12 / 09 / 2025, page 84 / 171 65 / 102 KD variant vs hTfRl (nM) h26D3 ~10 LC1 ~40 HC6 ~100 to 200 LC5 ~400 to 500 Example 10 Gen 2A bispecific binding protein design with scFv modules “VH first” or “VL first”

[0168] Fourteen different constructs of bispecific binding proteins were designed using the “Gen 2A” format originally described in WO2022 / 258841 (Figure 4, left panel), using the hTfR1 h26D3 HC6 linker scFv format described above in two different configurations, “VH first” (VH first) as represented by SEQ ID NO: 67 and “VL first” (VL first) as represented by SEQ ID NO: 68. In the language of the present disclosure, the “scBM” of WO2022 / 258841 represents the first M1 portion described herein. Seven of the constructs were designed using the “VH first” configuration (#1 to 7, with single-chain components represented by SEQ ID NO: 69 to 75, respectively), while another seven constructs were designed using the “VL first” configuration (#8 to 14, with single-chain components represented by SEQ ID NO: 76 to 82, respectively). The antibody heavy chain used in all these constructs was the same and is represented by SEQ ID NO: 83.

[0169] Figure 24 provides a schematic overview of the different constructs tested. Constructs #1 to 7 (VH first) and #8 to 14 (VL first) were produced as a series of combinations of different linker lengths, as shown in Table 11, in order to investigate the influence of linker length and the first VH vs. VL configuration in combination with the scFv of the h26D3 HC6 ligand on hTfR1 binding and antibody positioning when hTfR1 is expressed on the cell surface. Petition 870250082258, dated 12 / 09 / 2025, page 85 / 171 Table 11: Linker properties for constructs tested in Gen 2A format Construct # Connector 1 [L1] Required connector length measured [L1] (À) Connector 2 [L2] Required connector length measured [L2] (À) Total connector size (À) Total required size measured (À) First configuration of VH 2A-3 #1 1x G4S «44 4x G4S «40 «95 «88 2A-3 #2 2x G4S 4x G4S «114 «88 2A-3 #3 3x G4S 4x G4S «133 «88 2A-3 #4 1x G4S 5x G4S «114 «88 2A-3 #5 2x G4S 5x G4S «133 «88 2A-3 #6 3x G4S 5x G4S «154 «88 2A-3 #7 5x G4S 5x G4S «192 «88 First configuration of VL 2A-3 #8 1x G4S «46 4x G4S «60 «95 «106 2A-3 #9 2x G4S 4x G4S «114 106 2A-3 #10 3x G4S 4x G4S 5x G4S «154 «106 2A-3 #14 5x G4S 5x G4S «192 «106 66 / 102 Petition 870250082258, dated 12 / 09 / 2025, page 86 / 171 67 / 102 Example 11 Production and purification of engineered Gen 2A constructs

[0170] The fourteen constructs designed as described in Example 10 were functionally expressed by transient transfection of Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. Filtered supernatants from the cell culture were applied to a MabSelect SuRe column (Cytiva), which was subsequently washed with DPBS pH 7.4. The expressed binding proteins were eluted by applying 0.7% HAc pH 2.5, followed by neutralization of the sample to pH 7.5. Purified samples were subsequently polished by size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex 200; Cytiva) on DPBS pH 7.4. Each purified expressed construct was characterized using SDS-PAGE, size exclusion chromatography (Superdex 200 Increase 3.2 / 300; Cytiva), and protein concentration determination by UV.

[0171] The purification results are presented in Table 12. A representative SDS-PAGE analysis of the purified constructs is shown in Figure 25. The unreduced gel showed a band around 175 kDa. The reduced gel showed the expected two-band profile for the Gen 2A format, with the antibody heavy chain around 50 kDa and the single-chain component comprising two hTfR1-binding scFv light chains at an approximate molecular weight of 75 kDa. As shown in Table 12, the monomeric content of the bispecific binding proteins was high (generally >98%) and they were produced at low mg / l levels. Table 12: Purification of test constructs Construct # Lot % Monomer Quantity (mg) Yield (mg / l culture) 2A-3 #1 220909 96.3 1.0 2.8 2A-3 #2 220915 96.5 5.4 14.3 2A-3 #3 220811 99.8 1.6 8.2 Petition 870250082258, dated 12 / 09 / 2025, page 87 / 171 68 / 102 Construto # Lote % monômero Quantidade (mg) Rendimento (mg / l cultura) 2A-3 #4 220811 97.3 2.8 13.8 2A-3 #5 220812 98.6 1.1 5.3 2A-3 #5 220915 99.5 0.5 1.2 2A-3 #6 220812 99.2 0.4 2.2 2A-3 #6 220915 99.9 0.1 0.3 2A-3 #7 220829 99.9 0.5 2.6 2A-3 #7 220913 99.9 1.0 2.4 2A-3 #8 220915 97.9 0.2 0.5 2A-3 #9 220826 99.6 3.2 8.5 2A-3 #9 220915 99.9 0.5 1.3 2A-3 #10 220913 99.1 6.7 15.8 2A-3 #11 220916 93.6 0.6 1.5 2A-3 #12 220913 93.0 1.2 3.3 2A-3 #13 220829 99.9 0.7 3.6 2A-3 #13 220913 99.3 0.6 1.7 2A-3 #14 220829 99.7 0.7 3.6 2A-3 #14 220913 99.8 0.8 2.0 Exemplo 12 Download the Gen 2A title with hTfRl and SPR

[0172] The binding of bispecific binding proteins, expressed and purified as described in Example 11, to hTfR1 was evaluated using SPR (Biacore 8K, Cytiva). 2 pg / ml of hTfR1 were immobilized on a Cm5 sensor chip (Cytiva, #BR100399) using the type 2 amine coupling kit (Cytiva, #BR100633) according to the manufacturer's instructions. Bispecific binding proteins were injected onto the chip using a series of four-step double dilutions, starting with 200 nM. The interaction was measured using the single-cycle kinetic method with a contact time of 120 s at a flow rate of 30 µl / min, followed by a dissociation time of 600 s. Surface regeneration between cycles was performed by injecting 3 M MgCl2. The binding data were fitted to a 1:1 interaction model. Bispecific binding proteins were diluted in HBS-EP+ (Cytiva, #BR100669). Experiments were performed at 25 °C.The data in Figure 26 show that all Gen 2A constructs designed and produced bind to hTfR1. Petition 870250082258, dated 12 / 09 / 2025, page 88 / 171 69 / 102 All constructs show similar activation and deactivation rates when compared to a Fab construct controlling the hTfR1 h26D3 HC6 ligand. This illustrates that all expressed constructs are functional and that neither the linker lengths nor the VH-first / VL-first configuration directly influence the binding of the constructs to hTfR1. Example 13 Binding to hTfR1 expressed on cell surfaces

[0173] hTfRl binding in cells was measured in the immortalized human B lymphocyte cell line Ramos (Sigma, cat: 85030802). This cell line is known to express high levels of hTfRl on the cell surface. Fcy receptors were blocked using Fc receptor blocker (Innovex biosciences, #NB309-4X-40) for 30 min at 4 °C, after which the cells were washed in PBS. Cells were seeded in a 96-well V-bottom plate (#249570, Thermo Scientific Nunc), and Gen 2A constructs in serial dilution were added and the plate was incubated at 4 °C overnight. Cells were washed with PBS with 1% BSA and then fixed with 4% freshly prepared formaldehyde (Thermo Scientific Pierce, #28906) diluted in PBS for 15 minutes at room temperature. The cells were washed with PBS containing 1% BSA and then labeled.Bispecific binding proteins linked to hTfR1 on cell surfaces were detected via their shared IgG heavy chain component using a goat secondary antibody F(ab')2 anti-IgG(y)-Alexa fluor 488 (Invitrogen Life Technologies, #H10120). Labeling was performed for 30 minutes at 4 °C. After incubation with the detection reagent, cells were washed with PBS containing 1% BSA. Cells were then resuspended in 200 g / L of PBS with 1% BSA and acquired using a BD FACSLyric flow cytometer system (BD Biosciences). Samples were analyzed using flowJo software (BD Biosciences). Petition 870250082258, dated 12 / 09 / 2025, page 89 / 171 70 / 102 median fluorescence intensities (MFI) measured were plotted against binding protein concentration and displayed in Figure 27. The results show that all Gen 2A constructs tested, regardless of linker length and “VH first / VL first” configuration, bind similarly to hTfR1 expressed on cell surfaces. Example 14 Complement-dependent cytotoxicity (CDC) analysis

[0174] Complement activity is initiated by the binding of C1q to a portion of the Fc of, for example, an antibody, leading to the binding of other complement factors, which ultimately leads to cell death. To assess whether a given bispecific test construct gives rise to any CDC activity by allowing C1q to bind to the Fc, Ramos cells (Sigma, cat: 85030802) were used as target cells for CDC analysis. To measure cell death, Ramos cells were labeled with a cell viability dye, Calcein-AM (Sigma, 17783). These labeled cells were then treated with bispecific test constructs in serial dilution in the presence of pooled human complement serum (Innovative Research Inc, #39337) for 4 ha at 37 °C, 5% CO2. As a negative control, cells were treated with a similar concentration of bispecific constructs in the presence of C1q-depleted human serum (Sigma, #234401).As a positive control, the monoclonal antibody rituximab (MabThera; Roche) was also tested under both conditions (pooled complement serum and C1q-depleted serum).

[0175] Treated cells were acquired using a BD BDLyric flow cytometer (BD Biosciences). Samples were analyzed using flowJo software (BD Biosciences). The frequency of cell death was determined in calcein AM labeled with suppressed fluorescence selected by Petition 870250082258, dated 12 / 09 / 2025, pp. 90 / 171 71 / 102 gating, and plotted as a function of the concentration of the tested bispecific construct or the control. The results are shown in Figure 28 for constructs #1 to 7 (“VH first”) and in Figure 29 for constructs #8 to 14 (“VL first”). It was clearly observed that the bispecific binding proteins with the “VH first” configuration mediated CDC activity, leading to cell death (Figure 28). On the other hand, none of the bispecific binding proteins in the “VL first” configuration mediated any CDC activity (Figure 29), leading to the conclusion that hTfR1 binding mediated by the “VL first” configuration inhibits C1q binding to the antibody's Fc domain. Example 15 Plasma and brain exposure of Gen2A constructs with hTfR1 affinity variants

[0176] To evaluate brain and plasma exposure over time of bispecific binding protein constructs according to development, additional constructs based on the Gen 2A format were generated, in analogy to Example 10. This example investigates the mAb158 antibody in an hIgG1 format with the Fc K322A mutation, with or without three affinity variants of the h26D3 binding module for hTfR1 (see Example 9). The tested constructs and their amino acid sequences are presented in Table 13. Table 13: Test constructs and amino acid sequences Designation Heavy chain Light chain mAb158 hIgG1-K322A SEQ ID NO: 91 SEQ ID NO: 87 2A3#2-LC1-K322A SEQ ID NO: 91 SEQ ID NO: 92 2A3#2-HC6-K322A SEQ ID NO: 91 SEQ ID NO: 70 2A3#2-LC5-K322A SEQ ID NO: 91 SEQ ID NO: 93

[0177] The different affinity variants, as well as the mAb158 hIgG1 comparator, were injected intravenously (iv) into hTfR1 knock-in mice (hTfR1-KI), generated as described in Example 6 (n = 15 per test item) at equimolar doses of 40 nmol / kg (corresponding to Petition 870250082258, dated 12 / 09 / 2025, pp. 91 / 171 72 / 102 approximately 6 to 7 mg / kg). Plasma and brain exposure was assessed at five consecutive termination time points of 4, 24, 72, 168, and 240 h, respectively, with n = 3 mice per time point and test composite. Blood for assessment of continuous plasma concentration versus time profiles was collected at 0, 25, 4, 24, 48, 72, 120, 168, and 240 h after administration from the group of animals (n = 3) sacrificed at the 240 h time point. Live blood was collected from the saphenous vein in Sarstedt Microvette CB300 K2E tubes.

[0178] At individual termination points, animals were deeply anesthetized with isoflurane, and terminal blood samples were collected from the orbital plexus in BD Microtainer K2EDTA tubes. Samples were inverted and centrifuged at 2400 xg for 10 min at 4 °C. Plasma was extracted and transferred to Eppendorf tubes and frozen at -80 °C. Immediately after blood collection, the animals' abdomens were opened and a cannula (21 G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium and transcardiac perfusion was performed with chilled PBS. After perfusion, the brains were extracted and the olfactory bulbs removed.The brains were separated into left and right hemispheres, and the cerebellum was removed from the left hemisphere; then, the left hemispheres were weighed, flash-frozen in dry ice, and stored at -80 °C until further preparation and analysis of the concentrations of the injected test constructs, using a Meso Scale Discovery (MSD) based assay. The right hemispheres were placed in 4% formaldehyde and stored at 4 °C for 24 h, then rinsed in cold PBS, transferred to a cold 30% sucrose solution prepared in PBS, and stored at 4 °C for further processing for immunohistochemistry (IHC) (Example 16 below).

[0179] For brain concentration measurements, the frozen left hemispheres were thawed on ice and Petition 870250082258, dated 12 / 09 / 2025, page 92 / 171 73 / 102 homogenized in Tris-buffered saline (TBS) containing protease inhibitor cOmplete and phosphatase inhibitor phosSTOP (#11836145001 and #04906837001, Roche) by automated bead homogenization using the MP Biomedical FastPrep-24 5G system with Lysing Matrix D for 5 s a 6 m / s. Triton X-100 (#X100, Merck) was added to the homogenate, resulting in a final Triton X-100 concentration of 0.5% and a weight / volume ratio of 1:10. The homogenates were vortexed for 10 s and centrifuged at 16,000 x g for 1 h at 4 °C, and then the supernatants were collected and used for brain antibody exposure measurements.

[0180] Plasma and brain concentrations of 2A3#2LC1-K322A, 2A3#2-HC6-K322A, 2A3#2-LC5-K322A and mAb158 hIgG1-K322A were determined using a custom MSD assay for human Fc detection. A 96-well MSD plate (#L15XA-3) was coated overnight at 4 °C with 25 ng / well of goat anti-human IgG antibody, Fcy-specific fragment (#109-005098, Jackson Immuno Research Europe Ltd) diluted in 1x PBS (#099400-100, Medicago AB). The coating was removed and the wells were blocked with 1% Blocker A (#R93BA-4, MSD) in 0.05% PBS-T Tween20 (PBS-T) (#09-9410-100, Medicago AB). After 4 washes with 1x PBS-T, the samples and calibrators of the test constructs diluted in 1% Blocker A in PBS-T were added to the plate and incubated at room temperature (RT) for 2 hours at 900 rpm. Detection of bound antibodies was performed by sequential incubations for 1 hour at 900 rpm.with secondary antibody (anti-mouse human IgG #3850-1-1000, MT145, Mabtech), followed by 1 h incubation at 900 rpm RT with anti-mouse detection antibody conjugated with SULFO-TAG (R32AC-1, MSD). The secondary and detection antibodies were diluted to 25 ng / well in 1% Blocker A in PBS-T. Four washes with 1x PBS-T were performed between all incubation steps. After the last wash, 2X Read Buffer T (#R92TC, MSD) was added. Petition 870250082258, dated 12 / 09 / 2025, p. 93 / 171 74 / 102 was added before reading the plates on an MSD SECTOR Imager. The concentration of the test construct in the samples was evaluated using MSD Discovery Workbench software, employing a 4PL curve fitting algorithm and 1 / Y2 curve weighting for the calibration curve of the corresponding test construct.

[0181] The results are shown in Figures 30 and 31. As shown in the terminal samples in Figure 30, a higher peak concentration in the brain, as well as greater brain exposure over time, was observed for test constructs containing an hTfR1 binding module compared to mAb158 hIgG1-K322A alone. As shown in Figure 31, plasma exposure for test constructs carrying an hTfR1 binding module was lower compared to that of mAb158 hIgG1-K322A, indicating the involvement and scavenging of test constructs from plasma to tissues expressing hTfR1. Taken together, the data support the conclusion that test constructs undergo hTfR1-mediated transport across the BBB and that the affinity of h26D3 binding variants for hTfR1 influences both brain and plasma exposure profiles. Example 16 Immunohistochemistry of Gene 2A constructs with hTfR1 affinity variants

[0182] The in vivo involvement of hTfR1 by the test constructs of Example 15 (2A3#2-LC1-K322A, 2A3#2-HC6-K322A, 2A3#2LC5-K322A, and mAb158 hIgG1-K322A) was studied in more detail using a qualitative immunohistochemical (IHC) analysis. In summary, the sucrose-embedded right hemispheres of the animals sacrificed in Example 15 were embedded in the OCT compound (LAMB / OCT, Thermo Fisher Scientific) and flash-frozen in dry ice. The embedded right hemispheres were sectioned, and 20 µm sagittal slides were collected on Superfrost cryogenic slides (J1800AMNZ, Thermo Fisher Scientific) and air-dried before IHC. Brain sections were Petition 870250082258, dated 12 / 09 / 2025, page 94 / 171 75 / 102 cells were pre-treated with MOM mouse IgG blocking reagent (MKB-2213-1, Vector Laboratories) for 1 h at room temperature. Primary antibodies were diluted in 1X PBS with 0.1% Triton X-100 and incubated overnight at 4 °C, and secondary antibodies were diluted in 1X PBS and incubated for 1.5 h at room temperature. Blood vessels were visualized with IV anti-collagen (1:100) (2150-1470, Biorad) and Alexa488 anti-rabbit H+L IgG (1:500) (A21206, Invitrogen). Intravenously dosed constructs were visualized with Alexa647 anti-human H+L IgG (1:500) (A21206, Invitrogen). All incubations were conducted in a humidified chamber with PBS. The slides were washed in 1x PBS in a cuvette (usually 5x5 min) after incubation.The sections were mounted with Fluoromount-G (00-4958-02, Invitrogen, USA) and the images were captured using a Leica Stellaris 5 confocal system equipped with HC PL APO 40x / 1.25 GLYC motCORR CS2 (Figure 32) and HC PL APO 63x / 1.40 OIL CS2 (Figure 33) objectives (Leica, #11506423).

[0183] Brains 24 hours after dosing showed distinct immunofluorescence signals in capillaries using IHC for the LC1 and HC6 constructs (Figure 32). The LC5 variant and the control (mAb158 hIgG1-K322A) showed no detectable immunofluorescence signal in the capillaries (Figure 32). Because some variation was observed between brains from the same groups, a subjective score of 0 to 3 for macroscopic perfusion success was considered in the interpretation of the images. The score was established based on visual examination of the brain after extraction, where a score of 0 corresponded to a white brain with no visible signs of blood in any vessel, a score of 1 corresponded to a brain with a slightly pinkish coloration and almost invisible signs of blood in some vessels, a score of 2 corresponded to a brain with a pinkish coloration and visible signs of blood in the vessels, and a score of 3 corresponded to a red brain with pronounced signs and Petition 870250082258, dated 12 / 09 / 2025, page 95 / 171 76 / 102 visible remaining blood covering most of the brain or larger vessels. The perfusion score corresponds well to the images, as the brains that were injected with 2A3#2-LC5K322A and mAb158 hIgG1-K322A and exhibited an immunofluorescence signal in the capillaries were those that presented poor perfusion. Collagen IV staining was performed to visualize all capillaries on the slide and compare with the immunofluorescence signal of the dosed construct (Figure 33). The data indicate active uptake of the constructs comprising hTfR1 binding variants in cerebral capillaries when administered intravenously. Example 17 Investigation of infusion-related immune reactions

[0184] In vivo infusion of Fc-containing biomolecules brings with it the possibility of immunological reactions, for example, exhibiting acute clinical symptoms such as those described by Couch et al. (2013), Sci Transl Med 5(183):183ra57, 1-12). In order to evaluate the test constructs of the revelation with respect to such reactions, the test and control constructs provided in Table 14 were designed and expressed. Table 14: Test constructs and amino acid sequences Designation Heavy chain Light chain 2A2#2-8D3 SEQ ID NO: 83 SEQ ID NO: 94 2A2#2-15G11 SEQ ID NO: 83 SEQ ID NO: 95 2A2#2-8D3-K322A SEQ ID NO: 91 SEQ ID NO: 94 2A3#10-WT SEQ ID NO: 83 SEQ ID NO: 96 2A3#14-WT SEQ ID NO: 83 SEQ ID NO: 97 2A3#3-WT SEQ ID NO: 83 SEQ ID NO: 98 2A3#2-WT SEQ ID NO: 83 SEQ ID NO: 99

[0185] Constructs were generated with a murine (2A2#2-8D3) or human (2A2#2-15G11) high-affinity apical TfR1 ligand coupled to a fully effector IgG1 in the Fc domain. 2A2#2-8D3-K322A was generated as a resilient comparator complementary to 2A2#2-8D3. The “VL first” variants Gen 2A 2A3#10-WT and 2A3#14-WT and the “VH first” variants 2A3#3-WT and Petition 870250082258, dated 12 / 09 / 2025, page 96 / 171 77 / 102 2A3#2-WT, all comprising the h26D3 TfR1 binding module in the scFv format “VL first” or “VH first”, were generated to investigate whether infusion reactions could be mitigated by epitope binding and binding module orientation. All test constructs were administered as single intravenous (iv) injections in hTfR1 knock-in mice (hTfR1-KI) expressing murine and human TfR1 (Example 6) at doses of 2.5, 11, 40, or 60 nmol / kg (n = 1 to 3 mice per dose and test construct). Separate animals were used for each dose and each test construct. The first cohort of mice for each test construct received the 11 nmol / kg dose.

[0186] Progression to a subsequently higher or lower dose depended on the absence or presence of an infusion reaction observed at the previous dose level, with a thorough assessment of the degree and duration of observed symptoms performed in accordance with Swedish and EU animal welfare legislation, as well as ethical approval and guidelines. Observational symptoms were assessed as mild, moderate, or severe for each mouse. Symptoms ranged from absence of clinical symptoms to a hunched and listless appearance approximately 15 to 25 minutes post-dose, isolation and inactivity, profound post-dose lethargy, mild motor dysfunction, and increased heart and respiratory rate. Mild to moderate symptoms were completely reversed within a few hours, while severe or prolonged symptoms resulted in immediate sacrifice of the animal.The observational outcome, with first infusion reactions (FIRs) reported as none, mild, moderate, or severe, is presented in Table 15. Table 15: Initial infusion reactions after intravenous administration of test constructs Petition 870250082258, dated 12 / 09 / 2025, page 97 / 171 78 / 102 Dose (nmol / kg) Construct 2.5 11 40 60 2A2#2-8D3 Mild to moderate Mild to moderate Moderate 2A2#2-8D3-K322A None None 2A2#2-15G11 None Severe 2A3#10-WT None None None 2A3#14-WT None None None 2A3#3-WT None None None 2A3#2-WT None None None to mild

[0187] hTfR-KI mice express murine and human TfR1 and therefore may cross-react with mouse TfR1-specific 8D3 and human-specific 15G11 ligands, as well as with h26D3 WT variants. Mild to moderate FIR was observed for 2A2#2-8D3 up to doses of 60 nmol / kg. According to in vitro data, the response was abolished by the introduction of the K322A mutation in the Fc domain of the charging antibody, as implemented in 2A2#2-8D3-K322A, preventing a CDC response and thus omitting complement activation-dependent FIR. Administration of the human apical ligand 2A2#215G11 resulted in severe FIR even at a dose of 11 nmol / kg. No symptoms of FIR were observed after injection of 2A3#10-WT, 2A3#14WT or 2A3#3-WT up to a dose of 60 nmol / kg, while there were no symptoms or possibly mild symptoms after injection of 60 nmol / kg of 2A3#2-WT.This supports the hidden space hypothesis where the antibody (M2 portion) is positioned below the hTfR1-binding scFv domain (M1) and therefore closer to the plasma membrane. The results indicate that the binding epitope for scFv on hTfR1 and the orientation of the binding module together lead to an observational mitigation of FIR. In contrast, FIR was observed for both apical TfR1 ligands 8D3 and 15G11-1, regardless of whether they bind to murine or human TfR1 in the hTfR1-KI mouse model.

[0188] All mice were given a pre-dose blood sample and mice with mild infusion reactions to Petition 870250082258, dated 12 / 09 / 2025, p. 98 / 171 79 / 102 moderate reversible patients also underwent a 2-hour blood sampling. Both blood samples were collected live from the saphenous vein in Sarstedt Microvette CB300 K2E tubes for subsequent plasma processing and cytokine analysis. Brain and plasma exposure to test constructs was investigated at the 24-hour endpoint.

[0189] At the end of the procedure, and regardless of post-sample analysis, the animals were deeply anesthetized with isoflurane and terminal blood samples were collected from the orbital plexus in BD Microtainer K2EDTA tubes. The samples were inverted and centrifuged at 2400 xg for 10 min at 4 °C. Plasma was extracted and transferred to Eppendorf tubes and frozen at 80 °C. Immediately after blood collection, the abdomen of the animals was opened and a cannula (21 G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium and transcardiac perfusion was performed with chilled PBS. After perfusion, the brains were extracted and the olfactory bulbs removed.The brains were separated into left and right hemispheres, and the cerebellum was removed from the left hemisphere. Then, the left hemispheres were weighed and flash-frozen in dry ice and stored at -80 °C until further preparation and analysis of the concentrations of the injected test constructs, using a Meso Scale Discovery (MSD) based assay.

[0190] For brain concentration measurements, frozen left hemispheres were thawed on ice and homogenized in Tris-buffered saline (TBS) containing protease inhibitor cOmplete and phosphatase inhibitor phosSTOP (#11836145001 and #04906837001, Roche) by automated bead homogenization using the MP Biomedical FastPrep-24 5G system with Lysing Matrix D for 5 s a 6 m / s. Triton X-100 (#X100, Merck) was added to the homogenate, resulting in a final Triton X-100 concentration of 0.5% and a ratio Petition 870250082258, dated 12 / 09 / 2025, page 99 / 171 80 / 102 weight / volume ratio of 1:10. The homogenates were vortexed for 10 seconds and centrifuged at 16,000 x g for 1 hour at +4 °C, and then the supernatants were collected and used for brain antibody exposure measurements.

[0191] Plasma and brain concentrations of the test constructs 2A3#2-WT, 2A3#3-WT, 2A3#10-WT, 2A3#14-WT, and 2A2#2-8D3 were determined using a custom MSD assay detecting human Fc. A 96-well MSD plate (#L15XA-3) was coated overnight at 4°C with 25 ng / well of goat anti-human IgG antibody, Fcy-specific fragment (#109-005098, Jackson Immuno Research Europe Ltd) diluted in 1x PBS (#099400-100, Medicago AB). The coating was removed and the wells were blocked with 1% Blocker A (#R93BA-4, MSD) in 0.05% PBS-T Tween20 (PBS-T) (#09-9410-100, Medicago AB). After 4 washes with 1x PBS-T, the samples and test construct calibrators diluted in 1% Blocker A in PBS-T were added to the plate and incubated at room temperature (RT) for 2 hours at 900 rpm. Detection of bound antibodies was performed by sequential incubations for 1 hour at 900 rpm.with secondary antibody (anti-mouse human IgG #3850-1-1000, MT145, Mabtech), followed by 1 h incubation at 900 rpm RT with anti-mouse detection antibody conjugated with SULFO-TAG (R32AC-1, MSD). The secondary and detection antibodies were diluted to 25 ng / well in 1% Blocker A in PBS-T, and 4 washes with 1x PBS-T were performed between all incubation steps. After the last wash, 2X Read Buffer T (#R92TC, MSD) was added before reading the plates on an MSD SECTOR Imager. The concentration of the test construct in the samples was evaluated using MSD Discovery Workbench software, employing a 4PL curve fitting algorithm and 1 / Y2 curve weighting for the calibrator curve of the corresponding test construct. For the construct 2A2#2-8D3, 2A3#2-WT was used as a calibrator. Petition 870250082258, dated 12 / 09 / 2025, pp. 100 / 171 81 / 102

[0192] Plasma and brain concentrations 24 hours after dosing of the indicated test constructs are shown in Figure 34. As shown in Figure 34, the increased dose was reflected in increased plasma and brain exposure, demonstrating successful administration of the test items and engagement with TfR1 in vivo.

[0193] Plasma concentrations of ten different cytokines (IFNy, IL-Iβ, IL-2, IL-4, IL-5, IL-6, IL-10, IL12p70, KC / GRO, and TNF) were determined using the prefabricated V-PLEX Plus Proinflammatory Panel 1 Mouse Kit (K15048G, Meso Scale Discovery (MSD)) following the manufacturer's instructions. Briefly, plates were incubated with the calibrator, control samples and plasma samples were diluted 10x for 2 h, then a mixture of all 10 SULFO-TAG detection antibodies was added for 2 h. All incubations were performed at 900 rpm at room temperature. Before and after each incubation step, four consecutive washes were performed in PBS-0.05% Tween20 (PBS-T) (#099410-100, Medicago AB). After the last wash, 2x Read Buffer T (#R92TC, MSD) was added before reading the plates on an MSD SECTOR Imager.The concentration of analytes in the samples was evaluated using MSD Discovery Workbench software, employing a 4PL curve fitting algorithm and 1 / Y2 curve weighting for the corresponding calibrator standard curve.

[0194] The results for a subset of relevant cytokines tested are shown in Figure 35. The tested constructs induced different cytokine responses, with the 2A3#3-WT and 2A3#2-WT (“VH first”) constructs responding more strongly than the 2A3#10-WT and 2A3#14-WT (“VL first”) constructs. Reinforcing the general hidden space hypothesis, the results demonstrate that positioning the antibody portion (“M2”) under the h26D3 binding portion (“M1”) and closer to the plasma membrane induces lower levels of Petition 870250082258, dated 12 / 09 / 2025, pp. 101 / 171 82 / 102 cytokines and chemokines. The 2A2#2-8D3 construct, which binds to TfR1 in the apical domain and exhibited FIR based on the observational data above (Table 15), generated high levels of cytokines / chemokines, especially KC / GRO and IL-10. Example 18 Production and purification of engineered Gen 2D constructs

[0195] Two bispecific constructs designed as knob-into-hole antibody variants were expressed, each containing an h2 6D3 scFv as the M1 portion linked to the C-terminal amino acid residue of the M2 antibody heavy chain (see Figure 4, right panel). In the first variant, “mAb158-Gen2Dh26D3 VH first” (SEQ ID NO: 84), the N-terminal amino acid residue of the VH region of the M1 scFv was linked to the Fc. In the second variant, “mAb158-Gen2D-h26D3 VL first” (SEQ ID NO: 85), the N-terminal amino acid residue of the VL region of the M1 scFv was linked to the Fc. The complete heavy chain used in both constructs is represented by SEQ ID NO: 86, while the light chain present in two copies in each part of the “M2” antibody is represented by SEQ ID NO: 87.

[0196] The engineered constructs “Gen 2D” were expressed by transient transfection of Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer’s instructions. Filtered cell culture supernatants were applied to a MabSelect SuRe column (Cytiva), which was subsequently washed with DPBS pH 7.4. The expressed binding proteins were eluted by applying 0.7% HAc pH 2.5, followed by neutralization of the sample to pH 7.5. The purified samples were subsequently polished by size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex 200; Cytiva) on DPBS pH 7.4 or anion exchange with, for example, a HiTrap Q HP column (Cytiva) and 20 mM Trizma as binding buffer and eluted with NaCl. Each purified expressed construct was characterized using SDS-PAGE, chromatography of Petition 870250082258, dated 12 / 09 / 2025, pp. 102 / 171 83 / 102 size exclusion (Superdex 200 Increase 3.2 / 300; Cytiva) and determination of protein concentration by UV. An example of the purity of the different constructs is shown in Figure 36 from a representative SDS-PAGE analysis. The unreduced gel showed a band around 175 kDa, while the reduced gel shows the three expected bands of the Gen 2D format: the heavy chain “knob” fused to the scFv of h26D3 appearing around 75 kDa, the identical light chains at approximately 25 kDa and the heavy chain “hole” without scFv fused at approximately 50 kDa. Example 19 Analysis of bispecific Gen 2D binding proteins for hTfR1 using SPR

[0197] The binding of purified bispecific binding proteins to hTfR1 and cTfR1 was evaluated as described for the Gen 2A constructs in Example 12. The data in Figure 37 show that all designed and produced Gen 2D constructs bind to hTfR1. All constructs show similar activation and deactivation rates against hTfR1 (SEQ ID NO: 89) and cTfR1 (SEQ ID NO: 90) compared to a control Fab fragment of the h26D3 ligand for hTfR1. The results illustrate that all produced constructs are functional and that the “VH first / VL first” configuration does not influence the binding of the constructs to hTfR1. Example 20 ADCC Measurements

[0198] To investigate the effector function of Gen 2D constructs, an antibody-dependent cellular cytotoxicity (ADCC) assay was used. To assess ADCC activity, Jurkat effector cells (Promega; #G7018) were used. The cells stably express the FcyRIIIa receptor, the V158 variant (high affinity), and an NFAT response element that directs firefly luciferase expression as a measure of activity. Petition 870250082258, dated 12 / 09 / 2025, pp. 103 / 171 84 / 102 of ADCC. When an Fc portion is bound to FcyR, the activation signal for effector cells is triggered, leading to the death of target cells that are coated with antibodies on their surface. Branch cells (Sigma, cat: 85030802), which express high levels of hTfR1 on the cell surface, were used as target cells. Effector and target cells were used in a 6:1 effector:target ratio, with and without the test construct in serial dilutions. The controls used were isolated antibody, i.e., without the hTfR1-binding scFv, as a negative control, and rituximab as a positive control. Target cells with test constructs were placed in a 96-well assay plate (Corning, #3917), mixed with effector cells, and incubated for 6 ha at 37 °C with 5% CO2. When an Fc-containing protein forms a bridge between the target and the effectors (through the interaction between Fc and FcyR), this leads to luciferase activity.After 6 h of incubation, the BioGlo luciferase reagent was added and the luciferase signal was quantified using a SPARK plate reader (Tecan).

[0199] First, rituximab antibody was used as a positive control to verify ADCC activity and fold induction (Figure 38A). Rituximab is known to be a strong ADCC inducer, and this was verified in the assay setup. When the target cell is excluded, there is no ADCC induction. Importantly, the M2 portion antibody used in the tested Generation 2D constructs (mAb158) does not have ADCC activity when tested without M1 alone, which is also demonstrated by the absence of ADCC activity for mAb158 when target cells are excluded (Figure 38A). Next, the Gen 2D construct mAb158-Gen2D-h26D3 VL first”, expressed and analyzed as described in Examples 18 and 19, was investigated. Importantly, no ADCC activity was detected for this construct (Figure 38B), although it binds strongly to the target cell via the hTfR1 ligand. Petition 870250082258, dated 12 / 09 / 2025, pp. 104 / 171 85 / 102

[0200] Next, K562 cells (Sigma / ECACC) were used in a cell ligation experiment. Fcy receptors were blocked using Fc receptor blocker (Innovex biosciences, #NB309-4X-40) for 30 min at 4 °C, after which the cells were washed in PBS. Cells were seeded in a 96-well V-bottom plate (#249570, Thermo Scientific Nunc) and a serial dilution of the tested Gen 2D construct mAb158Gen2D-h26D3 VL first” was added. The plate was incubated at 4 °C overnight. Cells were washed with PBS containing 1% BSA and then fixed with freshly prepared 4% formaldehyde (Thermo Scientific™ Pierce™, #28906) diluted in PBS for 10 minutes at room temperature. Cells were washed with PBS containing 1% BSA and then stained with fluorescence-labeled goat F(ab')2 anti-IgG(Y)-Alexa fluor 488 secondary antibody (Invitrogen Life technologies, #H10120). Staining was performed for 30 minutes at 4 °C.After incubation with the detection reagent, the cells were washed with PBS and 1% BSA. The cells were then resuspended in 200 µl of PBS with 1% BSA and acquired using a BD FACSLyric flow cytometer system (BD Biosciences). Samples were analyzed using flowJo software (BD Biosciences). The measured median fluorescence intensities (MFI) were plotted against the binding protein concentration and displayed in Figure 39. The results show that the tested Gen 2D construct binds to hTfR1 on the cell surface.

[0201] Taken together, ADCC and cell binding experiments indicate that in the Gen 2D construct with the h26D3 scFv in the “VL first” configuration, the Fc portion present in the M2 binding protein cannot engage with Fcy receptors and lead to ADCC, despite being bound to the cell surface via hTfR1. Example 21 Plasma and brain exposure from Gen 2D construction in vivo Petition 870250082258, dated 12 / 09 / 2025, pp. 105 / 171 86 / 102

[0202] To better evaluate the h26D3 HC6 binding module as an M1 portion of scFv in Gen 2D format, along with a β-amyloid binding antibody, the cerebral and plasma exposure of such a test construct was investigated over time in hTfR1 knock-in mice (hTfR1-KI) (Example 6). The in vivo involvement of the target with cerebral β-amyloid pathology was investigated 72 h post-dose in 5xFAD x hTfR-KI crossbred mice. The 5xFAD x hTfR-KI mice were generated by crossing 5xFAD male mice in a C57BL / 6J genetic background (Northwestern University) with hTfRKI females. The 5xFAD mouse model is an Alzheimer's disease (AD) model with mice expressing human APP and PSEN1 transgenes with a total of five AD-linked mutations, including the Swedish (K670N / M671L), Florida (I716V), and London (V717I) mutations in APP, and the M146L and L286V mutations in PSEN1.

[0203] In the constructs tested in this example, the β-amyloid binding antibody used in the previous examples, mAb158, was replaced by another β-amyloid binding antibody referred to here as mAb000. The construct was created in the same format as “mAb158-Gen2D-h26D3 VH first” from Example 18 and denoted mAb000-Gen2D-h26D3-HC6. To examine the exposure of the test construct and the control antibody in the brain and plasma, mAb000-Gen2D-h26D3-HC6 and mAb000 were generated and injected intravenously (iv) into hTfR1-KI mice (n = 11 per test construct) at equimolar doses of 40 nmol / kg (corresponding to approximately 6 to 7 mg / kg). Terminal exposure in plasma and brain was assessed at three consecutive endpoints of 24, 72, and 336 h, respectively, with n = 3 to 5 mice per endpoint and test construct.Blood for evaluation of continuous plasma concentration versus time profiles was collected from animals sacrificed at the 336 h time point (n=5) and at the following time points: 0.25, 4, 24, 48, 72, 168, 240 and 336 h after construct administration. Petition 870250082258, dated 12 / 09 / 2025, pp. 106 / 171 87 / 102 test. Blood samples were collected during life from the saphenous vein in Sarstedt Microvette CB300 K2E tubes.

[0204] To investigate the engagement of the cerebral β-amyloid target, mAb000 and mAb000-Gen2D-h26D3-HC6 were injected intravenously (iv) into 5xFAD x hTfR-KI mice (n = 2 to 3 per test item) at equimolar doses of 40 nmol / kg (corresponding to approximately 6 to 7 mg / kg). The animals were then terminated 72 h after dosing.

[0205] At individual termination points, regardless of post-sample analysis, animals were deeply anesthetized with isoflurane and terminal blood samples were collected from the orbital plexus in BD Microtainer K2EDTA tubes. Samples were inverted and centrifuged at 2400 xg for 10 min at 4 °C. Plasma was extracted and transferred to Eppendorf tubes and frozen at -80 °C. Immediately after blood collection, the animals' abdomens were opened and a cannula (21 G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium and transcardiac perfusion was performed with chilled PBS. After perfusion, the brains were extracted and the olfactory bulbs removed.The brains were separated into left and right hemispheres, and the cerebellum was removed from the left hemisphere. The left hemispheres were then weighed and flash-frozen in dry ice and stored at -80 °C until further preparation and analysis of the concentrations of the injected test constructs using a Meso Scale Discovery (MSD) based assay. The right hemispheres were placed in 4% formaldehyde and stored at 4 °C for 24 h, then rinsed in cold PBS, transferred to a cold 30% sucrose solution prepared in PBS, and stored at 4 °C for further immunohistochemistry (IHC) processing (see Example 22 below).

[0206] For brain concentration measurements, the frozen left hemispheres were thawed on ice and Petition 870250082258, dated 12 / 09 / 2025, pp. 107 / 171 88 / 102 homogenized in Tris-buffered saline (TBS) containing protease inhibitor cOmplete and phosphatase inhibitor phosSTOP (#11836145001 and #04906837001, Roche) by automated bead homogenization using the MP Biomedical FastPrep-24 5G system with Lysing Matrix D for 5 s a 6 m / s. Triton X-100 (#X100, Merck) was added to the homogenate, resulting in a final Triton X-100 concentration of 0.5% and a weight / volume ratio of 1:10. The homogenates were vortexed for 10 s and centrifuged at 16,000 x g for 1 h at 4 °C, and then the supernatants were collected and used for brain antibody exposure measurements.

[0207] Plasma and brain concentrations of mAb000 and mAb000-Gen2D-h26D3-HC6 were determined using a custom MSD assay that detects human Fc. A 96-well MSD plate (#L15XA-3) was coated overnight at 4°C with 25 ng / well of goat anti-human IgG antibody, Fcy-specific fragment (#109-005-098, Jackson Immuno Research Europe Ltd) diluted in 1x PBS (#09-9400-100, Medicago AB). The coating was removed and the wells were blocked with 1% blocker A (#R93BA-4, MSD) in 0.05% Tween20 PBS (PBS-T) (#09-9410-100, Medicago AB). After 4 washes with 1x PBS-T, the samples and test construct calibrators diluted in 1% Blocker A in PBS-T were added to the plate and incubated at room temperature (RT) for 2 hours at 900 rpm. Detection of bound antibodies was performed by sequential incubations for 1 hour at 900 rpm.with secondary antibody (anti-mouse human IgG #3850-11000, MT145, Mabtech), followed by 1 h incubation at 900 rpm RT with anti-mouse detection antibody conjugated with SULFOTAG (R32AC-1, MSD). The secondary and detection antibodies were diluted to 25 ng / well in 1% blocker A in PBS-T and 4 washes with 1x PBS-T were performed between all incubation steps. After the last wash, 2X Read Buffer T (#R92TC, MSD) was added before reading the plates in an MSD SECTOR. Petition 870250082258, dated 12 / 09 / 2025, pp. 108 / 171 89 / 102 Imager. The concentration of the test construct in the samples was evaluated using MSD Discovery Workbench software, employing a 4PL curve fitting algorithm and 1 / Y2 curve weighting for the calibration curve of the corresponding test construct.

[0208] The results are shown in Figure 40. As shown in Figure 40A, a higher peak concentration in the brain, as well as greater brain exposure, considered as the area under the curve, were observed during the study period for mAb000-Gen2D-h26D3-HC6 compared to mAb000. As shown in Figure 40B, the plasma exposure of mAb000-Gen2Dh26D3-HC6 was lower compared to mAb000, indicating hTfR1 involvement and elimination of plasma test constructs for tissues expressing hTfR1. Taken together, the data corroborate the conclusion that the mAb000-Gen2Dh26D3-HC6 test construct undergoes hTfR1-mediated transport across the BBB. Example 22 Immunohistochemistry of the 2D gene construct in vivo

[0209] The in vivo engagement of both hTfRl and β-amyloid by the mAb000-Gen2D-h26D3-HC6 test construct and the mAb000 control antibody (see Example 21) was studied in more detail using a qualitative immunohistochemical (IHC) analysis. In summary, the sucrose-embedded right hemispheres of animals terminated in Example 21 were embedded in the OCT compound (LAMB / OCT, Thermo Fisher Scientific, USA) and flash-frozen in dry ice. The embedded right hemispheres were sectioned, and 20 gm sagittal slides were collected on Superfrost cryogenic slides (J1800AMNZ, Thermo Fisher Scientific) and air-dried before IHC. The brain sections were pretreated for 20 minutes with 4% PFA (HL96753.1000, HistoLab, Sweden), followed by 5 minutes of washing with dH2O and 5 minutes of incubation with 70% PFA for antigen recovery.After two 10-minute washes with 1X PBS, the slides were blocked with MOM Mouse IgG Blocking Reagent (MKB-2213 Petition 870250082258, dated 12 / 09 / 2025, p. 109 / 171). 90 / 102 1, Vector Laboratories) for 1 h at room temperature. Primary antibodies were diluted in 1X PBS with 0.1% Triton X-100 and incubated overnight at 4 °C, and secondary antibodies were diluted in 1X PBS and incubated for 1.5 h at room temperature.

[0210] β-amyloid was visualized with murine antibodies 6E10 (1 pg / ml) (803002, Biolegend) and 4G8 (1 pg / ml) (800702, Biolegend) and Alexa555 anti-mouse IgG H+L (1:500) (A21424, Invitrogen). The tested compounds were visualized with Alexa647 anti-human IgG H+L (1:500) (A21206, Invitrogen). All incubations were conducted in a humidified chamber with PBS. Slides were washed in 1x PBS in a cuvette (usually 5x for 5 min) after incubation. The sections were mounted with Fluoromount-G (00-4958-02, Invitrogen) and the images were captured using a Leica Stellaris 5 confocal system equipped with an HC PL APO 40x / 1.25 GLYC motCORR CS2 lens (Leica, #11506423).

[0211] The resulting images are shown in Figure 41. Colocalization of hIgG and antibodies against β-amyloid was observed, illustrating extensive β-amyloid target involvement for mAb000-Gen2D-h26D3-HC6 in the 7-month-old 5XFAD / hTfR-KI brain, 72 hours post-dose (Figure 41A). Central plaques were predominantly positive for mAb000-Gen2D-h26D3-HC6, while low colocalization of the β-amyloid plaque was observed for the mAb000 antibody without an hTfR1 binding module (Figure 41B). Example 23 Competition of Gen 2D constructs for hTfRl binding with the M-A712 antibody.

[0212] In a complementary experiment to the study described in Example 3, another method was used to study the competition of test constructs for hTfR1 binding. The test constructs investigated were “mAb158-Gen2D-h26D3 VH first” and Petition 870250082258, dated 12 / 09 / 2025, pp. 110 / 171 91 / 102 mAb158-Gen2D-h26D3 VL first” from Example 18 and mAb000-Gen2Dh26D3-HC6” from Example 21, in both the VH first” and VL first” configurations, denoted herein mAb000-Gen2D-h26D3-HC6 VH first” and mAb000-Gen2D-h26D3-HC6 VL first”, respectively.

[0213] The method in this example uses the anti-CD71 (anti-hTfR1) antibody M-A712 as a marker for a specific epitope in the apical domain of hTfR1. The M-A712 antibody is reported to bind to residues 208 to 212 of hTfR1 in the apical domain (Radoshitzky et al. (2008), PNAS 105(7):2664-2669, Maier et al. (2016), Molecular Therapy Nucleic Acids 5:e321). This site overlaps with the binding site described in human TfR1 for human ferritin (Montemiglio et al. (2019), Nat Commun. 10(1):1121). The binding of ligands to hTfR1, as revealed, i.e., binding to the protease-like domain of hTfR1 and identified as described in Example 1, was evaluated in competition with antibody M-A712. Furthermore, M-A712 was also investigated in competition with recombinant human ferritin heavy chain 1 (FTH1).

[0214] For the competition experiments, K562 lymphoblastic cells (Sigma) were used. To assess the competition between M-A712 and the revealed ligands and to confirm the binding of the labeled M-A712 antibody, the cells were first incubated with human Fc blocker (BD Pharmingen, 564220) for 30 min at 4 °C to block antibody binding mediated by the non-specific Fc receptor. The cells were subsequently incubated with serial dilution test constructs along with PE-conjugated M-A712 antibody (monoclonal, BD Pharmingen, 555537) and incubated for 1 ha at 4 °C. After incubation, the cells were washed 3 times in staining buffer (1% BSA, 0.1% sodium azide in 1X DPBS). The hTfR1-bound M-A712 antibody on cell surfaces was analyzed using flow cytometry, and the mean fluorescence intensity (MFI (PE)) was plotted. Figure 42 shows that there is no competition. Petition 870250082258, dated 12 / 09 / 2025, pp. 111 / 171 92 / 102 direct binding between the h26D3 ligand in the different constructs indicated and M-A712. When the unlabeled (unconjugated) M-A712 antibody was used as a positive control for competition, the binding of the labeled signal (PE) M-A712 was reduced in a concentration-dependent manner. The experiment illustrates that a ligand directed against the protease-like domain of TfR1 does not directly compete for the same epitope as the M-A712 antibody.

[0215] A similar experiment was conducted with human ferritin heavy chain 1 (FTH1; Sino Biologicals #13217HNAE, Lot LC15NO0415) labeled with Alexa647. As seen in Figure 43, competition was demonstrated at a higher concentration compared to the positive control for competition (M-A712). These data clearly demonstrate that hTfR1 ligands, as revealed, which bind to the protease-like domain of hTfR1, do not compete with the epitope described in the apical domain that corresponds to the binding site used by the ferritin protein. Detailed listing of modalities 1. Binding protein, comprising: - a first M1 portion, which is a human transferrin receptor 1 (hTfR1) binding portion comprising a variable immunoglobulin heavy chain (VH) region and a variable immunoglobulin light chain (VL) region, said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, wherein said antigen-binding surface provides the binding protein with the ability to selectively bind to an epitope located in the protease-like domain of hTfR1 defined by amino acid residues 121 to 183 and 384 to 605 in SEQ ID NO: 66, and - a second M2 portion comprising an antibody Fc domain, for example, being selected from the group consisting of an antibody and an Fc fusion protein, Petition 870250082258, dated 12 / 09 / 2025, pp. 112 / 171 93 / 102 wherein M1 and M2 are connected to each other by at least one linker peptide between M1 and M2, said linker being arranged in such a way that M2 induces a reduced Fc-mediated response when administered to a human and when M1 binds to hTfR1 present in a cell. 2. Binding protein according to item 1, wherein said M1 epitope located in the protease-like domain of hTfR1 comprises or consists of amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO: 66. 3. Binding protein according to any previous item, wherein said antigen-binding surface of M1 is composed of three complementarity-determining regions (CDRs) of said VH region and three CDRs of said VL region, and wherein the CDRs comprise the following: VHCDR1: X1X2NMX3 (SEQ ID NO: 1), where: X1 is selected from D and A; X2 is selected from Y and A; and X3 is selected from D and A; VHCDR2: X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO: 2), where: X4 is selected from D and A; X5 is selected from D and A; X6 is selected from Y and A; X7 is selected from D and A; and X8 is selected from Y and A; VHCDR3: GGX9SGSSX10X11HPMX12X13 (SEQ ID NO: 3) where: X9 is selected from Y and A; X10 is selected from Y and A; X11 is selected from Y and A; X12 is selected from D and A; and X13 is selected from Y and A; Petition 870250082258, dated 12 / 09 / 2025, pp. 113 / 171 94 / 102 VLCDR1: KSSQSLLX14STNQKNX15LA (SEQ ID NO: 4), where: X14 is selected from Y and A; and X15 is selected from Y and A; VLCDR2: X16ASTRES (SEQ ID NO: 5) where: X16 is selected from W and A; and VLCDR3: QQX17FIX18PRT (SEQ ID NO: 6) where: X17 is selected from Y and A; and X18 is selected from Y and A. 4. Binding protein according to item 3, wherein the amino acid sequence of said VHCDR1 is selected from the group consisting of SEQ ID NO: 7 and 13 to 15. 5. Binding protein according to any of items 3-4, wherein the amino acid sequence of said VHCDR2 is selected from the group consisting of SEQ ID NO: 8 and 16 to 20. 6. Binding protein according to any one of items 3-5, wherein the amino acid sequence of said VHCDR3 is selected from the group consisting of SEQ ID NO: 9 and 21 to 25. 7. Binding protein according to any one of items 3-6, wherein the amino acid sequence of said VLCDR1 is selected from the group consisting of SEQ ID NO: 10, 26 and 27. 8. Binding protein according to any one of items 3-7, wherein the amino acid sequence of said VLCDR2 is selected from the group consisting of SEQ ID NO: 11 and 28. 9. Binding protein according to any of items 3-8, wherein the amino acid sequence of said VLCDR3 is Petition 870250082258, dated 12 / 09 / 2025, pp. 114 / 171 95 / 102 selected from the group consisting of SEQ ID NO: 12, 29 and 30. 10. Binding protein according to any of items 3 to 9, wherein the amino acid sequences of the six CDRs are as follows: VHCDR1: DYNMD (SEQ ID NO: 7), VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO: 8), VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO: 9), VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO: 10), VLCDR2: WASTRES (SEQ ID NO: 11), VLCDR3: QQYFIYPRT (SEQ ID NO: 12). 11. Binding protein according to any of items 3-9, wherein the amino acid sequences of the six CDRs are as follows: VHCDR1: DYNMD (SEQ ID NO: 7), VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO: 18), VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO: 9). VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO: 10), VLCDR2: WASTRES (SEQ ID NO: 11). VLCDR3: QQYFIYPRT (SEQ ID NO: 12). 12. A binding protein in accordance with any of the preceding items, wherein the said VH region comprises or consists of an amino acid sequence selected from: (i) the group consisting of SEQ ID NO: 31 to 44, for example, the group consisting of SEQ ID NO: 31 and 37; and (ii) a sequence with at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of a sequence defined in (i). Petition 870250082258, dated 12 / 09 / 2025, pp. 115 / 171 96 / 102 13. A binding protein in accordance with any of the preceding items, wherein the said VL region comprises or consists of an amino acid sequence selected from: (i) the group consisting of SEQ ID NO: 45 to 51; and (ii) a sequence with at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of a sequence defined in (i). 14. Binding protein according to any of items 12-13, wherein the said VH region is as defined in item 12 and the said VL region is as defined in item 13. 15. Binding protein according to item 14, wherein said VH region comprises SEQ ID NO: 31 and said VL region comprises a sequence selected from SEQ ID NO: 45 to 51. 16. Binding protein according to item 14, wherein said VH region comprises a sequence selected from SEQ ID NO: 31 to 44 and said VL region comprises SEQ ID NO: 45. 17. Binding protein according to any one of items 15-16, wherein said VH region comprises SEQ ID NO: 31 and said VL region comprises SEQ ID NO: 45. 18. Binding protein according to any one of items 15-16, wherein said VH region comprises SEQ ID NO: 37 and said VL region comprises SEQ ID NO: 45. 19. A binding protein matching any previous item, in which the VH / VL pair of the first M1 portion forms part of an scFv, where the VH and VL regions are coupled by an scFv binding peptide. 20. Binding protein according to item 19, in which the aforementioned scFv linker is linked to the amino acid residue N Petition 870250082258, dated 12 / 09 / 2025, pp. 116 / 171 97 / 102 terminal of the VH region and to the C-terminal amino acid residue of the VL region. 21. Binding protein according to any of items 19-20, wherein the scFv linker is a flexible binding peptide consisting of 5 to 40 amino acid residues, for example, 10 to 30 amino acid residues, for example, 15 to 25 amino acid residues, for example, about 15 amino acid residues, for example, 15 amino acid residues, for example, comprising or consisting of the sequence (G4S)3(SEQ ID NO: 88). 22. Binding protein in accordance with any previous item, wherein the said Fc-mediated response induced by said second M2 portion is an Fc-mediated cytotoxic response, for example, selected from the group consisting of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and combinations thereof. 23. Binding protein according to item 22, wherein the aforementioned Fc-mediated cytotoxic response is selected from ADCC, CDC and combinations thereof. 24. Binding protein according to item 23, where the aforementioned Fc-mediated cytotoxic response is ADCC. 25. Binding protein according to item 23, where the aforementioned Fc-mediated cytotoxic response is CDC. 26. A linking protein according to any of the preceding items, in which said linking peptide between M1 and M2 is linked, on the M1 side, to the C-terminal amino acid residue of the VH region of M1 or to the N-terminal amino acid residue of the VL region of M1. 27. A linking protein according to item 26, in which the aforementioned linking peptide between M1 and M2 is linked, on the M2 side, to the C-terminal residue of a CH3 region of the aforementioned Fc domain. Petition 870250082258, dated 12 / 09 / 2025, pp. 117 / 171 98 / 102 and, on the M1 side, to the N-terminal amino acid residue of the VL region of M1. 28. A binding protein according to any one of items 1-25, wherein M2 comprises an antibody with two antibody light chains and wherein M1 and M2 are connected to each other by means of two linker peptides, the first linker being attached, on the M2 side, to the C-terminal amino acid residue of the first light chain of M2 and, on the M1 side, to the N-terminal amino acid residue of the VL region of M1, and the second linker being attached, on the M2 side, to the N-terminal amino acid residue of the second light chain of M2 and, on the M1 side, to the C-terminal amino acid residue of the VH region of M1. 29. Binding protein in accordance with any previous item, wherein the aforementioned at least one binding peptide between M1 and M2 is a flexible binder. 30. Binding protein according to item 29, wherein the said flexible linker(s) comprise(s) glycine, serine, alanine and / or threonine residues. 31. Binding protein according to item 30, wherein the said binder(s) have a general formula selected from (GnSm)pe (SnGm)p, wherein, independently, n = 1 to 7, m = 0 to 7, n + m < 8 and p = 1 to 10. 32. A linking protein according to any of the preceding items, wherein said linker has between 10 and 50 amino acid residues in length, such as between 10 and 30 amino acid residues in length, such as between 15 and 25 amino acid residues in length, or between 10 and 20 amino acids in length. 33. A binding protein according to any one of items 29-32, wherein M1 and M2 are connected to each other by means of two linker peptides, and both of said linkers are as defined in any one of items 29-32. Petition 870250082258, dated 12 / 09 / 2025, pp. 118 / 171 99 / 102 34. Binding protein according to item 33, where both of the aforementioned linkers have the same length. 35. Binding protein according to item 33, where both of the aforementioned linkers are of different lengths. 36. Binding protein according to any previous item, where M2 is an antibody capable of selectively binding to a target present in the brain of a mammal. 37. Binding protein according to item 36, wherein the said target is selected from the group consisting of β-amyloid peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, DNA-binding protein TAR 43 (TDP-43) or derivatives or fragments thereof, myeloid cell-expressed triggered receptor 2 (TREM2), beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, Tau, phosphorylated Tau or fragments thereof, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, β-amyloid precursor protein, p75 neurotrophin receptor, neuregulin and caspase 6. 38. Binding protein according to item 37, wherein the said target is selected from the group consisting of β-amyloid peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, DNA-binding protein TAR 43 (TDP-43) or derivatives or fragments thereof, receptor-triggered myeloid cell-expressed 2 (TREM2), Tau, phosphorylated Tau or fragments thereof and apolipoprotein E4. 39. Binding protein according to item 38, wherein the said target is selected from the group consisting of β-amyloid peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof and protein of Petition 870250082258, dated 12 / 09 / 2025, pp. 119 / 171 100 / 102 binding to TAR 43 (TDP-43) DNA or derivatives or fragments thereof. 40. Binding protein according to any of items 36-39, wherein the said antibody capable of selectively binding to a target present in the brain of a mammal is an anti-Αβ antibody, for example, an antibody selected from the group consisting of lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06 and KHK6640. 41. Binding protein according to any of items 36-39, wherein said antibody capable of selectively binding to a target present in the brain of a mammal is an anti-alpha-synuclein antibody, for example, an antibody selected from the group consisting of prasinezumab, UCB7853, Lu AF82422, TAK-341 and BAN0805. 42. Pharmaceutical composition comprising a binding protein according to any of the preceding items and a pharmaceutically acceptable vehicle or excipient. 43. A binding protein according to any of items 1-41 or a composition according to item 42 for use in treatment, such as for use in therapeutic treatment or for use in prophylactic treatment. 44. A binding protein according to any one of items 1-41 or a composition according to item 42 for use in in vivo diagnosis or in vivo prognosis. 45. A binding protein or composition for use in accordance with any of items 43-44, wherein said therapy, prophylaxis, in vivo diagnosis or in vivo prognosis is with respect to a neurodegenerative disorder, for example, a disorder selected from Alzheimer's disease and other disorders associated with Aβ protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Petition 870250082258, dated 12 / 09 / 2025, pp. 120 / 171 101 / 102 Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy. 46. ​​A binding protein or composition for use in accordance with item 45, wherein the said therapy, prophylaxis, in vivo diagnosis or in vivo prognosis is with respect to a selected disorder from Alzheimer's disease and other disorders associated with Aβ protein aggregation, Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, Parkinson's disease (PD), Parkinson's disease dementia (PDD) and Lewy body variant of Alzheimer's disease. 47. A binding protein or composition for use in accordance with item 46, wherein the said therapy, prophylaxis, in vivo diagnosis or in vivo prognosis is with respect to a disorder selected from Alzheimer's disease and other disorders associated with Aβ protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). 48. A binding protein or composition for use in accordance with item 47, wherein the said therapy, prophylaxis, in vivo diagnosis or in vivo prognosis relates to Alzheimer's disease. 49. A binding protein or composition for use in accordance with any of items 43-44, wherein the said therapy, prophylaxis, in vivo diagnosis or in vivo prognosis is with respect to a disorder selected from brain cancer, multiple sclerosis and lysosomal storage diseases. 50. A method of therapeutic or prophylactic treatment of a mammal that has, or is at risk of developing, a disorder, said method comprising administering to said mammal a therapeutically effective amount of a molecule of Petition 870250082258, dated 12 / 09 / 2025, pp. 121 / 171 102 / 102 connection according to any of items 1-41 or a composition according to item 42. 51. A method in accordance with item 50, wherein the disorder in question is a neurodegenerative disorder, for example, a neurodegenerative disorder as defined in any of items 45-48. 52. A method in accordance with item 51, wherein the aforementioned disorder is as defined in item 49. Petition 870250082258, dated 12 / 09 / 2025, pp. 122 / 171

Claims

1 / 6 CLAIMS 1. A binding protein, characterized in that it comprises: - a first portion M1, which is a human transferrin receptor 1 (hTfR1) binding portion comprising a variable immunoglobulin heavy chain (VH) region and a variable immunoglobulin light chain (VL) region, said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, wherein said antigen-binding surface provides the binding protein with the ability to selectively bind to an epitope located in the protease-like domain of hTfR1 defined by amino acid residues 121 to 183 and 384 to 605 of SEQ ID NO: 66, and - a second portion M2 comprising an antibody Fc domain, for example, being selected from the group consisting of an antibody and an Fc fusion protein, wherein M1 and M2 are connected to each other by at least one peptide linker between M1 and M2,the aforementioned linker is arranged in such a way that M2 induces a reduced Fc-mediated response when administered to a human, and when M1 binds to hTfR1 present in a cell.

2. Binding protein according to claim 1, characterized in that said M1 epitope located in the protease-like domain of hTfR1 comprises or consists of amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO:

66.

3. Binding protein according to claim 1 or 2, characterized in that said antigen-binding surface of M1 is composed of three complementarity-determining regions (CDRs) of said VH region and three CDRs of Petition 870250082258, dated 12 / 09 / 2025, p. 159 / 171 2 / 6 referred CDRs comprise the aforementioned VL region, and in which the following: 1) VHCDR1: X1X2NMX3 (SEQ ID NO: VHCDR2: where: X1 is selected from D and A; X2 is selected from Y and A; and X3 is selected from D and A; X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO: 2) VHCDR3: where: X4 is selected from D and A; X5 is selected from D and A; X6 is selected from Y and A; X7 is selected from D and A; and X8 is selected from Y and A; is selected from Y and A; X11 is selected from Y and A;and X13 is selected from Y and A; KSSQSLLX14STNQKNX15LA (SEQ ID NO: 4), VLCDR2: where: X14 is selected from Y and A; and X15 is selected from Y and A; X16ASTRES (SEQ ID NO: 5) VLCDR3: where: X16 is selected from W and A; and QQX17FIX18PRT (SEQ ID NO: 6) where: X17 is selected from Y and A; and X18 is selected from Y and A. Petition 870250082258, dated 12 / 09 / 2025, pp. 160 / 171 3 / 6; 4. Binding protein according to claim 3, characterized in that the amino acid sequences of the six CDRs are as follows: VHCDR1: DYNMD (SEQ ID NO: 7), VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO: 8), VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO: 9), VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO: 10), VLCDR2: WASTRES (SEQ ID NO: 11), VLCDR3: QQYFIYPRT (SEQ ID NO: 12).

5. Binding protein according to claim 3, characterized in that the amino acid sequences of the six CDRs are as follows: VHCDR1: DYNMD (SEQ ID NO: 7), VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO: 18), VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO: 9), VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO: 10), VLCDR2: WASTRES (SEQ ID NO: 11), VLCDR3: QQYFIYPRT (SEQ ID NO: 12).

6. Binding protein according to claim 4, characterized in that said VH region comprises SEQ ID NO: 31 and said VL region comprises SEQ ID NO:

45.

7. Binding protein according to claim 5, characterized in that said VH region comprises SEQ ID NO: 37 and said VL region comprises SEQ ID NO:

45.

8. A binding protein according to any one of claims 1 to 7, characterized in that the VH / VL pair of the first M1 portion forms part of an scFv, in which the VH and VL regions are coupled by an scFv binding peptide.

9. Binding protein according to claim 8, characterized in that said scFv linker is Petition 870250082258, dated 12 / 09 / 2025, p. 161 / 171 4 / 6 linked to the N-terminal amino acid residue of the VH region and to the C-terminal amino acid residue of the VL region.

10. Binding protein according to any one of claims 1 to 9, characterized in that said Fc-mediated response induced by said second fraction M2 is an Fc-mediated cytotoxic response, for example, selected from the group consisting of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and combinations thereof.

11. A linking protein according to any one of claims 1 to 10, characterized in that said at least one linking peptide between M1 and M2 is linked, on the M1 side, to the C-terminal amino acid residue of the VH region of M1 or to the N-terminal amino acid residue of the VL region of M1.

12. A linking protein according to claim 11, characterized in that said linking peptide between M1 and M2 is linked, on the M2 side, to the C-terminal residue of a CH3 region of said Fc domain and, on the M1 side, to the N-terminal amino acid residue of the VL region of M1.

13. A linker protein according to any one of claims 1 to 11, characterized in that M2 comprises an antibody with two antibody light chains and in which M1 and M2 are connected to each other by means of two linker peptides, the first linker being attached, on the M2 side, to the C-terminal amino acid residue of the first light chain of M2 and, on the M1 side, to the N-terminal amino acid residue of the VL region of M1, and the second linker being attached, on the M2 side, to the N-terminal amino acid residue of the second light chain of M2 and, on the M1 side, to the C-terminal amino acid residue of the VH region of M1.

14. Binding protein according to any one of claims 1 to 14, characterized in that M2 is an antibody capable of selectively binding to a target present in the brain of a mammal.

15. A binding protein according to claim 14, characterized in that said target is selected from the group consisting of β-amyloid peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, TAR 43 DNA-binding protein (TDP-43) or derivatives or fragments thereof, myeloid cell-expressed triggered receptor 2 (TREM2), beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, Tau, phosphorylated Tau or fragments thereof, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, β-amyloid precursor protein, neurotrophin receptor p75, neuregulin and caspase 6.

16. Pharmaceutical composition, characterized in that it comprises a binding protein, as defined in any one of claims 1 to 15, and a pharmaceutically acceptable carrier or excipient.

17. Binding protein according to any one of claims 1 to 15 or composition according to claim 16, characterized in that it is for use in treatment, either for use in therapeutic treatment or for use in prophylactic treatment.

18. Binding protein or composition for use according to claim 17, characterized in that said therapy or prophylaxis is with respect to a neurodegenerative disorder, for example, a disorder selected from Alzheimer's disease and other disorders associated with Aβ protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), Petition 870250082258, dated 12 / 09 / 2025, p. 163 / 171 6 / 6 Amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy.

19. Use of the binding protein, as defined in any one of claims 1 to 15, or of the pharmaceutical composition, as defined in claim 16, said use being characterized by being for the manufacture of a medicament for the therapeutic or prophylactic treatment of a neurodegenerative disorder, for example, a disorder selected from Alzheimer's disease and other disorders associated with Aβ protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease and familial amyloid neuropathy.

20. Invention of a product, composition, kit or use, characterized by comprising one or more elements disclosed in this patent application. Petition 870250082258, dated 12 / 09 / 2025, pp. 164 / 171

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