New regulations combined with molecules

Abstract

This disclosure relates to binding molecules, such as antibodies or antigen-binding fragments thereof that bind to the protease-like domain of human transferrin receptor 1 (TfR1), and their therapeutic and diagnostic applications.

Description

[Technical Field]

[0001] This disclosure relates to binding molecules, such as antibodies or antigen-binding fragments thereof that bind to the protease-like domain of human transferrin receptor 1 (TfR1), and their therapeutic and diagnostic uses. [Background technology]

[0002] Because cerebral blood vessels are impermeable to most substances transported by the bloodstream, treatment options for brain and neurological diseases are limited (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 compared to the blood vessels found in the periphery of the body. When BBB endothelial cells (ECs) adhere tightly to nerve cells, such as astrocytes, pericytes, and neurons, phenotypic characteristics are induced, contributing to the observed impermeability. Tight junctions between ECs within the BBB restrict intercellular transport, while the absence of passive vesicles and windows restricts nonspecific intercellular transport. These factors combined restrict molecular flux from blood to brain to lipophilic molecules, generally smaller than 500 Da in size. Therefore, the surface area for mass transport in blood flow (from 600 km of capillaries in the human brain to 20 m) 2 The inherently promising prospect of using the above as a delivery vehicle becomes virtually impossible, except when a drug with desirable pharmacological properties happens to possess the size and lipophilicity attributes that allow it to cross the blood-brain barrier (BBB). Due to these constraints, it is estimated that over 98% of all small molecule drugs, and nearly 100% of emerging protein and gene therapies, cannot cross the BBB.

[0003] International Publication No. 91 / 03259 proposes a principle for transporting neuropharmaceuticals across the blood-brain barrier (BBB), demonstrating the conjugation of a drug to an antibody that reacts with a transferrin receptor. According to this disclosure, once the conjugate binds to the transferrin receptor, it is actively transported across the BBB. This concept has been further developed in subsequent studies, for example, in International Publication Nos. 2012 / 075037, 2014 / 033074, 2018 / 011353, and 2022 / 258841. All of these documents describe different forms of transporting biopharmaceuticals across the BBB using the transferrin receptor.

[0004] Human transferrin receptors exist in two forms. Transferrin receptor 1 (TfR1) is the target of the binding molecule of this disclosure. TfR1 is an iron transporter protein that recognizes the iron transporter proteins transferrin (Tf) and ferritin (Ft) and maintains intracellular iron concentration by internalizing transferrin (Tf) and ferritin (Ft) into the cell via clathrin-coated vesicle endocytosis. TfR1 is expressed in many cells and organs, but at varying levels. Importantly, TfR1 is expressed more highly in BBB endothelial cells than in other endothelial cells, making this receptor a target for neuropharmaceutical delivery. Structurally, TfR1 is a dimeric transmembrane glycoprotein containing the amino acid sequence of SEQ ID NO: 85, which has a large ectodomain (residues 89-760), an intramembrane domain (residues 62-88), and a cytoplasmic domain (residues 1-61). Next, the ectodomain has three distinct domains separated from the cell surface by the stalk region (residues 89-120). These three parts of the ectodomain are the helical domain (residues 606-760), the protease-like domain (residues 121-183, 384-605), and the apical domain (residues 184-383) (Lawrence et al (1999), Science 286:779-782).

[0005] In the context of TfR1-mediated BBB transport, antibodies and fragments thereof that have affinity for TfR1 have been reported. For example, International Publication 2014 / 189973 discloses numerous TfR1-binding antibodies, which are classified into classes I-IV based on epitope specificity (see, for example, Figure 3 and the description of the relevant figure on lines 11-15 of page 30). Classes I-III in International Publication 2014 / 189973 are labeled as "apical conjugates," while class IV antibodies are labeled as "non-apical conjugates." Other TfR1-binding antibodies are disclosed in European Patent Application Publication 3088518, European Patent Application Publication 3315606, and European Patent Application Publication 3560958, but information regarding the epitope specificity of these disclosed antibodies is not provided.

[0006] Therefore, most studies using TfR1 as a target for binding and BBB transport have focused on apical conjugates. This is thought to be because the apical domain is the structure within TfR1 that, when used as an immunogen, is thought to induce a potent immune response and thus cause antibody production in animals. Consequently, most known antibody conjugates for TfR1 have epitopes located within the apical domain. Another indication that the apical domain contains structures that readily bind to various ligands is the reported use of epitopes within the apical domain by viruses to enter cells (Cohen-Dvashi et al (2020), Nat Commun 11:67).

[0007] Furthermore, the detailed structure of the TfR1-ferritin complex has recently been determined (Montemiglio et al (2019), Nat Commun 10:1121), showing that the TfR1-ferritin interface is located within the apical domain. This suggests that if a TfR1 apical binder is used for BBB transport, it may interfere with ferritin's binding to TfR1, thus potentially affecting ferritin's normal function in iron transport. It has also been shown that H-ferritin binding and uptake are mediated by TfR1 (Li et al (2010), Proc Natl Acad Sci USA 107(8):3505-10). Therefore, there is reason to conclude that binders targeting the apical domain of TfR1, and in particular binding to the binding site utilized by ferritin, may adversely affect the important function of ferritin in transporting iron via binding to TfR1.

[0008] TfR1 apical binders have been reported to induce both acute clinical signs and a decrease in circulating reticulocytes (Couch et al (2013), Sci Transl Med 5:183ra57). TfR1 has also been described in association with anemia and iron deficiency (Braga et al (2014), Clin Chim Acta 431:143-147). Furthermore, anemia caused by autoantibodies against TfR1 has also been described (Hyman et al (1984), N Engl J Med 311:214-218). These data, taken together, suggest that when TfR1 binds to and inhibits iron transporters such as transferrin and / or ferritin, safety issues such as a decrease in reticulocyte count and anemia may occur.

[0009] To date, this field has focused on avoiding interference with one of the described TfR1 ligands, namely transferrin. Therefore, the field has guided the use of binding sites located in the apical domain of TfR1, away from the transferrin binding site. However, such apical binding agents still interfere with another important TfR1 ligand, ferritin, potentially affecting iron transport and function.

[0010] Against this backdrop, there remains a need in this field for antibodies and other binding molecules that have binding affinity to TfR1 but do not exhibit the drawbacks and risks associated with conventionally known binding molecules.

[0011] Disclosure of the invention The purpose of this disclosure is to address this need by providing a TfR1-binding molecule that utilizes a binding site on TfR1 that is different from naturally occurring ligands. One such objective is to provide a TfR1-binding molecule that utilizes a binding site on TfR1 different from that of transferrin. Another such objective is to provide a TfR1-binding molecule that utilizes a different binding site on TfR1 than ferritin. Another objective is to provide a TfR1-binding molecule that utilizes a different binding site than HFE (heat-sensitive iron regulator) on TfR1. A related objective of this disclosure is to provide a TfR1-binding molecule that interacts with TfR1 in a manner that minimizes interference with TfR1 itself and / or its normal function. Another object of this disclosure is to provide a TfR1-binding molecule suitable for use as a fusion partner in a construct configured for transport through the BBB. One or more of these objectives, and other objectives that are apparent to those skilled in the art from the teachings herein, are achieved by various aspects of this disclosure.

[0012] Accordingly, in a first embodiment, the present disclosure provides a transferrin receptor 1 (TfR1) binding molecule capable of selectively binding to an epitope located in the protease-like domain of TfR1 as defined by amino acid residues 121-183 and 384-605 of SEQ ID NO: 85. Although not bound by theory, the binding of TfR1 to an epitope or binding site within the protease-like domain is considered useful in that it avoids the drawbacks associated with known binders for TfR1, particularly those known to have affinity for an epitope or binding site located in the apical domain of TfR1.

[0013] In a specific embodiment, the epitope or binding site of the TfR1 binding molecule of the present disclosure includes amino acid residues 150, 151, 154, 158, 159, 161, 163, and 385 of SEQ ID NO: 85. In another embodiment, the epitope or binding site of the TfR1 binding molecule of the present disclosure consists of amino acid residues 150, 151, 154, 158, 159, 161, 163, and 385 of SEQ ID NO: 85. In another specific embodiment, the epitope or binding site of the TfR1 binding molecule includes 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 of the amino acid residues 150, 151, 154, 158, 159, 161, 163, and 385 of SEQ ID NO: 85. As shown in the following examples, for example in relation to Figure 12, this embodiment of the epitopes of the binding molecules identified and disclosed herein ensures non-interfering binding to the natural TfR1 ligands transferrin and ferritin.

[0014] As is known to those skilled in the art, an epitope (or “antigenic determinant”) is a group of amino acids or other chemical groups exposed on the surface of a molecule (often a protein, here hTfR1) that can generate an antigenic response and bind to an antibody. An epitope is a local region on the surface of an antigen that is recognized by the immune system, particularly an antibody. A conformational epitope consists of adjacent amino acid residues located on the surface structure of an antigen protein. A conformational epitope binds to a complementary paratope in a B cell receptor and / or antibody. In one embodiment of this disclosure, the epitope to which the binding molecule binds is a conformational epitope.

[0015] In one embodiment of the binding molecule, the binding molecule comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the VH and VL regions form a VH / VL pair including an antigen-binding surface, which imparts to the binding molecule the ability to selectively bind to the epitope. The VH / VL pair may be provided, for example, in the form of a full-length conventional antibody, or in an antibody fragment selected from the group consisting of Fab fragments, single-chain Fab (scFab) fragments, Fv fragments, and single-chain (scFv) fragments. In a specific embodiment, the VH / VL pair forms part of the scFv. The designation "VH / VL" used with respect to the VH / VL pair is not used to limit the construct to a specific order of the VH and VL regions in its polypeptide chain, but is used solely to indicate that both the VH and VL regions are present and, when paired, can form an Ig domain with an antigen-binding surface. Therefore, the term "VH / VL pair" encompasses, for example, constructs in which the VL region precedes the VH region in a single strand of Fv, constructs in which the VH region precedes the VL region in a single strand of Fv, and constructs in which the VH region and the VL region are associated with each other non-covalently.

[0016] With respect to the antigen-binding surface of the VH / VL pair, it may appropriately consist of three complementarity-determining regions (CDRs) derived from the VH region and three CDRs derived from the VL region. In one embodiment, the CDRs include: 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: X4INPX5X6X7TTSX8X9X10KFKG (SEQ ID NO: 2), where X4 is selected from D and A, X5 is selected from D, N, and A, X6 is selected from Y and A, X7 is selected from D and A, X8 is selected from Y and A, X9 is selected from N and S, and X10 is selected from E and Q. VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID NO: 4) where X11 is selected from Y and A, X12 is selected from T and S, X13 is selected from Q and R, and X14 is selected from Y and A. VLCDR2: X15ASTRES (SEQ ID NO: 5) where X15 is selected from W and A, and VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO: 6) where, X16 is selected from Y and A, X17 is selected from F and Y, X18 is selected from I and N, X19 is selected from Y and A, and X20 is selected from R and Y.

[0017] In one embodiment, the CDR further includes the following: VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO: 3) where X21 is selected from Y and A. X22 is selected from Y and A. X23 is selected from Y and A. X24 is selected from D and A, and X25 is selected from Y and A.

[0018] In another embodiment, the CDR further includes: VHCDR3:SEAGNYYWYFDV (Sequence ID 35).

[0019] As defined herein, embodiments of the binding molecules of the first aspect of this disclosure, including VH / VL pairs, have a specific amino acid sequence in the region that determines its binding ability (e.g., the CDR of the heavy and / or light chain variable domain, or actually the VL and / or VH domain or the entire region). Non-limiting examples of such specific amino acid sequences are provided herein for specific antibodies produced and characterized as described in Examples 1-9. The specific sequence information provided for the produced antibodies will enable those skilled in the art to define combinations and variations of these sequences within the scope of the invention (including combinations and variations provided by variations of common CDR sequences, provided as SEQ ID NOs: 1-6).

[0020] In one embodiment, the CDR further includes: VHCDR3:GGX21SGSSX22X23HPMX24X25 (Sequence ID 3) Here, X21 is selected from Y and A. X22 is selected from Y and A. X23 is selected from Y and A. X24 is selected from D and A, and X25 is selected from Y and A.

[0021] In one embodiment, the VHCDR2 is as follows: VHCDR2:X4INPX5X6X7TTSX8NEKFKG (Sequence ID 7) Here, 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.

[0022] In one embodiment, the VLCDR1 is as follows: VLCDR1:KSSQSLLX11STNQKNX14LA (Sequence ID 8) Here X11 is selected from Y and A, and X14 is selected from Y and A.

[0023] In one embodiment, the VLCDR3 is as follows: VLCDR3:QQX16FIX19PRT (Sequence ID 9) Here X16 is selected from Y and A, and X19 is selected from Y and A.

[0024] In one embodiment, the amino acid sequence of VHCDR1 is selected from the group consisting of SEQ ID NOs: 10 and 16-18. In one embodiment, the amino acid sequence of VHCDR2 is selected from the group consisting of SEQ ID NOs: 11, 19-23, and 34, for example, from the group consisting of SEQ ID NOs: 11 and 19-23. In one embodiment, the amino acid sequence of VHCDR3 is selected from the group consisting of SEQ ID NOs: 12, 24-28, and 35, for example, from the group consisting of SEQ ID NOs: 12 and 24-28. In one embodiment, the amino acid sequence of VLCDR1 is selected from the group consisting of SEQ ID NOs: 13, 29, 30, and 36, for example, from the group consisting of SEQ ID NOs: 13, 29, and 30. In one embodiment, the amino acid sequence of VLCDR2 is selected from the group consisting of SEQ ID NOs: 14 and 31. In one embodiment, the amino acid sequence of VLCDR3 is selected from the group consisting of SEQ ID NOs: 15, 32, 33, and 37, for example, from the group consisting of SEQ ID NOs: 15, 32, and 33.

[0025] In some embodiments, the CDR sequences can be freely combined among the options listed above. Such embodiments include, but are not limited to, the combinations illustrated in Example 9 for the alanine-substituted variant of the h26D3 embodiment of the binding molecule of this disclosure.

[0026] In a particular embodiment of the binding molecule of this disclosure, including a VH / VL pair, the amino acid sequences of the six CDRs are as follows: VHCDR1:DYNMD (Sequence ID 10), VHCDR2:DINPDYDTTSYNEKFKG (Sequence ID 11), VHCDR3:GGYSGSSYYHPMDY (Sequence ID 12), VLCDR1:KSSQSLLYSTNQKNYLA (Sequence ID 13), VLCDR2:WASTRES (SEQ ID NO: 14), VLCDR3:QQYFIYPRT(Sequence ID 15)

[0027] In another specific embodiment of the binding molecule of this disclosure, which includes a VH / VL pair, the amino acid sequences of the six CDRs are as follows: VHCDR1:DYNMD (Sequence ID 10), VHCDR2:DINPDADTTSYNEKFKG (Sequence ID 21), VHCDR3:GGYSGSSYYHPMDY (Sequence ID 12), VLCDR1:KSSQSLLYSTNQKNYLA (Sequence ID 13), VLCDR2:WASTRES (SEQ ID NO: 14), VLCDR3:QQYFIYPRT(Sequence ID 15)

[0028] In another specific embodiment of the binding molecule of this disclosure, which includes a VH / VL pair, the amino acid sequences of the six CDRs are as follows: VHCDR1:DYNMD (Sequence ID 10), VHCDR2:DINPNYDTTSYSQKFKG (Sequence ID 34), VHCDR3:SEAGNYYWYFDV (Sequence ID 35), VLCDR1:KSSQSLLYSSNRKNYLA (Sequence ID 36), VLCDR2:WASTRES (SEQ ID NO: 14), VLCDR3:QQYYNYPYT(Sequence ID 37)

[0029] In another specific embodiment of the binding molecule of this disclosure, which includes a VH / VL pair, the amino acid sequences of the six CDRs are as follows: VHCDR1:NYWLG (Sequence ID 38), VHCDR2:DIFPGSDNTYYNEKFKG (Sequence ID 39), VHCDR3:SGNFYAMDY (Sequence ID 40), VLCDR1:SASSSVNYMN (Sequence ID 41), VLCDR2:DTSKLAS (SEQ ID NO: 42), VLCDR3:FQGSGYPFT(SEQ ID NO: 43)

[0030] In one embodiment, the CDR sequence of the antigen-binding interface contained in the binding molecule of this disclosure is defined using Kabat rules 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 from the US Department of Health and Human Services).

[0031] In one embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VH region comprises or consists of an amino acid sequence selected from the following: (i) The group consisting of sequence numbers 44-57, 65, and 67, for example the group consisting of sequence numbers 44-57, for example the group consisting of sequence numbers 44 and 50, and (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 the sequence defined in (i) (provided that the sequence of the CDR region is 100% identical with the sequence defined in (i)).

[0032] In one embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VL region comprises or consists of an amino acid sequence selected from the following: (i) A group consisting of sequence numbers 58-64, 66, and 68, for example, a group consisting of sequence numbers 58-64, and (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 the sequence defined in (i) (provided that the sequence of the CDR region is 100% identical with the sequence defined in (i)).

[0033] In such a specific embodiment, both the VH region and the VL region are as defined immediately above, namely, VH contains or consists of sequences selected from sequence numbers 44 to 57 and sequences having at least 80% sequence identity thereto, and VL contains or consists of sequences selected from sequence numbers 58 to 64 and sequences having at least 80% sequence identity thereto.

[0034] In another embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VH region comprises or comprises an amino acid sequence selected from SEQ ID NO: 69 and 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 SEQ ID NO: 69 (provided that the sequence of the CDR region is 100% identical to the sequence of SEQ ID NO: 69).

[0035] In another embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VL region comprises or consists of an amino acid sequence selected from SEQ ID NO: 70 and 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 SEQ ID NO: 70 (provided that the sequence of the CDR region is 100% identical to the sequence of SEQ ID NO: 70).

[0036] In such a specific embodiment, both the VH region and the VL region are as defined immediately above, namely, VH includes or consists of the sequence of sequence number 69 and a sequence having at least 80% sequence identity thereto, and VL includes or consists of the sequence of sequence number 70 and a sequence having at least 80% sequence identity thereto.

[0037] In one embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VH region comprises SEQ ID NO: 44, and the VL region comprises a sequence selected from SEQ ID NOs: 58-64. In one embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VH region comprises a sequence selected from SEQ ID NOs: 44-57, and the VL region comprises SEQ ID NO: 58. In one embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VH region comprises SEQ ID NO: 44, and the VL region comprises SEQ ID NO: 58. In one embodiment of the binding molecule of the present disclosure comprising a VH / VL pair, the VH region comprises SEQ ID NO: 50, and the VL region comprises SEQ ID NO: 58.

[0038] In some embodiments, the VH sequence and the VL sequence, when present in the binding molecule, are selected from the enumerated sequences and one of the sequences 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%, at least 99%, or at least 100% identity thereto.

[0039] In one embodiment of the binding molecule of the present disclosure, which includes a VH / VL pair, the VH / VL pair forms part of an antibody construct.

[0040] In one such embodiment, the antibody construct has two or more binding specificities. For example, it may be bispecific or triplicate, or it may have four or more binding specificities. In a specific embodiment, the binding molecule is bispecific, for example, a bispecific antibody construct.

[0041] In one embodiment of the binding molecule of the present disclosure, which includes a VH / VL pair, the VH / VL pair is present in an antibody fragment selected from the group consisting of a Fab fragment, a single-stranded Fab (scFab) fragment, an Fv fragment, and a single-stranded (scFv) fragment. In a specific embodiment of such a binding molecule, the antibody fragment is scFv.

[0042] In key embodiments of the binding molecules disclosed herein, this further comprises an antibody or its antigen-binding fragment, in addition to a VH / VL pair that provides selective binding to hTfR1. In one such embodiment, this additional antibody or fragment can selectively bind to a target present in the mammalian brain. In some embodiments, the target is selected from the group consisting of amyloid-beta peptide or its derivatives or fragments, alpha-synuclein or its derivatives or fragments, TAR DNA-binding protein 43 (TDP-43) or its derivatives or fragments, trigger receptor 2 (TREM2) expressed in bone marrow cells, beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, tau, phosphorylated tau or its fragments, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma-secretase, death receptor 6, amyloid-beta precursor protein, p75 neurotrophic factor receptor, neuregulin, and caspase 6. In a more specific embodiment, the target is selected from the group consisting of amyloid-beta peptide or its derivatives or fragments, alpha-synuclein or its derivatives or fragments, TAR DNA-binding protein 43 (TDP-43) or its derivatives or fragments, trigger receptor 2 (TREM2) expressed in bone marrow cells, tau, phosphorylated tau or its fragments, and apolipoprotein E4. In an even more specific embodiment, the target is selected from the group consisting of amyloid-beta peptide or its derivatives or fragments, alpha-synuclein or its derivatives or fragments, and TAR DNA-binding protein 43 (TDP-43) or its derivatives or fragments.

[0043] In one embodiment, the antibody or antigen-binding fragment thereof, which is selectively capable of binding to a target present in the mammalian brain, is an antibody selected from the group consisting of anti-Aβ antibodies, such as lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06, and KHK6640.

[0044] In another embodiment, the antibody or antigen-binding fragment thereof, which is selectively capable of binding to a target present in the mammalian brain, is an antibody selected from the group consisting of anti-alpha-synuclein antibodies, such as pracinezumab, UCB7853, LuAF82422, TAK-341, and BAN0805.

[0045] Affinity for the target As used herein, the terms “specific binding to X,” “selective binding to X,” and “affinity for X” [where X is the target (e.g., an antigen or epitope, e.g., TfR1 to which the VH / VL pair of the binding molecule defined above binds)] refer to the properties of the binding molecule, e.g., an antibody or its antigen-binding fragment, or the properties of a bispecific or multispecific construct incorporating such an antibody or its antigen-binding fragment, which can be tested, for example, by ELISA, surface plasmon resonance (SPR), or bio-layer interferometry (BLI). Those skilled in the art are familiar with these and other methods.

[0046] For example, the binding affinity to a target, antigen, or epitope X can be tested by capturing the binding molecule of test on an ELISA plate coated with X or a molecule containing epitope X, adding a biotinylated detection antibody, and then adding streptavidin-conjugated horseradish peroxidase (HRP). Alternatively, the detection antibody may be directly conjugated to the HRP. A tetramethylbenzidine (TMB) substrate is added, and the absorbance at 450 nm is measured using an ELISA multiwell plate reader. Those skilled in the art can establish at least a qualitative measure of the binding affinity of the binding molecule to X by interpreting the results obtained by such experiments. For example, if a quantitative measure is needed to determine the EC50 value (half of the maximum effective concentration) of the interaction, ELISA can also be used. The response of the binding molecule to a dilution series of X can be measured using ELISA as described above. Those skilled in the art can interpret the results obtained by such experiments and calculate the EC50 value from them using, for example, GraphPad Prism v.9 and nonlinear regression.

[0047] As used herein, the term "EC50" refers to half of the maximum effective concentration of the binding molecule that induces an intermediate response between the baseline and the maximum value after a given exposure time.

[0048] Alternatively, an inhibitory ELISA can be used to obtain a quantitative measure of the interaction by measuring the "IC50" (half of the maximum inhibitory concentration). In an inhibitory ELISA, the concentration of target X in a liquid sample is measured by detecting interference with the expected signal output. In principle, a multi-well plate is coated with a known target or epitope-containing substance. Simultaneously, a binding molecule presumed to have affinity for X is added and incubated with a solution containing various concentrations of the target. After standard blocking and purification steps, a sample containing the mixture of the binding molecule and the target is added to the wells. Next, a labeled detection antibody having affinity for the binding molecule is used for detection using a suitable substrate (e.g., TMB). In principle, if a high concentration of the target is present in the liquid sample, a significant decrease in signal output is observed. In contrast, if there is little or no target in the fluid sample, the expected decrease in signal output will be very small. Those skilled in the art will understand that the signal output also depends on the affinity of the binding molecule for the target.

[0049] As used herein, the term "IC50" refers to half of the maximum inhibitory concentration of the binding molecule that induces an intermediate response between baseline and maximum inhibition after a specific exposure time. In this specification, a lower IC50 value, compared to a higher IC50 value, indicates that a lower target concentration is required to interfere with the binding of the detection antibody to a known target coated on a plate. Therefore, a lower IC50 value generally corresponds to higher affinity.

[0050] The binding affinity of a binding molecule can also be tested by surface plasmon resonance (SPR). For example, affinity can be tested in an experiment in which a target or epitope X is immobilized on a sensor chip of the instrument, and a sample containing the binding molecule of test is passed over the chip. Alternatively, the binding molecule of test is immobilized on a sensor chip of the instrument, and a sample containing X is passed over the chip. A person skilled in the art can then interpret the results obtained in such an experiment to establish at least a qualitative measure of the binding affinity of the binding molecule to X. For example, the K of the interactionD If a quantitative measure is needed to determine the value, SPR can also be used. Binding values ​​can be defined using instruments such as Biacore (Cytiva) or ProteOn XPR 36 (Bio-Rad). The target or epitope is properly immobilized on the instrument's sensor chip, and a sample of the binding molecule whose affinity is to be determined is prepared by serial dilution and injected. Then, using, for example, the 1:1 Langmuir binding model in Biacore Insight Evaluation Software 2.0, or other appropriate software typically provided by the instrument manufacturer, the results are analyzed from K D The value can be calculated.

[0051] Binding affinity can also be measured by biolayer interferometry (BLI), a label-free technique for measuring biomolecular interactions within an interactome. This is an optical analysis technique that analyzes the interference patterns of white light reflected from two surfaces: a protein layer immobilized on a biosensor chip and an internal reference layer. The binding of a ligand (target or epitope X) immobilized on the biosensor chip surface to an analyte in solution (such as a binding molecule presumed to have affinity for X) results in an increase in the optical thickness of the biosensor chip, resulting in a wavelength shift (Δλ), which is a direct measure of the change in the thickness of the biological layer. The interaction is measured in real time, providing the ability to track binding specificity, association and dissociation rates, or concentrations with high precision and accuracy.

[0052] Those skilled in the art are aware of the above-mentioned method and other methods for measuring the affinity of a binding molecule to a target or epitope X qualitatively, quantitatively, or both.

[0053] Pharmaceutical composition In a second aspect, the Disclosure provides a pharmaceutical composition comprising a binding molecule described herein and at least one pharmaceutically acceptable excipient or carrier.

[0054] The technology for formulating polypeptides such as antibodies and their derivatives for human therapeutic use is well known in this field and is outlined, for example, in Wang et al (2007), J Pharm Sci, 96:1-26, the entire content of which is incorporated herein by reference.

[0055] 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 (e.g., human serum albumin), buffers (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0056] In some embodiments, the pharmaceutical composition is formulated via any suitable route of administration (including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, oral (e.g., sublingual), and transdermal administration) for administration to a subject. In preferred embodiments, the composition is formulated for intravenous or subcutaneous administration.

[0057] Methods of prevention, treatment, diagnosis, prognosis, and detection The conjugated molecules of this disclosure may be useful as therapeutic agents, prophylactic agents, diagnostic agents, and / or prognostic agents.

[0058] Accordingly, in further embodiments of this disclosure, a conjugated molecule according to the first embodiment or a pharmaceutical composition according to the second embodiment is provided for use as a pharmaceutical.

[0059] In yet another aspect of this disclosure, a conjugated molecule according to the first embodiment or a pharmaceutical composition according to the second embodiment is provided for use as a diagnostic agent.

[0060] In yet another aspect of this disclosure, a conjugating molecule according to a first embodiment or a pharmaceutical composition according to a second embodiment is provided for use as a prognostic agent.

[0061] Furthermore, methods for preventing, treating, or diagnosing diseases, or for evaluating the prognosis of diseases, are also provided, wherein the conjugated molecules disclosed herein are administered to subjects requiring them, typically human subjects.

[0062] The use of the disclosed conjugating molecules for the manufacture of compositions (e.g., pharmaceuticals) for use in the prevention, treatment, diagnosis, and / or prognosis of any one of the listed diseases is also provided.

[0063] Accordingly, in one embodiment, a binding molecule, or a pharmaceutical composition containing the same, is useful for the treatment, prevention, diagnosis, and / or prognosis of a disease selected from neurodegenerative diseases, such as Alzheimer's disease and other diseases 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-associated dementia (PDD), Lewy body Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy.

[0064] In a more specific embodiment, the disease is selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, Parkinson's disease (PD), Parkinsonian dementia (PDD), and Lewy body Alzheimer's disease.

[0065] In a more specific embodiment, the disease is selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD), particularly Alzheimer's disease.

[0066] In another embodiment, the binding molecule or a pharmaceutical composition containing the same is useful for the treatment, prevention, diagnosis, and / or prognosis of other disorders, such as brain cancer, multiple sclerosis, and lysosomal storage disorders.

[0067] In another embodiment, a method is provided for treating, preventing, diagnosing, and / or determining the prognosis of any of the disorders listed above, the method comprising administering to the mammal a certain amount, for example, a therapeutically effective amount of a binding molecule or a pharmaceutical composition containing the same.

[0068] Built-in by reference This application cites various publications, and each of these documents is incorporated herein by reference in its entirety. [Brief explanation of the drawing]

[0069] [Figure 1] Figure 1 shows the results of binding screening of the IgG antibodies described in Example 1, obtained by immunization, against human (hTfR1), cynomolgus monkey (cTfR1), and mouse (mTfR1)TfR1 in crude hybridoma supernatant, performed by biolayer interferometry (BLI). [Figure 2] Figure 2 shows the results of the BLI binding analysis described in Example 2, performed on the Fab fragments of the mouse antibodies 24B4, 26D3, and 37D10, as well as the Fab fragment of the control antibody 8D3. [Figure 3]Figure 3 shows the mapping of antibody-binding epitopes to the protease-like domain of hTfR1, as described in Example 2, by selective antibody binding to ELISA plates coated with human, mouse, or one of three different chimeric human / mouse TfR1 receptors. Antibodies 24B4, 26D3, and 37D10 bind to hTfR1 (A) but not to mTfR1 (B). Furthermore, 24B4, 26D3, and 37D10 also bind to the h / m protease-like domain chimeric (D) but not to any of the plates coated with the other chimeric receptors (C and E). [Figure 4] Figure 4 illustrates the epitope binning assay described in Example 2, consisting of the following four main steps: Step 1 - Immobilization of bio-TfR1 onto the sensor chip; Step 2 - Washing of the unbound material; Step 3 - Binding of competing binders to TfR1; Step 4 - Binding of the binder to the TfR1:binder complex formed in Step 3. The data from Step 4 determines whether the two binders under consideration compete for binding to hTfR1. [Figure 5] Figure 5 shows the results of the epitope binning assay performed as described in Example 2, illustrating the degree of competition among antibodies for co-binding to hTfR1. The images show the binding of (A) antibody 26D3, (B) antibody 24B4, and (C) control antibody 15G11-1 to pre-formed complexes of hTfR1 with any of the antibodies listed. Binding responses for all antibodies are normalized to the binding response measured against free hTfR1 (without competing antibodies). [Figure 6] Figure 6 shows the binding of the above binder to hTfR1 on the cell surface, as tested as described in Example 2. The Y-axis in both figures shows the mean fluorescence intensity when cells were stained with (A) the total antibody and (B) the Fab fragment of the above binder. No background staining was detected with the negative isotype control IgG (A) or the unrelated Fab fragment Ly128 (B). [Figure 7]Figure 7 shows the results of a competitive analysis of the described binder against ferritin and transferrin as described in Example 3. The figure shows (A) the MFI of the described binder that binds to TfR1 expressed on the surface of THP-1 cells, (B) the MFI of ferritin on the cell surface when exposed to the described binder (positive control antibody MA-712 competes with ferritin), and (C) the MFI of transferrin on the cell surface when exposed to the described binder. [Figure 8] Figure 8 is a collection of sensorgrams showing the SPR analysis results of the original Fab form of 26D3 and the humanized 26D3 (h26D3) as described in Example 4, when bound to hTfR1 and cTfR1 as described. [Figure 9] Figure 9 shows the results of BLI and ELISA binding tests performed on the mouse and humanized forms of 26D3 in the scFv form described in Example 4. (A) Sensorgram obtained by BLI measurement of the binding of the indicated construct to hTfR1. (B) Binding response obtained by ELISA measurement of the binding of the indicated construct to coated TfR1. [Figure 10] Figure 10 is a depiction of the X-ray structure of the 26D3-Fab and hTfR1 complex determined as described in Example 5. The chain names used in the correspondence file are shown. (A) A precise structure showing the overall folding of the three independent complexes within the asymmetric unit. (B) An example of electron density (2m|Fo|-D|Fc|) outlined at the 1σ level. The protein chain is depicted as a cartoon, while the sugar portion is shown as a bar. [Figure 11] Figure 11 shows the ribbon representation of the h26D3-Fab human TfR1 complex determined by X-ray crystallography described in Example 5. h26D3-Fab is shown in dark gray, and hTfR1 is shown in white. The binding interfaces (epitope / paratope) are circled. [Figure 12] Figure 12 shows the surface region of hTfR1 having the binding sites for the described natural ligands ferritin and transferrin, and the epitope of the binder 26D3 of this disclosure. Different binding sites and epitopes are indicated by circling their respective specific sites. [Figure 13] Figure 13 shows the preparation and characterization of the hTfR1-KI mouse model described in Example 6. (A) Schematic diagram of the transgenic hTfR1-KI mouse construct. The extracellular domain of human TFRC was inserted into the mouse Tfrc gene by homologous recombination. (B) Quantitative reverse transcription PCR (RT-qPCR) analysis of mouse Tfrc and human TFRC gene expression in the brain (N=3 / genotype). hTfR1-KI mice (gray circles) express human TFRC and mouse Tfrc in whole-brain homogeneous mice, while wild littermates express only mouse Tfrc (white). (C) Western blot analysis of hTfR1, whole TfR1, and hTfR1-KI in the brain. hTfR1-KI animals aged 6-8 months (N=5) and 15 months (N=4) express equivalent levels of hTfR1 protein. Total TfR1 levels were comparable between hTfR1-KI transgenic mice and wild-type littermates (N=3). [Figure 14] Figure 14 shows the results of in vivo brain and plasma exposure analysis of various described hTfR1 binding molecules in hTfR1-KI transgenic mice described in Example 7. (A) Brain exposure 24 hours after intravenous administration of the described hTfR1 binding agent. (B) Plasma exposure 24 hours after intravenous administration of the described hTfR1 binding agent. (C) Brain:plasma ratio 24 hours after intravenous administration of the described hTfR1 binding agent. Negative controls are indicated by "158" and positive controls by "15G11-1". [Figure 15] Figure 15 shows the results of an analysis of in vivo brain exposure to various described hTfR1 binding molecules in hTfR1-KI mice by immunohistochemistry as described in Example 8. Staining of cerebral capillaries was observed for several binding molecules, including h26D3. The reference hTfR1 binding agent "15G11-1" and the non-TfR1 binding agent "158" were used as positive and negative controls, respectively. [Figure 16] Figure 16 shows the BLI sensorgrams of the alanine variants of h26D3 described in Example 9. Each variant exhibits a different dynamic profile, illustrating that introducing specific mutations into the CDR region of the heavy or light chain may generate variants with different affinities to human TfR1. [Figure 17] Figure 17 shows a representative SPR sensorgram of the interaction between h26D3, which has hTfR1 and cTfR1, as measured as described in Example 9. [Figure 18] Figure 18 shows the results of indirect ELISA analysis of the binding of the indicated alanine variant of h26D3 having hTfR1 and cTfR1, as measured as described in Example 9. [Figure 19] Figure 19 shows an SPR sensorgram of the interaction between shown alanine variants of h26D3, which was tested as an scFv component within a bispecific protein morphology as described in Example 9. [Figure 20] Figure 20 shows two different Gen2A structures designed and manufactured as described in Example 10. [Figure 21] Figure 21 shows the purification results of different Gen2A constructs prepared and purified as described in Example 11. The monomer content of the bispecific binding protein was high (generally over 98%), and these were prepared at low mg / l levels. Purity was analyzed using Coomassie blue SDS-PAGE staining. [Figure 22] Figure 22 shows sensorgrams obtained from SPR binding analysis of the 14 Gen2A-binding protein constructs and controls described in Example 12. One sensorgram is shown for each variant, illustrating that all constructs functioned and bound to hTfR1. [Figure 23] Figure 23 shows the cell binding data for the Gen2A constructs shown, measured as described in Example 13. All constructs tested bound to cells expressing hTfR1 in the same way. [Figure 24] Figure 24 is a collection of figures showing the results of CDC measurements performed in Ramos cells using the indicated test constructs #1 to #7 ("VH-first" configuration) as described in Example 14. [Figure 25] Figure 25 is a collection of figures showing the results of CDC measurements performed in Ramos cells using the indicated test constructs #8-14 ("VL-first" configuration) as described in Example 14. [Figure 26] Figure 26 is a collection of figures showing the results of the in vivo pharmacokinetic study described in Example 15. It shows the plasma and brain exposure levels of euthanized mice (n=3 mice used for each time point and each test component) collected at 4, 24, 72, 168, and 240 hours after intravenous administration of the indicated test components. Data are shown as mean ± SD. [Figure 27] Figure 27 shows the plasma concentration versus time profiles obtained from continuous sampling using n=3 mice for each test component after intravenous administration as described in Example 15. The data are shown as mean ± SD. [Figure 28] Figure 28 shows 40x Z-stack images of the cerebral cortex of hTfR-KI mice stained for hIgG 24 hours after administration, as described in Example 16. Triple measurements (n=3) are shown for each group (LC1, HC6, and LC5). The perfusion score (0-3) for each brain is shown in the white square in the lower left corner. Blood vessels are indicated by arrows. [Figure 29] Figure 29 shows 63x Z-stack images of each group using hIgG staining versus collagen IV, as described in Example 16. The perfusion score (0-3) for each brain is shown in the white square in the lower left corner. [Figure 30] Figure 30 is a pair of figures showing the results of the in vivo study described in Example 17. It shows the plasma (A) and brain (B) concentrations at 24 hours for three indicated dose test constructs: 11 nmol / kg (rhomboid), 40 nmol / kg (triangle), and 60 nmol / kg (inverted triangle). The concentrations of 2A2#2-8D3 are based on a calibration substance prepared from 2A3#2-WT. [Figure 31] Figure 31 shows the results of the cytokine response analysis described in Example 17, and shows the individual plasma levels and median plasma levels of the indicated cytokines (A: TNF and KC / GRO; B: IL-6 and IL-10) before administration and 2 hours after administration for three test constructs with doses of 11 nmol / kg (diamond), 40 nmol / kg (triangle), and 60 nmol / kg (inverted triangle). [Figure 32]Figure 32 shows the results of the preparation and purification of the Gen2D construct described in Example 18 after gel analysis of the purified construct using in-gel protein detection staining with Coomassie blue. The lanes are as follows: (1) Marker; (2) mAb158-2D-h26D3-HC6, unreduced; (3) mAb158-2D-h26D3-HC6, reduced; (4) mAb158-2D-h26D3, unreduced; (5) mAb158-2D-h26D3, reduced. [Figure 33] Figure 33 shows sensorgrams obtained from SPR binding analysis of the Gen2D binding protein constructs and controls described in Example 19. Sensorgrams showing the binding of each variant shown to both hTfR1 and cTfR1 are shown, indicating that all constructs function and bind to both hTfR1 and cTfR1. The Fab fragment of h26D3 was included as a positive control. [Figure 34] Figure 34 shows the results of the ADCC assay described in Example 20, using rituximab as a positive control. (A) Verification of the assay configuration using rituximab, which shows strong polyploidy induction ability in the presence of target cells (Figure labeled "Rituximab"). In the absence of target cells, no ADCC induction by rituximab was observed (Figure labeled "Rituximab (no target cells)"). Similarly, the mAb158 antibody (i.e., antibody without the hTfR1 binding site M1) was tested with and without target cells. (B) No ADCC induction was observed in the analysis of Gen2D morphology with h26D3scFv in a "VL-first" configuration as M1 and mAb158 as M2. In the same assay, rituximab showed strong induction, but mAb158 antibody alone showed no signs of ADCC activity. [Figure 35] Figure 35 shows the cell-binding data of the indicated Gen2D construct on K562 cells, measured as described in Example 20. [Figure 36]Figure 36 shows the results of the in vivo pharmacokinetic study described in Example 21. A: Mean (±SD) euthanasia plasma concentration (black fill) and brain concentration (white space)-time profiles of mAb000-Gen2D-h26D3-HC6 (diamond) and mAb000 (square) in hTfR-KI mice after a single intravenous administration of 40 nmol / kg (n=3-4 per sampling time). B: Mean (±SD) plasma concentration-time profiles of mAb000-Gen2D-h26D3-HC6 (diamond) and mAb000 (square) from continuous sampling in hTfR-KI mice after a single intravenous administration of 40 nmol / kg (n=4). [Figure 37] Figure 37 shows 40x Z-stack images of the frontal cortex of 5XFAD×hTfR-KI mice 72 hours after injection of mAb000-Gen2D-h26D3-HC6(A) or mAb000(B), as described in Example 22. Representative images of total amyloid beta and hIgG from duplicate measurements (n=2) per group. [Figure 38] Figure 38 shows the competition among the indicated test constructs for hTfR1 binding by the M-A712 antibody on the cell surface, as measured by flow cytometry as described in Example 23. [Figure 39] Figure 39 shows the competition by human ferritin for hTfR1 binding by the M-A712 antibody on the cell surface, as measured by flow cytometry as described in Example 23. [Examples]

[0070] While the present invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various modifications can be made and elements can be replaced with equivalents without departing from the scope of the invention. Furthermore, many modifications can be made to adapt specific situations or molecules to the teachings of the invention without departing from the essential scope of the invention. Accordingly, the present invention is not limited to specific embodiments, and is intended to encompass all embodiments included in the appended claims.

[0071] The present invention is further illustrated by the following non-limiting embodiments. These embodiments are for illustrative purposes only and do not limit the invention in any way. Those skilled in the art will readily recognize a variety of non-essential parameters that can be changed or modified to obtain essentially the same results. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantities, temperatures, etc.), some degree of experimental error and bias may exist. Unless otherwise specified, the implementation of the present invention involves conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the art. Such techniques are described in detail in existing literature. Furthermore, those skilled in the art will see that the protein engineering methods applied herein can also be applied to other constructs described herein and that the inventors consider to be within the scope of this disclosure.

[0072] Example 1 Identification of human TfR1 binders by immunization and screening. Immunology and Hybridoma Screening 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 immunogen and an adjuvant. The hTfR1 immunogen was fused at the N-terminus of a T cell epitope derived from tetanus toxin P2 (Kovacs-Nolan and Mine (2006), Biochim Biophys Acta 1760:1884-1893) via a GSS linker, along with the ectodomain of the human TfR1 protein and the N-terminal 10× histidine tag (His 10 Designed to include -P2-hTfR1 (SEQ ID NO: 71). After gene construction, Hek293 cells were transiently transfected using the Expi293™ expression system (Gibco), purified with a nickel column (HisTrapFF, catalog no. 17-5255-01, GE Healthcare), buffered with PBS, concentrated to 1 mg / ml, and recombinant His 10The -P2-hTfR1 protein was prepared. The expressed TfR1 immunogen was aliquoted and stored at -80°C until use. Qui-A adjuvant (vac-quil, InvivoGen) was used for all immunizations, except for the final booster immunization injection, which did not contain the adjuvant. For use, Qui-A was resuspended twice in distilled water at a concentration of 1 mg / ml, filtered under sterile conditions, and aliquoted into 0.1 ml portions and stored at -80°C. Qui-A was administered at a dose of 10 μg / mouse.

[0073] His, a recombinant immunogen 10 Animals were immunized monthly by co-administering P2-hTfR1 mixed with Qui-A. Three weeks after each immunization, blood samples were collected, and the plasma was analyzed for the presence of antibodies reactive to recombinant human TfR1 and mouse TfR1. Antibody titers were considered sufficiently high if the ELISA response at a 1 / 100,000 dilution exceeded [mean value of the blank (i.e., background) + standard deviation of the blank × 3]. The four mice used in this study each received 4 to 6 immunizations.

[0074] Three days prior to fusion, mice received a final intraperitoneal booster immunization injection in the absence of adjuvants. Mice were anesthetized with isoflurane at euthanasia. The entire spleen was removed and dissected. Briefly, a single-cell suspension was prepared from the immunized mouse spleen and mixed with Sp2 / 0 cells in a 3:1 ratio. The cells were fused using PEG and added to a bottle of ClonaCell®-HY medium D (STEMCELL Technologies). 60–70 μl per well was then dispensed into a 96-well plate. After 6–7 days, 150 μl of HAT medium was added to each well of the 96-well plate in semi-solid form. The following day, 120 μl of supernatant was discarded from each well and 100 μl of fresh HAT medium was added. The next day, 100 μl of supernatant was taken from each well, transferred to a storage plate, and tested for the presence of antibodies against mouse TfR1 using indirect ELISA on a nickel-coated plate according to the following protocol. On day 12, 120 μl of HAT medium was added to the hybridoma plates for repeated screening, and 25 μl of the supernatant was transferred to an ELISA plate by day 3 for screening of reactivity to mouse TfR1 (both screenings are referred to as "primary screening"). Clones that were positive for mouse TfR1 with an OD > 0.2 were transferred to 24-well plates and cultured for at least 3 days for secondary screening using biolayer interferometry (BLI) to examine reactivity to mouse, human, and cynomolgus monkey TfR1 in solution (referred to as "secondary screening"). Binding to both hTfR1 and cynomolgus monkey TfR1 was shown, but in the secondary screening, only very weak binding or no binding at all was detected for mTfR1. The supernatant from the 24-well plates was also screened for binding to His-tagged hTfR1 and the absence of binding to His-tagged amyloid-beta precursor protein (APP, negative control) using both directly coated TfR1 plates and nickel-coated plates as described below. Binding to cynomolgus monkey TfR1 (cTfR1) was also analyzed using direct TfR1 coating.In particular, the ELISA response (OD450 value) of mTfR1 was very low compared to hTfR1 and cTfR1, indicating that the binding to mTfR1 for all positive clones was weak compared to the binding to hTfR1 and cTfR1.

[0075] The selected clones were diluted using limiting dilution assay (LDA) to obtain monoclonality. The reactivity against mouse TfR1 and human TfR1 was reexamined by ELISA for the LDA and the monoclonal cultures after growth.

[0076] Indirect ELISA screening An ELISA assay was performed according to the standard ELISA protocol to screen plasma samples after immunization for reactivity against the target antigen or to identify hybridoma clones producing antibodies with reactivity against the TfR1 target protein. Briefly, a 96-well half-area plate (Corning) was coated with 1 μg / ml of His 10 -mTfR1 (SEQ ID NO: 72) or His 10 -hTfR1 (SEQ ID NO: 73). His 10 -mTfR1 and His 10 -hTfR1 are His 10The -P2-hTfR1 immunogen was recombinantly produced and purified using the procedure described above. The plates were blocked with 150 μl / well of protein-free blocking solution (Pierce) at room temperature for 1 hour with shaking (600-900 rpm). The plates were washed four times with PBS containing 0.1% Tween®-20 and Kathon®. Plasma samples, serially diluted from an initial dilution of 1 / 450, or hybridoma supernatant diluted to 1 / 2 were added to the plates (50 μl / well; dilution buffer: PBS containing 0.1% BSA and 0.05% Tween®-20), incubated at room temperature for 2 hours, and then washed four times. Detection antibody (HRP-conjugated anti-mouse IgG, Southern Biotech, catalog no. 1030-05, diluted to 1 / 5000 with dilution buffer) was added at 50 μl / well, and the plates were incubated at room temperature for 1 hour. After one more wash (see above), 50 μl / well of TMB substrate (K-Blue® Aqueous, Neogen) was added, and after 10-15 minutes, 50 μl / well of 0.5 M H2SO4 was added to stop the reaction. The optical density at 450 nm was measured using a plate reader (Tecan). The endpoint titer was defined as a dilution exceeding [mean value of blank wells (background) + standard deviation of blank wells × 3].

[0077] Primary screening of hybridoma clones producing antibodies reactive to the target protein was performed using nickel-coated ELISA plates. Briefly, 96-well Ni-coated plates (PIERCE) supplied pre-blocked with BSA were ELISAd with 3 μg / ml (100 μl) of His 10The plates were incubated overnight at 4°C without shaking, along with -mTfR1. The plates were washed four times with PBS containing 0.1% Tween®-20 and Kathon®. The 1 / 4 diluted hybridoma supernatant was added to the plates (dilution buffer: PBS containing 0.1% BSA and 0.05% Tween®-20), incubated at room temperature for 2 hours, and then washed four times. 100 μl / well of detection antibody (HRP-conjugated anti-mouse IgG, Southern Biotech, catalog no. 1030-05, diluted 1 / 5000 with dilution buffer) was added, and the plates were incubated at room temperature for 1 hour. After one more wash (see above), 100 μl / well of K-Blue® aqueous substrate (Neogen) was added, and the reaction was stopped after 10-15 minutes with 100 μl / well of 0.5 M H2SO4. The absorbance at 450 nm was measured using an ELISA plate reader (Tecan).

[0078] Table 1 shows examples of clones that were considered positive for binding to mouse TfR1 and human TfR1. These clones were confirmed by ELISA to bind to both His-tagged hTfR and cTfR, and not to His-tagged APP (negative control). The selected clones were further characterized using various assays. [Table 1]

[0079] Measurement using biolayer interferometry The selected clones were measured by biolayer interferometry (BLI) using an Octet instrument (Octet Red384, ForteBio). The configuration used involved capturing IgG from each clone onto individual sensor chips, allowing for the detection of antibodies binding to targets in solution. In addition to providing a measure of binding, BLI measurements also include estimates of binding and dissociation rates, thus providing further detailed information about the overall binding characteristics.

[0080] Figure 1 shows the results of BLI measurements for three selected clones provided as an example, where binding was measured directly in crude hybridoma supernatant. Briefly, mouse IgG antibody clones in hybridoma supernatant diluted 1:1 with running buffer (PBS, 0.02% Tween®-20, 0.01% BSA) were captured with an anti-mouse capture biosensor (anti-mouse capture, AMC, Molecular devices, catalog no. 18-5580). The IgG-immobilized sensor was then briefly washed for 10 seconds and incubated in running buffer to establish a baseline signal. Binding to target antigens was measured by incubating the sensor for 120 seconds in wells of an assay plate containing each target antigen at the following concentrations (500 nM mTfR1, 250 nM hTfR1, and 250 nM cTfR1). All proteins were diluted in running buffer. Target dissociation was measured by incubating the biosensor in running buffer for 90 seconds. All clones tested (i.e., 24B4, 26D3, and 37D10) bound to both human and cynomolgus monkey TfR1, but very weakly to mouse TfR1. Overall, most clones showed higher cross-reactivity to human and cynomolgus monkey TfR1 than to mouse TfR1.

[0081] Sequence determination of selected clones The target clones were cryopreserved and sequenced using whole transcriptome shotgun sequencing. Among the sequenced hybridoma clones were those named 26D3, 24B4, and 37D10. The amino acid sequences obtained for the heavy chain variable region (VH) and light chain variable region (VL) of each of these antibodies are shown in Table 2 below. [Table 2]

[0082] The complementarity-determining regions (CDRs) of these antibodies were identified based on Kabat's definition and are shown in Table 3 below. [Table 3]

[0083] Example 2 In vitro binding to human and cynomolgus monkey TfR1 and epitope screening Further detailed binding analysis was performed on purified and selected antibodies using the BLI method. The binding of Fab fragments of mouse antibodies 26D3, 24B4, and 37D10 to human TfR1 and cynomolgus monkey TfR1 was investigated. For example, the binding of immobilized TfR1 to the tested Fab fragments in solution was measured using the Octet Red384 BLI instrument. Antibody binding to TfR1 was measured using TfR1 complexed with human transferrin ligand (Tf). Tf / TfR1 complexes were formed on the treptoavidin biosensor by first loading the sensor with biotinylated human holotransferrin, followed by capturing hTfR1 or cTfR1 onto the sensor. The final complex density on the sensor was similar for both hTfR1 and cTfR1. Antibody binding to TfR1 was measured during a 120-second binding phase and a 300-second dissociation phase. Figure 2 shows sensorgrams of 15 nM 24B4-Fab, 26D3-Fab, and 37D10-Fab, as well as Fab derived from the known TfR1-binding antibody 8D3 (Boado et al (2009), Biotechnol Bioeng 102:1251-1258). The data showed that both 24B4-Fab and 26D3-Fab exhibited similar binding profiles to human and cTfR1, and cross-reactive binding to both isoforms was detected for 37D10-Fab, whereas no significant binding was detected for human or cynomolgus monkey TfR1 from 8D3-Fab. Importantly, this experiment demonstrates that all three Fabs—24B4-Fab, 26D3-Fab, and 37D10-Fab—bind to TfR1 when the natural ligand transferrin is complexed with TfR1.

[0084] Next, ELISA experiments showed that antibodies 26D3, 24B4, and 37D10 bind to the protease-like domain of TfR1. In the ELISA experiments, ELISA plates were coated using human, mouse, or three different chimeric TfR1 receptors (Figure 3). The ELISA protocol was slightly modified from the indirect ELISA described in Example 1 as follows: Briefly, ELISA plates were coated with the following His-tagged antigen at 1 μg / ml: the ectodomain of human TfR1 (His 10Chimeric TfR1 consisting of -hTfR1 (SEQ ID NO: 74), the ectodomain of mouse TfR1 (His10-mTfR1 (SEQ ID NO: 75) and a human apical domain transplanted into the mouse TfR1 ectodomain (h / m apical domain chimera, mhHD_TfR1 (SEQ ID NO: 76)), chimeric TfR1 consisting of a human helical domain transplanted into the mouse TfR1 ectodomain (h / m helical domain chimera, mhHD_TfR1 (SEQ ID NO: 77)), or chimeric TfR1 consisting of a human protease-like domain transplanted into the mouse TfR1 ectodomain (h / m protease-like domain chimera, mhPLD_TfR1 (SEQ ID NO: 78)). Next, the coated plates were blocked. Dilution series of mouse IgG of the antibodies to be analyzed were prepared in PBS and incubated on ELISA plates. After washing away unbound antibodies, the wells were incubated with HRP-conjugated secondary anti-mouse IgG for 1 hour. Next, after washing the plate again, the HRP substrate TMB was added to induce color development, and antibodies bound to the wells were detected. The TMB color development was stopped by adding 0.5 M H2SO4 to the wells, and the ELISA response was measured as OD at 450 nm using an ELISA plate reader. As shown in Figure 3, 26D3, 24B4, and 37D10 bind only to hTfR1 (A) and not to mTfR1 (B). 26D3, 24B4, or 37D10 do not bind to constructs in which the human apical domain is transplanted to the rest 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, binds to the h / m apical domain chimera as expected (C). Furthermore, 26D3, 24B4, and 37D10 bind to the h / m protease-like domain chimera (D), but do not bind to any plates coated with other chimeric receptors (C and E). In addition, the control antibody 8D3, which has an epitope in the apical domain of mTfR1, binds to all plates coated with TfR1 antigens containing this domain [i.e., mTfR1 (B), the h / m protease-like domain chimera (D), and the h / m helical domain chimera (E)].In summary, this experiment demonstrates that one or more epitopes, 26D3, 24B4, and 37D10, are primarily located within the protease-like domain of hTfR1, which is in contrast to the control antibodies 15G11-1 and 8D3.

[0085] Further BLI experiments conducted for epitope binning (binding competition) showed that binding by both 26D3 and 24B4 targeted the same or overlapping region of hTfR1 via an epitope located outside the apical domain (Figure 4). Epitope binning experiments using BLI were performed using an Octet Red384 instrument (ForteBio). First (Step 1), biotinylated hTfR1 was immobilized on a streptavidin biosensor (high-precision biosensor, ForteBio), and then (Step 2) a washing step was performed. Subsequently (Step 3), the hTfR1-filled sensor was incubated with either a buffer (non-competitive reference) or 200 nM of each antibody (Ab) to form an hTfR1:Ab complex on the sensor. Finally (Step 4), free hTfR1 (reference) or each pre-formed hTfR1:Ab complex was incubated in 200 nM antibody to measure binding to hTfR1 complexed with the competing antibody. Figure 4 shows representative BLI sensorgrams obtained during the indicated main assay steps. The signal in Step 4 indicates the degree of competition between the two analyzed antibodies. If the antibodies compete for binding to the same or overlapping epitopes, the signal in the Step 4 sensorgram does not increase. Conversely, if the two tested antibodies bind to distinctly different epitopes, the signal from Step 4 increases.

[0086] Figure 5 shows the results of competitive screening of antibody binding to epitopes on hTfR1 by the epitope binning described above. Antibodies 26D3 (dark gray bars) and 24B4 (light gray bars) were shown to bind to overlapping epitopes different from those of the control antibody 15G11-1 (black bar) hTfR1 apical domain epitope. Figure 5A shows that when hTfR1 is complexed with 24B4, the binding response to 26D3 is reduced by more than 70%. As expected, the binding of 26D3 to the pre-formed hTfR1:26D3 complex is almost completely inhibited, indicating that the complex blocks itself. Similarly, Figure 5B shows that when hTfR1 is complexed with 26D3, the binding response of 24B4 is reduced by 70%, and is almost completely inhibited by itself. When hTfR1 is complexed with control antibody 15G11-1, which has its binding epitope within the apical domain of hTfR1, both 24B4 and 26D3 maintain a complete binding response to hTfR1 (Figures 5A and 5B, black bars). As shown in Figure 5C, control antibody 15G11-1 shows a similar binding response to the apical domain of hTfR1 regardless of whether it is tested against hTfR1 without a competing antibody, or when the receptor is complexed with 24B4 or 26D3. In Figure 5, all responses are normalized to the maximum binding response of each antibody to free hTfR1.

[0087] Furthermore, the binding of antibodies to endogenous hTfR1 on brain endothelial cells was also tested. Binding to endogenous hTfR1 on the cell surface was tracked using flow cytometry and human hCMEC / D3 cells (Weksler et al (2013), Fluids Barriers CNS 10:16). Human hCMEC / D3 cells are known to express significant levels of hTfR1 on their cell surface. Positively stained cells were plotted, and the mean fluorescence intensity (MFI) is shown in Figure 6. Figures 6A (IgG1 antibody) and 6B (Fab fragment) both show that cells were positively stained to a similar degree (MFI) for hTfR1 in 24B4 and 26D3 compared to the positive control antibody 15G11-1, which has high affinity for hTfR1, and with higher staining intensity than the low-affinity control antibody 15G11-2 (Yu et al (2014), mentioned above). No background staining was detected in the negative isotype control (Figure 6A) and the unrelated Fab fragment Ly128 (Figure 6B). These data indicate that both 24B4 and 26D3 bind to hTfR1 expressed on the cell surface.

[0088] Example 3 Competition for hTfR1 binding with ferritin and transferrin The intrinsic binding of the binders disclosed herein to hTfR1 (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 transferrin (Tf). To test competition between ferritin and antibodies, the human monocyte cell line THP-1 (Sigma / ECACC) was used. Binding of the scFv-Fc form (see Example 4 below) and the control antibody (M-A712) to hTfR1 on the surface of THP-1 cells was confirmed, as shown in Figure 7A. To evaluate competition between ferritin and the disclosed binders, cells were incubated with serially diluted test binders and human liver-derived ferritin (BioRad, 4420-4804) at 4°C for 1 hour. After incubation, ferritin bound to hTfR1 on the cell surface was captured using a sheep primary antibody against human liver ferritin (BioRad, AHP2179G) and analyzed using flow cytometry. The results are shown in Figure 7B, showing that 26D3 scFv-Fc does not compete with cell surface ferritin, while anti-CD71 (clone M-A712) (Maier et al (2016), Mol Ther Nucleic Acids 5:e321), a control antibody known to bind to the same epitope as Ft on hTfR1, clearly competes with Ft binding. The identified 26D3 hTfR1 conjugate also had a much smaller effect on Ft binding, indicating that 26D3 has an epitope on hTfR1 different from the Ft binding site (Figure 7B).

[0089] K562 lymphoblastic cells (Sigma / ECACC) were used for the transferrin competition study. Cells were incubated at 4°C for 1 hour with serially diluted test constructs and human holotransferrin (ThermoFisher; T13342) conjugated with Alexa Fluor 488. Transferrin bound to hTfR1 on the cell surface was captured by flow cytometry, and the mean fluorescence intensity was plotted. Figure 7C shows no competition between the 26D3 conjugate and transferrin. When unlabeled (unbound) Tf was used as a positive control for competition, the binding of the labeled (AF488) Tf signal decreased in a concentration-dependent manner. This experiment demonstrates that conjugates targeting the protease-like domain of TfR1 do not directly compete with transferrin for the same epitope.

[0090] Overall, this example demonstrates that the binding of 26D3 to hTfR1 does not adversely affect the binding ability of the two endogenous ligands to the receptor, ferritin and transferrin.

[0091] Example 4 Humanization of hTfR1 binder 26D3 The Fab sequences of the mouse antibody 26D3, identified and characterized as described in Examples 1-3, were analyzed, and a computer model of the 26D3 Fab3D structure was created using Bioluminate software (Schrodinger). This mouse Fab model was used as input for humanization. In this process, the CDRs of the VH and VL regions of 26D3 (see Table 3, SEQ ID NOs. 10-15) were computer-transplanted into various human variable domains, and some residues were reverse-mutated at several positions to conform to the mouse framework. From the software, three variants with the fewest reverse mutations and otherwise desirable characteristics were generated and extracted. One of these humanized variants was selected for expression and named h26D3. h26D3 has a VH region sequence defined by SEQ ID NO: 44 and a VL region sequence defined by SEQ ID NO: 58. The humanized h26D3 and the original mouse sequence 26D3 were both expressed as His-tagged Fab cells by transient transfection of Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. The collected supernatant was purified using HiTrap IMAC Sepharose FF (Cytiva) followed by size exclusion chromatography using 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): 1 × dPBS (Thermo Fisher).

[0092] The binding of purified Fab to human and cynomolgus monkey TfR1 was evaluated by surface plasmon resonance (SPR) using a Biacore 8K instrument (Cytiva), and the results are shown in Figure 8. 1 μg / ml of human TfR1 (end-cleaved hTfR1, SEQ ID NO: 86) or cynomolgus monkey TfR1 (end-cleaved cTfR1, SEQ ID NO: 87) was immobilized on a Cm5 sensor chip (Cytiva, #BR100399) using an amine coupling kit type 2 (Cytiva, #BR100633) according to the manufacturer's instructions. h26D3 and 26D3Fab were injected onto the chip in a five-step 2x dilution series starting at 25 nM. Interactions were measured using a single-cycle kinetic method with a contact time of 120 seconds, a flow rate of 30 μl / ml, followed by a dissociation time of 600 seconds. Surface regeneration between cycles was performed by injecting 3 M MgCl2. The binding data was fitted to a 1:1 interaction model. Fab was diluted with HBS-EP+ (Cytiva, #BR100669). The experiment was conducted at 25°C. The data confirm that the humanized variant of 26D3 (i.e., h26D3) retains binding ability to human and cynomolgus monkey TfR1 (Figure 8). The dynamic parameters obtained from the experiment are shown in Table 4 below. [Table 4]

[0093] Both mouse 26D3 and the humanized variant h26D3 were converted to the scFv form and confirmed to maintain target binding as scFv (Figure 9). Mouse and humanized 26D3 were again converted to the scFv form (sequence number 79 and sequence number 80, respectively) and prepared as monovalent Fc-fused scFv antibody fragments using the knob-into-hole (KiH) technique. In this form, one scFv fragment is fused only to the knob-side half of the Fc (sequence number 81), while the hole-side half of the Fc (sequence number 82) remains unfused. The resulting antibody form is a single-arm scFv-Fc. The 26D3 scFv fused to the knob-side half of the Fc has sequence number 83, which is a complete (cOmplete) amino acid sequence, while the h26D3 scFv fused to the knob-side half of the Fc has sequence number 84, which is a complete (cOmplete) amino acid sequence. The binding profiles of the scFv form of mouse and humanized 26D3 are similar, confirming the binding activity in the scFv form. The binding response is consistent with the response of the Fab form of the antibody. This was confirmed by several methods, including dynamic experiments using BLI (results shown in Figure 9A) and ELISA (results shown in Figure 9B). The binding rates of mouse and humanized 26D3-scFv-Fc were first measured by immobilizing biotinylated hTfR1 on a streptavidin biosensor (Fortebio) using BLI. Next, the sensor was washed with buffer (Kinetics buffer, Fortebio), and the binding of 26D3-scFv-Fc (mouse) and h26D3-scFv-Fc (humanized) was measured at a concentration of 25 nM, followed by a 500-second dissociation period. In the ELISA experiment, a standard binding ELISA plate was coated with hTfR1 using the indirect ELISA protocol described in Example 1.

[0094] Example 5 Crystallization and structural determination of h26D3-Fab complexed with hTfR1 This example describes the crystallization of the h26D3-Fab and hTfR1 complex and the measurement of the binding interface. The ectodomain of human TfR1 (SEQ ID NO: 74) was expressed by transient transfection into human embryonic kidney cells (Expi297, Thermo Fisher Scientific) according to the manufacturer's instructions. The collected supernatant was purified using HiTrap IMA Sepharose FF (Cytiva) followed by size exclusion chromatography using HiLoad Superdex 200pg 26 / 600 (Cytiva). The buffer used and the purification method for humanized Fab are as described in Example 4.

[0095] The complex of humanized h26D3-Fab and hTfR1 was formed by mixing the two components in a 1:1 molar ratio in 1×dPBS solution and incubating at room temperature for 1 hour. The complex was then purified by size exclusion chromatography using HiLoad Superdex 200pg 26 / 600 (Cytiva), as described in Example 4.

[0096] Crystallization was performed using a 15 mg / ml stock solution of hTfR1-h26D3 in PBS, which was then diluted to 4 mg / ml in PBS supplemented with 4 mM β-mercaptoethanol. 100+100 nl droplets were prepared using additive screening in a reservoir (0.1 M potassium sodium phosphate (pH 6.5), 10% PEG3000, 0.05% dichloromethane, and 2 mM β-mercaptoethanol). The crystals were rapidly frozen in a reservoir solution supplemented with 8% glycerol and 16% PEG400.

[0097] X-ray data acquisition and refinement were performed as follows. Data was acquired down to 3.87 Å at the diamond light source beamline I04. 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) and STARANISO anisotropic scaling (Tickle et al (2018), Global Phasing Ltd) to obtain the reciprocal lattice c * Along the direction up to 3.87 Å, and a * / b * Diffraction was performed along the plane to 4.82 Å. Three complexes were found in the asymmetric unit. The structure was refined using Buster refinement software, and the model was built using Coot. Data acquisition and refinement parameters and statistics are shown in Table 5 below. [Table 5]

[0098] The final refined structure of the complex, showing the overall folded structure, is shown in Figure 10. As shown in Figure 10A, there were three independent complexes in the asymmetric unit. The chain names used in the correspondence file are indicated. Figure 10B shows an example of electron density along the heavy / light chain interface between hTfR1 and h26D3-Fab. Protein chains are shown as sketches, and glycans as lines. The interaction of the binding interface between h26D3 and human TfR1 is derived from the X-ray structure and is described below to provide information on the precise binding of h26D3 to human TfR1.

[0099] The binding interfaces between hTfR1 and h26D3-Fab are shown in Figures 10 and 11, and interactions were observed between the amino acid residues shown in Table 6. [Table 6]

[0100] Table 6 shows the major residues on both sides involved in the epitope / paratope interface as determined from the crystal structure. Additional residues in the vicinity are also likely to be important for the binding of h26D3 to hTfR1. Furthermore, as described in Example 9 below, several positions outside the location where the binding interaction was observed also show important involvement in the binding of h26D3 to hTfR1.

[0101] Table 7 below shows the amino acids of human TfR1 involved in the interactions with h26D3, Ft, and Tf, respectively. Notably, the amino acids involved in the binding of h26D3 are not present at any of the binding interfaces of the endogenous ligands. This indicates that the binders of this disclosure, exemplified by h26D3, bind to hTfR1 outside the binding sites where Ft and Tf are used. [Table 7] * Montemiglio et al (2019), Nat Commun 10:1121 # Eckenroth et al (2011), Proc Natl Acad Sci USA 108:13089

[0102] Various epitopes on the hTfR1 structure (pdb:1SUV) are further illustrated in Figure 12. As shown in Figure 12, the Ft binding site is located in the apical domain of hTfR1, the Tf binding site is mainly located in the helical domain of hTfR1, and the h26D3 epitope is located in the protease-like domain of hTfR1. This structure demonstrates that different ligands and binders utilize different specific surface regions on the hTfR1 structure. hTfR1 is a homodimer with two identical chains, and the epitopes are shown on only one of these chains.

[0103] Example 6 Production and characterization of hTfR1 knock-in mice Human TfR1 knock-in (hTfR1-KI; TFR1C-KI) mice were generated by homologous recombination (experiments were performed at Cyagen US). A cDNA vector containing the TFR1C (NCBI reference sequence: NM_001128148.3) ectodomain and mouse Tfrc transmembrane domain and intracellular domain was introduced into C57BL / 6N ES cell Tfrc cells by pronuclear microinjection. The coding region of Tfrc exon 2 and partial intron 2 was replaced with a TFR1C chimeric cassette (Figure 13A). The correct insertion of the hTfR1 cDNA was confirmed by Southern blotting and PCR. Transgene expression in hTfR1-KI mice was confirmed in brain tissue by qRT-PCR (Figure 13B) and Western blotting (Figure 13C), showing endogenous expression levels. hTfR1-KI mice were maintained in a C57BL / 6N background, and only heterozygous hTfR1-KI mice were used in the experiments.

[0104] Example 7 In vivo uptake of hTfR1-binding constructs into the brain. To evaluate in vivo uptake into the brain via hTfR1, monovalent Fc-scFv constructs (see Example 4) were prepared for four different binding proteins. As a control, 15G11-1, a known conjugate for hTfR1, was used (Yuetal (2014), op. cit.). This hTfR1 conjugate has been reported to be active in vivo and is used as a positive reference control for brain uptake. Furthermore, a construct containing a non-hTfR1 scFv conjugate based on the anti-amyloid beta antibody mAb158 was designed and included as a negative control in the form of an Fc fusion construct (Fc-scFv158, also simply referred to as "158" in the text and figures). Different Fc-scFv constructs were intravenously (iv) administered at an equimolar dose of 30 nmol / kg (approximately 2.3 mg / kg) to hTfR1 knock-in (hTfR1-KI) mice (n=4 per construct) prepared as described in Example 6. Plasma and brain exposure were evaluated 24 hours after administration.

[0105] Animals were anesthetized with isoflurane, and peripheral blood samples were collected from the orbital plexus into BD Microtainer K2EDTA tubes. The samples were inverted and centrifuged at 2400 × g for 10 minutes at 4°C. Plasma was extracted, transferred to Eppendorf tubes, and frozen at 80°C. Immediately after blood collection, the animal's abdomen was incised, and a cannula (21G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium, and transcardiac perfusion was performed using at least 50 ml of cold PBS. After perfusion, the brain was removed, and the olfactory bulb was removed. The brain was separated into left and right hemispheres, the cerebellum was removed from the left hemisphere, and then the left hemisphere was weighed, rapidly frozen on dry ice, and stored at -80°C until the concentration of the injected construct was further prepared and analyzed using a Meso Scale Discovery (MSD) based assay. The right hemisphere was immersed in 4% formaldehyde and stored at 4°C for 24 hours. After washing with cold PBS, it was transferred to a cold 30% sucrose solution prepared in PBS and stored at 4°C for immunohistochemistry (IHC) (see Example 8 below).

[0106] To measure brain concentration, the frozen left hemisphere was thawed on ice and homogenized in TBS using an automated bead homogenization method. Triton was added to the homogenate to a final triton concentration of 0.5%, and the mixture was centrifuged at 16,000 × g. The supernatant was then collected.

[0107] Brain and plasma concentrations of anti-hTfR1 Fc-scFv were measured using a custom-designed MSD assay for detecting human Fc. Standard 96-well MSD plates (MSD, #L15XA-3) were coated with 0.5 μg / ml goat anti-human IgG, Fcγ fragment-specific antibody (Jackson Immuno Research Europe Ltd, #109-005-098) diluted in 1×PBS (Medicago AB, #09-9400-100). After incubation overnight at 4°C, the plates were washed four times per well with 1×PBS-Tween (Fisher Scientific, #09-9410-100) and blocked with 150 μl of 1% Blocker A in PBS-Tween (MSD, #R93BA-4). Samples and corresponding standards in the 400 pM to 0.1 pM range were added in a 1:4 dilution step and incubated at room temperature for 2 hours at 900 rpm. This was followed by a 1-hour incubation step using mouse anti-human IgG (Mabtech, 3850-1-1000, MT145) diluted to 0.5 μg / ml, and then incubation with SULFO-TAG-conjugated anti-mouse antibody (MSD, R32AC-1) diluted to 0.5 μg / ml for 1 hour, after which the plates were incubated at room temperature at 900 rpm for a further 1 hour. After adding 150 μl of MSD read buffer (MSD, #R92TC) per well, the plates were read using an MSD SECTOR image sensor. Between each incubation step, the plates were washed four times with 1×PBS-Tween. All antibodies except the coated antibody and the samples were diluted in 1% blocker A in PBS-Tween and added at 50 μl / well. The concentrations of analytes in the samples were evaluated using the 4PL curve fitting algorithm and a standard curve weighting of 1 / Y2 with MSD Workbench software. Statistical analysis was performed using GraphPad Prism (v.9.0.0) with one-way ANOVA and Tukey's post-hoc test.

[0108] The results are shown in Figure 14. As shown in Figure 14A, 24 hours after administration, substantially higher brain concentrations were observed in the two test constructs and the positive control 15G11-1 compared to the negative control (158). As shown in Figure 14B, the plasma concentrations of the two test constructs and the positive control 15G11-1 were lower than those of 158 after 24 hours, indicating that activation of hTfR1 leads to faster clearance from plasma. The brain-to-plasma concentration ratio is shown in Figure 14C. Compared to the negative control, the two test constructs and the positive control 15G11-1 showed significantly increased exposure to the brain compared to exposure to plasma. In summary, these data support the idea that the novel hTfR1 conjugates tested in this experiment are responsible for hTfR1-mediated blood-brain barrier transport.

[0109] Example 8 Immunohistochemical staining data on brain exposure The in vivo involvement of hTfR1 by the Fc-scFv construct was further investigated using qualitative immunohistochemistry (IHC) analysis. Briefly, 20 μm thick coronal brain sections were prepared from PBS-perfused mouse cerebral hemispheres as described in Example 7 using a cryostat (Microm NX50 CryoStar, Epredia). The sections were collected on Superfrost plus slides (Menzel-Glaser, #J1800AMNZ), air-dried, and then subjected to 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) at room temperature for 2 hours. To visualize the intravenously administered construct, brain sections were incubated with secondary goat anti-human IgG (heavy and light chain specific) conjugated to Alexa Fluor 488 (Invitrogen, #A11013) at room temperature for 120 minutes, followed by three 15-minute washes with PBS. Slides were mounted on Fluoromount-G (Invitrogen, #00-4958-02) for image analysis. Confocal images from the cerebral cortex were captured using a Leica Stellaris 5 confocal system fitted with an HC PL APO 40x / 1.25 GLYC motCORR CS2 objective lens (Leica, #11506423).

[0110] Clear IHC immunofluorescence signals were observed in cerebral capillaries using the positive reference module 15G11-1, while minimal IHC signals were detected in brain sections of mice injected with the negative control 158 (Figure 15). IHC signals were observed in cerebral capillaries for two test constructs, h26D3 and 37D10, with h26D3 showing the strongest immunofluorescence signal, comparable to the positive control 15G11-1. Taken together, MSD (Example 7) and IHC (This Example) analyses demonstrate that the scFv form of the hTfR1 conjugate of this disclosure leads to increased brain exposure in hTfR1-KI mice.

[0111] Example 9 Creation of affinity variants and affinity measurement Several variants of the parent antibody h26D3 were prepared by substituting one alanine residue each with tyrosine, tryptophan, and aspartic acid residues in the CDR. The resulting variant VH regions are denoted as HC1-HC13, and their amino acid sequences are listed in the sequence listing as SEQ ID NOs. 45-57, respectively. The variant CDR sequences contained within these variant VH regions are listed as SEQ ID NOs. 16-28, respectively. The resulting variant VL regions are denoted as LC1-LC6, and their amino acid sequences are listed in the sequence listing as SEQ ID NOs. 59-64, respectively. The mutant CDR sequences contained within these variant VL regions are listed as SEQ ID NOs. 29-33, respectively. Table 8 below shows an overview of the specific mutations in each alanine variant. [Table 8]

[0112] The prepared alanine variants were expressed as single-mutant His-tagged Fab cells by transient transfection of Chinese hamster ovary cells (ExpiCHO, Thermo Fisher Scientific) according to the manufacturer's instructions. Binding of BLI (Octet Red 384, ForteBio) to hTfR1 was evaluated using clarified medium secreted by Fab. Expressed Fab cells were packed into anti-Fab biosensors from the cell supernatant for 240 seconds. Binding of the ectodomain of hTfR1 (SEQ ID NO: 74), diluted to 3.75 μg / ml in 1× Kinetics buffer (ForteBio), to the packed sensors was measured for 300 seconds, followed by dissociation for 300 seconds. All variants were confirmed to bind to hTfR1, but the degree of influence varied (Figure 16).

[0113] Varieties whose binding to hTfR1 was affected during screening were selected for further characterization. Furthermore, double mutants were created by combining heavy and light chains using alanine substitution. Table 9 below summarizes the specific mutations in each selected alanine variant. [Table 9]

[0114] The selected variants were expressed as His-tagged Fab by transient transfection into Chinese hamster ovary cells (ExpiCHO, Thermo Fisher Scientific) according to the manufacturer's instructions. Fab was purified on a small scale using HisPur® Ni-NTA magnetic beads (Thermo Scientific) according to the manufacturer's instructions, followed by buffer exchange to DPBS (pH 7.4). The selected variants were purified on a HisTrap Excel column (Cytiva) and washed with 20 mM Tris, 200 mM NaCl, and 5 mM imidazole. The proteins were eluted with 20 mM Tris, 200 mM NaCl, and 500 mM imidazole, followed by buffer exchange to DPBS (pH 7.4) using a HiPrep 26 / 10 desalting column (Cytiva). The proteins were concentrated using an Amicon ultracentrifuge (30MWCO, Millipore). The selected variants were further purified by size exclusion chromatography (SEC, HiLoad 26 / 600 Superdex200, Cytiva) in DPBS (pH 7.4). Analytical characterization of the proteins was performed by UV protein quantification, SDS-PAGE, and HPLC-SEC. sfjj

[0115] The binding of purified Fab to human and cynomolgus monkey TfR1 was evaluated using SPR (Figure 17) or indirect ELISA (Figure 18). A Biacore 8K instrument (Cytiva) was used for SPR. 1 μg / ml of hTfR1 (SEQ ID NO: 86) or cTfR1 (SEQ ID NO: 87) was immobilized on a Cm5 sensor tip (Cytiva, #BR100399) using an amine coupling kit type 2 (Cytiva, #BR100633) according to the manufacturer's instructions. Fab was injected into the tip using a four-step 2x dilution series starting at 100 nM. Interactions were measured using a single-cycle kinetic method with a contact time of 120 seconds, a flow rate of 30 μl / min, followed by a dissociation time of 1000 seconds. Surface regeneration between cycles was performed by injecting 3 M MgCl2. Binding data were fitted to a 1:1 interaction model. Fab was diluted with HBS-EP+ (Cytiva, #BR100669). The experiment was conducted at 25°C. The results are shown in Figure 17, and the calculated K D The values ​​are shown in Table 10 below. [Table 10]

[0116] In indirect ELISA, half of a 96-well plate (Corning, #3690) was incubated overnight at 4°C with 1 μg / ml recombinant hTfR1 ectodomain (SEQ ID NO: 74) in PBS. The coated plate was blocked with Pierce protein-free blocking solution (Thermo Fisher Scientific, #37572) with shaking at room temperature for 1 hour and washed four times with PBS containing 0.1% Tween 20. Serial dilutions (1:3) of various expression constructs in incubation buffer (PBS with 1% BSA and 0.1% Tween 20) were incubated at room temperature for 1 hour. After the four washing steps, the conjugated test constructs were detected with anti-human IgG F(ab')2-HRP antibody (Jackson Immuno Research, #109-036-003) diluted 1:5000 in incubation buffer (1 hour, room temperature). After four washing steps, K-Blue® aqueous TMB substrate (Neogen, #331177) was added to the wells and reacted at room temperature for 15 minutes. The reaction was then stopped with a 1:1 dilution of 0.5 M H2SO4. The optical density at 450 nm was recorded (Spark, Tecan), and the analysis was performed after subtracting the background signal. The results are shown in Figure 18.

[0117] Based on Biacore and ELISA measurements, several variants exhibiting a broad affinity range for human TfR1 were identified. Many variants retained cross-reactivity with cynomolgus monkey TfR1.

[0118] Finally, the morphology of the selected variant was again converted to scFv and used in the bispecific binding molecule form disclosed in International Publication No. 2022 / 258841. The bispecific binding molecule, containing the scFv module constructed from h26D3 and the selected alanine variant, was expressed in ExpiCHO cells as described above. The filtered supernatant was applied to a MabSelect SuRe column (Cytiva) and subsequently washed with DPBS (pH 7.4). The expressed binding molecule was eluted with 0.7% HAc (pH 2.5), and the sample was rapidly neutralized to pH 7.5. The purified sample was further purified (polished) by size exclusion chromatography (SEC, HiLoad 26 / 600 Superdex 200, Cytiva) in DPBS (pH 7.4). The purified construct was concentrated using a centrifugal concentrator Amicon Ultra (30MWCO, Millipore). Each purified expression construct was characterized by SDS-PAGE, size exclusion chromatography (Superdex 200 increase 3.2 / 300; Cytiva), and UV protein assay. Binding to hTfR1 was evaluated using SPR as described above, with the concentration range adjusted according to the variant. As shown in Figure 19 and Table 11 below, the various variants tested showed a wide range of affinities to the hTfR1 target. [Table 11]

[0119] Example 10 Design of Gen2A2 bispecific binding proteins using the "VH-first" or "VL-first" scFv module Using the "Gen2A" form first described in International Publication No. 2022 / 258841 (Figure 4, left panel), 14 different bispecific binding protein constructs were designed using the scFv form of the above hTfR1 conjugate h26D3 HC6 in two different configurations: "VH-first" represented by SEQ ID NO: 88 and "VL-first" represented by SEQ ID NO: 89. Seven constructs were designed using the "VH-first" configuration (#1-7, each having a single-chain component represented by SEQ ID NOs: 90-96), while the other seven constructs were designed using the "VL-first" configuration (#8-14, each having a single-chain component represented by SEQ ID NOs: 97-103). The antibody heavy chain used in all of these constructs is identical, represented by SEQ ID NO: 104.

[0120] Figure 20 shows schematic diagrams of the different constructs tested. Constructions #1-7 (VH-first) and #8-14 (VL-first) were prepared as a series of combinations with different linker lengths, as shown in Table 12, and the effects of linker length and VH-first vs. VL-first configuration combined with the h26D3 HC6 scFv binder on binding to hTfR1 and antibody placement were investigated when hTfR1 was expressed on the cell surface. [Table 12]

[0121] Example 11 Fabrication and refinement of the designed Gen2A construct 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. The supernatant of the filtered cell culture was added to a MabSelect SuRe column (Cytiva) and then washed with DPBS (pH 7.4). The expressed binding proteins were eluted using 0.7% HAc (pH 2.5), and the sample was then neutralized to pH 7.5. The purified samples were further purified in DPBS (pH 7.4) by size exclusion chromatography (SEC, HiLoad 26 / 600 Superdex 200; Cytiva). Each purified expression construct was characterized using SDS-PAGE, size exclusion chromatography (Superdex 200 Increase 3.2 / 300; Cytiva), and UV protein concentration measurement.

[0122] The purification results are shown in Table 13. Representative SDS-PAGE analyses of the purified constructs are shown in Figure 21. In the non-reducing gel, one band was observed at approximately 175 kDa. In the reducing gel, two bands were observed, as expected for the Gen2A morphology: an antibody heavy chain at approximately 50 kDa and a single-chain component containing two light chains linked to hTfR1-binding scFv at approximately 75 kDa. As shown in Table 13, the monomer content of the bispecific binding protein was high (generally over 98%), and these were produced at low mg / l levels. [Table 13]

[0123] Example 12 Bounding analysis of Gen2A to hTfR1 using SPR The binding of the bispecific binding protein expressed and purified as described in Example 11 to hTfR1 was evaluated using SPR (Biacore 8K, Cytiva). 2 μg / ml of hTfR1 was immobilized on a Cm5 sensor chip (Cytiva, #BR100399) using an amine coupling kit type 2 (Cytiva, #BR100633) according to the manufacturer's instructions. The bispecific binding protein was injected onto the chip using a 2-fold dilution series in four steps, starting at 200 nM. The interaction was measured using a single-cycle kinetic method with a contact time of 120 seconds, a flow rate of 30 μl / ml, followed by a dissociation time of 600 seconds. Surface regeneration between cycles was performed by injecting 3 M MgCl2. Binding data were fitted to a 1:1 interaction model. The bispecific binding protein was diluted with HBS-EP+ (Cytiva, #BR100669). Experiments were performed at 25°C. The data in Figure 22 show that all designed and fabricated Gen2A constructs bound to hTfR1. All constructs showed similar binding and dissociation rates compared to the control Fab construct with the hTfR1 binding agent h26D3 HC6. This illustrates that all expressed constructs were functional and that neither linker length nor "VH-first" / "VL-first" configuration directly affects the binding of the construct to hTfR1.

[0124] Example 13 Binding to hTfR1 expressed on the cell surface Binding to hTfR1 on cells was measured in the immortalized human B lymphocyte cell line Ramos (Sigma, catalog number: 85030802). This cell line is known to express high levels of hTfR1 on its cell surface. After blocking the Fcγ receptor with an Fc receptor blocker (Innovex biosciences, #NB309-4X-40) at 4°C for 30 minutes, the cells were washed with PBS. The cells were inoculated into a 96-well V-bottom plate (#249570, Thermo Scientific Nunc), serially diluted Gen2A constructs were added, and the plates were incubated overnight at 4°C. The cells were washed with PBS containing 1% BSA and then fixed at room temperature for 15 minutes using 4% formaldehyde (Thermo Scientific Pierce, #28906) diluted in freshly prepared PBS. The cells were washed with PBS containing 1% BSA and then stained. Bispecific binding proteins bound to hTfR1 on the cell surface were detected via a shared IgG heavy chain component using fluorescently labeled secondary goat F(ab')2 anti-human IgG(γ)-Alexa fluor 488 (Invitrogen Life Technologies, #H10120). Staining was performed at 4°C for 30 minutes. After incubation with the detection reagent, cells were washed with PBS containing 1% BSA. Finally, cells were resuspended in 200 μl of PBS containing 1% BSA and acquired using a BD FACSLyric flow cytometer system (BD Biosciences). Samples were analyzed using flowJo software (BD Biosciences). The median fluorescence intensity (MFI) measured was plotted against the binding protein concentration and is shown in Table 23. The results demonstrated that all tested Gen2A constructs bound similarly to hTfR1 expressed on the cell surface, regardless of linker length or "VH-first" / "VL-first" configuration.

[0125] Example 14 Complement-dependent cell-mediated cytotoxicity (CDC) analysis Complement activity is initiated, for example, by the binding of C1q to the Fc portion of an antibody, followed by the binding of other complement factors, ultimately leading to cell death. To evaluate whether a specific bispecificity construct induces CDC activity by enabling C1q binding to Fc, Ramos cells (Sigma, catalog number: 85030802) were used as target cells for CDC analysis. To measure cell death, Ramos cells were labeled with the cell viability stain Calcein-AM (Sigma, #17783). These labeled cells were then treated with serially diluted bispecificity constructs in the presence of pooled human complement serum (Innovative Research Inc, #39337) at 37°C and 5% CO2 for 4 hours. As a negative control, cells were treated with the same concentration of bispecificity 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).

[0126] Treated cells were collected using a BD BDLyric flow cytometer (BD Biosciences). Samples were analyzed using flowJo software (BD Biosciences). The frequency of cell death in gated cells quenched with calcein AM was measured and plotted against the concentrations of the tested bispecific constructs or controls. The results for constructs #1-7 ("VH-first") are shown in Figure 24, and for constructs #8-14 ("VL-first") are shown in Figure 25. It was clearly observed that bispecific binding proteins with the "VH-first" configuration mediated CDC activity and led to cell death (Figure 24). On the other hand, none of the bispecific binding proteins with the "VL-first" configuration mediated CDC activity (Figure 25), leading to the conclusion that hTfR1 binding mediated by the "VL-first" configuration inhibits the binding of C1q to the antibody's Fc domain.

[0127] Example 15 Exposure to plasma and brain of Gen2A constructs with hTfR1 affinity variants To evaluate the time-dependent changes in exposure of the bispecific binding protein constructs of this disclosure to brain and plasma, additional constructs based on the Gen2A morphology were prepared, similar to Example 10. This example examines the antibody mAb158 in the hIgG1 morphology with the Fc mutation K322A, with or without three affinity variants of the hTfR1 binding module h26D3 (see Example 9). The tested constructs and their amino acid sequences are shown in Table 14. [Table 14]

[0128] hTfR1 knock-in (hTfR1-KI) mice (n=15 per test substance) prepared as described in Example 6 were intravenously (iv) injected with different affinity variants and the comparison substance mAb158 hIgG1 at equimolar doses of 40 nmol / kg (equivalent to approximately 6-7 mg / kg). Plasma and brain exposure were evaluated at five consecutive termination time points of 4, 24, 72, 168, and 240 hours, using n=3 mice for each time point and test compound. Blood for evaluation of the consecutive plasma concentration versus time profile was collected from the group of animals (n=3) that were terminated at 240 hours at 0.25, 4, 24, 48, 72, 120, 168, and 240 hours after administration. Live blood was collected from the saphenous vein into a Sarstedt Microvette CB300 K2E tube.

[0129] At each euthanasia point, the animals were deeply anesthetized with isoflurane, and blood samples were collected from the orbital plexus into BD Microtainerr K2EDTA tubes. The samples were inverted and centrifuged at 2400 × g at 4°C for 10 minutes. Plasma was extracted, transferred to Eppendorf tubes, and frozen at -80°C. Immediately after blood sampling, the animals' abdomens were incised, and a cannula (21G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium, and transcardiac perfusion was performed using ice-cold PBS. After perfusion, the brain was collected, and the olfactory bulb was removed. The brain was separated into left and right hemispheres, the cerebellum was removed from the left hemisphere, and then the left hemisphere was weighed, rapidly frozen on dry ice, and stored at -80°C until further preparation of the injected test constructs and analysis of their concentrations using a Meso Scale Discovery (MSD) based assay. The right hemisphere was placed in 4% formaldehyde and stored at 4°C for 24 hours. After that, it was rinsed with cold PBS, transferred to a cold 30% sucrose solution prepared in PBS, and then stored at 4°C for immunohistochemistry (IHC) (see Example 16 below).

[0130] For intracerebral concentration measurements, the frozen left hemisphere was thawed on ice and homogenized for 5 seconds at 6 m / s using an automated bead homogenization method with MP Biomedical's FastPrep-24 5G system with Lysing Matrix D in Tris-buffered saline (TBS) containing the cOmplete protease inhibitor and the phoSTOP phosphatase inhibitor (#11836145001 and #04906837001, Roche). Triton X-100 (#X100, Merck) was added to the homogenate to obtain a final Triton X-100 concentration of 0.5% and a weight-to-volume ratio of 1:10. The homogenate was vortex-mixed for 10 seconds, centrifuged at 16,000 × g at 4°C for 1 hour, and the supernatant was collected and used to measure antibody exposure in the brain.

[0131] The brain and plasma concentrations of 2A3#2-LC1-K322A, 2A3#2-HC6-K322A, 2A3#2-LC5-K322A, and mAb158 hIgG1-K322A were measured using a custom MSD assay for detecting human Fc. 96-well MSD plates (#L15XA-3) were coated overnight at 4°C with 25 ng / well of goat anti-human IgG, Fcγ fragment-specific antibody (#109-005-098, Jackson Immuno Research Europe Ltd) diluted in 1×PBS (#09-9400-100, Medicago AB). The coating was removed, and the wells were blocked with 1% Blocker A (#R93BA-4, MSD) in PBS-0.05% Tween 20 (PBS-T) (#09-9410-100, Medicago AB). After washing four times with 1×PBS-T, the sample and the test construct calibrator diluted with 1% Blocker A in PBS-T were added to the plate and incubated at room temperature (RT) at 900 rpm for 2 hours. Detection of bound antibodies was performed by a continuous incubation: secondary antibody (mouse anti-human IgG #3850-1-1000, MT145, Mabtech) at 900 rpm at room temperature for 1 hour, followed by SULFO-TAG-conjugated anti-mouse detection antibody (R32AC-1, MSD) at 900 rpm at room temperature for 1 hour. Both the secondary antibody and the detection antibody were diluted to 25 ng / well in 1% Blocker A in PBS-T. Between all incubation steps, the plate was washed four times with 1×PBS-T. After the final wash, 2X Read Buffer T (#R92TC, MSD) was added, and the plate was read using an MSD SECTOR imaging system. The concentration of the test construct in the sample was evaluated using the MSD Discovery Workbench software, employing the 4PL (4-parameter logistic) curve fitting algorithm and a curve weighting of 1 / Y2 for the corresponding test construct calibration curve.

[0132] The results are shown in Figures 26 and 27. As shown in Figure 26 for the euthanasia samples, the test construct with the hTfR1 binding module was observed to have higher peak concentrations in the brain and higher brain exposure over time compared to mAb158 hIgG1-K322A alone. As shown in Figure 27, plasma exposure of the test construct with the hTfR1 binding module was lower than that of mAb158 hIgG1-K322A, indicating the involvement of hTfR1 and the transfer of the test construct from plasma to hTfR1-expressing tissues. Taken together, the data support the conclusion that the test construct undergoes blood-brain barrier transport via hTfR1 and that the affinity of the hTfR1-binding h26D3 variant influences both the brain and plasma exposure profiles.

[0133] Example 16 Immunohistochemical staining of Gen2A constructs with hTfR1 affinity variants The in vivo involvement of hTfR1 using the test constructs from Example 15 (2A3#2-LC1-K322A, 2A3#2-HC6-K322A, 2A3#2-LC5-K322A, and mAb158 hIgG1-K322A) was further investigated using qualitative immunohistochemistry (IHC) analysis. Briefly, the right hemisphere of animals euthanized in Example 15 was embedded in sucrose in OCT compound (LAMB / OCT, Thermo Fisher Scientific) and rapidly frozen in dry ice. The embedded right hemisphere was sectioned, and 20 μm sagittal slides were taken onto Superfrost cryoslides (J1800AMNZ, Thermo Fisher Scientific), air-dried, and then subjected to IHC. Brain sections were pre-treated at room temperature for 1 hour using MOM mouse IgG blocking reagent (MKB-2213-1, Vector Laboratories). The primary antibody was diluted in 1×PBS 0.1% Triton X-100 and incubated overnight at 4°C. The secondary antibody was diluted in 1×PBS and incubated at room temperature for 1.5 hours. Blood vessels were visualized using anti-collagen IV (1:100) (2150-1470, Biorad) and Alexa488 anti-rabbit IgG H+L (1:500) (A21206, Invitrogen). Constructs administered intravenously were visualized using Alexa647 anti-human IgG H+L (1:500) (A21206, Invitrogen). All incubations were performed in a PBS humidified chamber. After incubation, slides were washed in a cuvette with 1×PBS (usually 5×5 minutes). Sections were mounted using Fluoromount-G (00-4958-02, Invitrogen, USA), and images were acquired using a Leica Stellaris 5 confocal system equipped with HC PL APO 40x / 1.25 GLYC motCORR CS2 (Figure 28) and HC PL APO 63x / 1.40 OIL CS2 (Figure 29) objective lenses (Leica, #11506423).

[0134] Brains 24 hours post-administration showed clear IHC immunofluorescence signals for LC1 and HC6 constructs in the capillaries (Figure 32). The LC5 variant and control (mAb158 hIgG1-K322A) did not show immunofluorescence signals in the capillaries (Figure 28). Some variability was observed even among brains in the same group, so macroscopic perfusion success was assessed using a subjective score of 0 to 3 in interpreting the images. The score was established based on visual inspection of the excised brain, with a score of 0 corresponding to a white brain with no signs of blood in any vessels, a score of 1 corresponding to a slightly pink brain with faint signs of blood in a few vessels, a score of 2 corresponding to a pink brain with signs of blood in the vessels, and a score of 3 corresponding to a red brain with significant visible signs of residual blood in the remaining vessels or major vessels covering most of the brain. Brains injected with 2A3#2-LC5-K322A and mAb158 hIgG1-K322A exhibited immunofluorescence signals in the capillaries, indicating poor perfusion, and the perfusion score was in good agreement with the imaging. Staining with collagen IV visualized all capillaries on the slide, and the immunofluorescence signals were comparable to those of the administered constructs (Figure 29). This data indicates that constructs containing hTfR1-binding variants are actively taken up by cerebral capillaries upon intravenous administration.

[0135] Example 17 Investigation of injection-related immune responses In vivo injection of Fc-containing biomolecules may induce immune responses, such as acute clinical symptoms, as described, for example, by Couch and his collaborators (Couch et al (2013), Sci Transl Med 5(183):183ra57, 1-12). To evaluate the test constructs of this disclosure in relation to such responses, the test constructs and control constructs shown in Table 15 were designed and expressed. [Table 15]

[0136] Constructs were prepared that were apical TfR1 conjugates bound to IgG1 with complete effector function in the Fc domain, exhibiting high affinity for mouse (2A2#2-8D3) or human (2A2#2-15G11). 2A2#2-8D3-K322A was prepared as a complement resistance comparison material for 2A2#2-8D3. "VL-first" Gen2A variants 2A3#10-WT and 2A3#14-WT, and "VH-first" variants 2A3#3-WT and 2A3#2-WT (all of which contain the h26D3 TfR1 binding module of this disclosure in "VL-first" or "VH-first" scFv format) were prepared to investigate whether the injection reaction was mitigated by epitope binding and binding module orientation. All test components were administered as a single intravenous (iv) infusion to hTfR1 knock-in (hTfR1-KI) mice (Example 6) expressing both mouse and human TfR1, at doses of 2.5, 11, 40, or 60 nmol / kg (n=1-3 mice per dose and test component). Separate animals were used for each dose and each test component. The first mouse cohort for each test component was administered a dose of 11 nmol / kg.

[0137] Subsequent transitions to higher or lower doses depended on the presence or absence of an infusion response observed at the previous dose level, and a thorough evaluation of the severity and duration of observed symptoms was carried out in accordance with Swedish and EU animal welfare laws, ethical approvals, and guidelines. Observed symptoms were rated as mild, moderate, or severe for each individual mouse. Symptoms ranged from no clinical symptoms at all to a slumped, hunched appearance, isolation and lethargy, marked post-administration apathy, mild motor dysfunction, and increased heart rate and respiratory rate within approximately 15–25 minutes after administration. Mild to moderate symptoms resolved completely within a few hours, but in cases of severe or prolonged symptoms, the animals were immediately euthanized. Observations of first infusion response (FIR) reported as no response, mild, moderate, and severe are shown in Table 16. [Table 16]

[0138] hTfR-KI mice express both mouse and human TfR1 and can therefore cross-react with mouse-specific 8D3 and human-specific 15G11-1TfR1 conjugates, as well as the h26D3WT variant. In 2A2#2-8D3, mild to moderate FIR was observed at doses up to 60 nmol / kg. Consistent with in vitro data, this response was abolished by introducing the K322A mutation into the Fc domain of the cargo antibody, as performed with 2A2#2-8D3-K322A, disrupting the CDC response and thus eliminating complement activation-dependent FIR. When administered with the human apical conjugate 2A2#2-15G11, severe FIR was already observed at a dose of 11 nmol / kg. No FIR symptoms were observed after injecting 2A3#10-WT, 2A3#14-WT, or 2A3#3-WT up to 60 nmol / kg, but no symptoms or mild symptoms were observed after injecting 2A3#2-WT at 60 nmol / kg. This supports the hypothesis that the antibody portion is located below the hTfR1-binding scFv domain and therefore close to the plasma membrane. The results indicate that the orientation of the scFv binding epitope and binding module on hTfR1 together mitigates the observed FIR. In contrast, in the hTfR1-KI mouse model, FIR was observed regardless of whether the apical TfR1 conjugates 8D3 or 15G11-1 were used.

[0139] Pre-administration blood samples were collected from all mice, and blood samples were also collected 2 hours later from mice that recovered a mild to moderate infusion response. The two pre-mortem blood samples were collected from the saphenous vein into Sarstedt Microvette CB300 K2E tubes and subjected to plasma treatment and cytokine analysis. Exposure of the test components to the brain and plasma was examined at euthanasia at 24 hours.

[0140] Separately from euthanasia and sample analysis, animals were deeply anesthetized with isoflurane, and peripheral blood samples were collected from the orbital plexus into BD MicrotainerrK2EDTA tubes. The samples were inverted and centrifuged at 2400 × g at 4°C for 10 minutes. Plasma was extracted, transferred to Eppendorf tubes, and frozen at -80°C. Immediately after blood collection, the animal's abdomen was incised, and a cannula (21G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium, and transcardiac perfusion was performed using ice-cold PBS. After perfusion, the brain was removed, and the olfactory bulb was extracted. The brain was separated into left and right hemispheres, the cerebellum was removed from the left hemisphere, and then the left hemisphere was weighed, rapidly frozen on dry ice, and stored at -80°C until the concentration of the injected test construct was further prepared and analyzed using a Meso Scale Discovery (MSD) based assay.

[0141] For intracerebral concentration measurements, the frozen left hemisphere was thawed on ice and homogenized for 5 seconds at 6 m / s using automated bead homogenization with MP Biomedical's FastPrep-24 5G system with Lysing Matrix D in Tris-buffered saline (TBS) containing the cOmplete protease inhibitor and the phoSTOP phosphatase inhibitor (#11836145001 and #04906837001, Roche). Triton X-100 (#X100, Merck) was added to the homogenate to obtain a final Triton X-100 concentration of 0.5% and a weight-to-volume ratio of 1:10. The homogenate was vortex-mixed for 10 seconds, centrifuged at 16,000 × g at 4°C for 1 hour, and the supernatant was collected and used to measure antibody exposure in the brain.

[0142] The intracerebral and plasma concentrations of test constructs 2A3#2-WT, 2A3#3-WT, 2A3#10-WT, 2A3#14-WT, and 2A2#2-8D3 were measured using a custom MSD assay for detecting human Fc. 96-well MSD plates (#L15XA-3) were coated overnight at 4°C with 25 ng / well of goat anti-human IgG, Fcγ fragment-specific antibody (#109-005-098, Jackson Immuno Research Europe Ltd) diluted in 1×PBS (#09-9400-100, Medicago AB). After removing the coating, the wells were blocked with 1% Blocker A (#R93BA-4, MSD) in PBS-0.05% Tween 20 (PBS-T) (#09-9410-100, Medicago AB). After washing four times with 1×PBS-T, the sample and the test construct calibrator diluted with 1% Blocker A in PBS-T were added to the plate and incubated at room temperature (RT) at 900 rpm for 2 hours. Detection of bound antibodies was performed using a continuous incubation method: a secondary antibody (mouse anti-human IgG #3850-1-1000, MT145, Mabtech) incubated at 900 rpm at RT for 1 hour, followed by a SULFO-TAG-conjugated anti-mouse detection antibody (R32AC-1, MSD) incubated at 900 rpm at RT for 1 hour. Both the secondary and detection antibodies were diluted to 25 ng / well in 1% Blocker A in PBS-T, and washed four times with 1×PBS-T between each incubation step. After the final wash, 2× Read Buffer T (#R92TC, MSD) was added, and the plate was read using an MSD SECTOR imaging system. The concentration of the test construct in the sample was evaluated using the 4PL curve fitting algorithm and a curve weighting of 1 / Y2 on the corresponding test construct calibration curve, using MSD Discovery Workbench software. For construct 2A2#2-8D3, 2A3#2-WT was used as the calibration material.

[0143] Figure 30 shows the intracerebral and plasma concentrations of the described test construct 24 hours after administration. As shown in Figure 30, the increase in dose is reflected in the increase in plasma and cerebral exposure, indicating that the test substance was successfully administered and bound to TfR1 in vivo.

[0144] Plasma concentrations of 10 different cytokines (IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC / GRO, and TNF) were measured using a pre-built V-PLEX Plus Proflammatory Panel 1 mouse kit (K15048G, Meso Scale Discovery (MSD)) according to the manufacturer's instructions. Briefly, plates were incubated for 2 hours with calibrator, control sample, and 10-fold diluted plasma sample, followed by incubation for 2 hours with a mixture of all 10 SULFO-TAG detection antibodies. All incubations were performed at 900 rpm and room temperature. Before and after each incubation step, the plates were washed four times with PBS-0.05% Tween 20 (PBS-T) (#09-9410-100, Medicago AB). After the final wash, 2×Read Buffer T (#R92TC, MSD) was added, and the plate was then read using the MSD SECTOR imaging system. The concentration of the analyte in the sample was evaluated using the MSD Discovery Workbench software, employing the 4PL curve fitting algorithm and a 1 / Y² curve weighting against the corresponding calibration material standard curve.

[0145] The results for a subset of relevant test cytokines are shown in Figure 31. The tested constructs induced different cytokine responses, with constructs 2A3#3-WT and 2A3#2-WT ("VH first") showing a stronger response than constructs 2A3#10-WT and 2A3#14-WT ("VL first"). This result indicates that positioning the antibody moiety below the h26D3 conjugate moiety and closer to the cell membrane induces low levels of cytokines and chemokines. Construct 2A2#2-8D3, which bound to TfR1 at the apical domain and showed FIR based on the observational data above (Table 16), generated high levels of cytokines / chemokines, particularly KC / GRO and IL-10.

[0146] Example 18 Creation and refinement of designed Gen2D constructs Two bispecific constructs designed as knob-into-hole antibody variants were expressed, each containing an h26D3 scFv ligated to the C-terminal amino acid residue of the antibody knob heavy chain. In the first variant, "mAb158-Gen2D-h26D3 VH-first" (SEQ ID NO: 105), the N-terminal amino acid residue of the VH region of the scFv was ligated to the Fc. In the second variant, "mAb158-Gen2D-h26D3 VL-first" (SEQ ID NO: 106), the N-terminal amino acid residue of the VL region of the scFv was ligated to the Fc. The hole heavy chain used in both constructs is shown in SEQ ID NO: 107, while the light chain, which is present in two copies in each antibody portion, is shown in SEQ ID NO: 108.

[0147] The designed "Gen2D" construct was expressed by transient transfection into Chinese hamster ovary cells (ExpiCHO, Thermo Fisher Scientific) according to the manufacturer's instructions. The filtered cell culture supernatant was added to a MabSelect SuRe column (Cytiva) and subsequently washed with DPBS (pH 7.4). The expressed binding protein was eluted with 0.7% HAc (pH 2.5), and the sample was then neutralized to pH 7.5. The purified sample was further purified by size exclusion chromatography (SEC, HiLoad 26 / 600 Superdex 200, Cytiva) in DPBS (pH 7.4), or by anion exchange and elution with NaCl using, for example, a HiTrap Q HP column (Cytiva) with 20 mM Trizma as the binding buffer. Each purified expression construct was characterized by SDS-PAGE, size exclusion chromatography (Superdex 200 increase 3.2 / 300; Cytiva), and UV protein concentration measurement. Examples of the purity of different constructs obtained from representative SDS-PAGE analysis are shown in Figure 32. The non-reduced gel showed one band at approximately 175 kDa, while the reduced gel showed three bands as expected from the Gen2D morphology: a "knob" heavy chain fused to the h26D3 scFv appearing at approximately 75 kDa, the same light chain at approximately 25 kDa, and a "whole" heavy chain without fused scFv at approximately 50 kDa.

[0148] Example 19 Binding analysis using SPR of Gen2D2 bispecific binding protein to hTfR1 The binding of purified bispecific binding proteins to hTfR1 and cTfR1 was evaluated as described for the Gen2A construct in Example 12. The data in Figure 33 show that all designed and fabricated Gen2D constructs bound to hTfR1. All constructs showed similar binding and dissociation rates to hTfR1 (SEQ ID NO: 86) and cTfR1 (SEQ ID NO: 87) compared to the Fab fragment of the control hTfR1 binding agent h26D3. This result indicates that all fabricated constructs are functional and that the "VH-first" / "VL-first" configuration does not affect the binding of the constructs to hTfR1.

[0149] Example 20 ADCC measurement To investigate the effector function of the Gen2D construct, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used. Jurkat effector cells (Promega, #G7018) were used to evaluate ADCC activity. These cells stably express the FcγRIIIa receptor, V158 (high affinity) variant, and an NFAT response element that induces the expression of firefly luciferase as an indicator of ADCC activity. Binding of the Fc moiety to FcγR induces an activation signal in the effector cells, leading to the death of target cells whose surfaces are coated with the antibody. Ramos cells (Sigma, catalog: 85030802) expressing high levels of hTfR1 on their cell surface were used as target cells. Effector cells and target cells were used in a ratio of effector cells:target cells = 6:1, with or without serially diluted test constructs. The controls used were an antibody alone without hTfR1-binding scFv as a negative control, and rituximab as a positive control. Target cells containing the test construct were seeded in a 96-well assay plate (Corning, #3917), mixed with effector cells, and incubated at 37°C and 5% CO2 for 6 hours. Luciferase activity is generated when Fc-containing proteins form a bridge between the target and effector cells (via the interaction of Fc and FcγR). After 6 hours of incubation, Bio-Glo luciferase reagent was added, and the luciferase signal was quantified using a SPARK plate reader (Tecan).

[0150] First, ADCC activity and induction ratio were confirmed using the antibody rituximab as a positive control (Figure 34A). Rituximab is known to be a potent inducer of ADCC, which was validated in the assay configuration. When target cells were eliminated, ADCC was not induced. Importantly, the antibody used on the construct (mAb158) tested with Gen2D did not show ADCC activity when tested alone, which is evident from the fact that mAb158 does not have ADCC activity when target cells are eliminated (Figure 34A). Next, the Gen2D construct "mAb158-Gen2D-h26D3VL first," which was expressed and analyzed as described in Examples 18-19, was examined. Importantly, despite this construct strongly binding to target cells via an hTfR1 binder, no ADCC activity was detected (Figure 34B).

[0151] Next, K562 cells (Sigma / ECACC) were used in the cell binding experiment. The Fcγ receptor was blocked for 30 minutes at 4°C using an Fc receptor blocker (Innovex biosciences, #NB309-4X-40), and then the cells were washed with PBS. The cells were seeded into a 96-well V-bottom plate (#249570, Thermo Scientific Nunc), and serial dilutions of the tested Gen2D construct "mAb158-Gen2D-h26D3VL fast" were added. The plate was incubated overnight at 4°C. The cells were washed with PBS containing 1% BSA and fixed at room temperature for 10 minutes with freshly prepared 4% formaldehyde (Thermo Scientific® Pierce®, #28906) diluted with PBS. Cells were washed with PBS containing 1% BSA and then stained with fluorescently labeled secondary goat F(ab')2 anti-human IgG(γ)-Alexa fluor 488 (Invitrogen Life Technologies, #H10120). Staining was performed at 4°C for 30 minutes. After incubation with the detection reagent, the cells were washed with PBS containing 1% BSA. Finally, the cells were resuspended in 200 μl of PBS containing 1% BSA and collected using the BD FACSLyric flow cytometry system (BD Biosciences). The samples were analyzed using flowJo software (BD Biosciences). The median fluorescence intensity (MFI) measured was plotted against the binding protein concentration and is shown in Figure 35. This result indicates that the tested Gen2D construct binds to hTfR1 on the cell surface.

[0152] In summary, ADCC and cell binding experiments show that the Gen2D construct with h26D3scFv in a "VL-first" configuration fails to induce ADCC because the Fc portion of the binding protein cannot bind to the Fcγ receptor, even though it is bound to the cell surface via hTfR1.

[0153] Example 21 In vivo exposure of Gen2D constructs to plasma and brain To further evaluate the h26D3 HC6-binding module as the scFv portion in Gen2D form, along with an antibody that binds to amyloid-beta, exposure of such test constructs to brain and plasma was investigated over time in hTfR1 knock-in (hTfR1-KI) mice (Example 6). In the crossed 5×FAD×hTfR-KI mice, invincible target binding to brain amyloid-beta lesions was examined 72 hours after administration. 5×FAD×hTfR-KI mice were produced by crossing a 5×FAD male mouse from the C57BL / 6J lineage (Northwestern University) with an hTfR-KI female mouse. The 5×FAD mouse model is an Alzheimer's disease (AD) model using mice that express human APP and PSEN1 transgenes, each containing a total of five AD-related mutations [including the Swedish (K670N / M671L), Florida (I716V), and London (V717I) mutations in APP, and the M146L and L286V mutations in PSEN1].

[0154] In the construct tested in this example, the amyloid-beta binding antibody mAb158 used in the previous example was replaced with another amyloid-beta binding antibody, designated herein as mAb000. This construct was prepared in the same form as "mAb158-Gen2D-h26D3 VH first" in Example 18 and was designated mAb000-Gen2D-h26D3-HC6. To investigate the exposure of the test construct and control antibody to the brain and plasma, mAb000-Gen2D-h26D3-HC6 and mAb000 were prepared and intravenously (iv) injected into hTfR1-KI mice (n=11 per test construct) at equimolar doses of 40 nmol / kg (approximately 6-7 mg / kg). Plasma and brain exposure during euthanasia was evaluated at three consecutive euthanasia time points: 24 hours, 72 hours, and 336 hours, with n=3 to 5 mice used for each time point and test construct. Blood samples for evaluating continuous plasma concentrations over time were collected from animals euthanized at 336 hours after administration of the test construct (n=5) and at the following time points: 0.25, 4, 24, 48, 72, 168, 240, and 336 hours. Live blood samples were collected from the saphenous vein into Sarstedt Microvette CB300 K2E tubes.

[0155] To investigate the binding of cerebral amyloid beta to its target, mAb000 and mAb000-Gen2D-h26D3-HC6 were administered intravenously (iv) at equimolar doses of 40 nmol / kg (approximately 6-7 mg / kg) to 5×FAD×hTfR-KI mice (n=2-3 per test substance). The animals were euthanized 72 hours after administration.

[0156] At each euthanasia point, separate from subsequent sample analysis, animals were deeply anesthetized with isoflurane, and peripheral blood samples were collected from the orbital plexus into BD MicrotainerrK2EDTA tubes. The samples were inverted and centrifuged at 2400 × g at 4°C for 10 minutes. Plasma was extracted, transferred to Eppendorf tubes, and frozen at -80°C. Immediately after blood collection, the animal's abdomen was incised, and a cannula (21G) was inserted into the left ventricle of the heart. A small incision was made in the right atrium, and transcardiac perfusion was performed using ice-cold PBS. After perfusion, the brain was removed, and the olfactory bulb was extracted. The brain was separated into left and right hemispheres, the cerebellum was removed from the left hemisphere, and then the left hemisphere was weighed, rapidly frozen on dry ice, and stored at -80°C. Further preparation and concentration analysis of the injected constructs were performed using a Meso Scale Discovery (MSD) based assay. The right hemisphere was immersed in 4% formaldehyde and stored at 4°C for 24 hours. After washing with cold PBS, it was transferred to a cold 30% sucrose solution prepared in PBS and stored at 4°C for further immunohistochemistry (IHC) treatment (see Example 22 below).

[0157] For intracerebral concentration measurements, the frozen left hemisphere was thawed on ice and homogenized for 5 seconds at 6 m / s using an automated bead homogenization method with MP Biomedical's FastPrep-24 5G system with Lysing Matrix D in Tris-buffered saline (TBS) containing the cOmplete protease inhibitor and the phoSTOP phosphatase inhibitor (#11836145001 and #04906837001, Roche). Triton X-100 (#X100, Merck) was added to the homogenate to obtain a final Triton X-100 concentration of 0.5% and a weight-to-volume ratio of 1:10. The homogenate was vortex-mixed for 10 seconds, centrifuged at 16,000 × g at 4°C for 1 hour, and the supernatant was collected and used to measure intracerebral antibody exposure.

[0158] The intracerebral and plasma concentrations of mAb000 and mAb000-Gen2D-h26D3-HC6 were measured using a custom MSD assay for detecting human Fc. 96-well MSD plates (#L15XA-3) were coated overnight at 4°C with 25 ng / well of goat anti-human IgG, Fcγ fragment-specific antibody (#109-005-098, Jackson Immuno Research Europe Ltd) diluted in 1×PBS (#09-9400-100, Medicago AB). The coating was removed, and the wells were blocked with 1% Blocker A (#R93BA-4, MSD) in PBS-0.05% Tween 20 (PBS-T) (#09-9410-100, Medicago AB). After washing four times with 1×PBS-T, the sample and the test construct calibrator diluted with 1% Blocker A in PBS-T were added to the plate and incubated at room temperature (RT) at 900 rpm for 2 hours. Detection of bound antibodies was performed using a continuous incubation method: a secondary antibody (mouse anti-human IgG, #3850-1-1000, MT145, Mabtech) was incubated at 900 rpm, RT for 1 hour, followed by a SULFO-TAG-conjugated anti-mouse detection antibody (R32AC-1, MSD) incubated at 900 rpm, RT for 1 hour. Both the secondary and detection antibodies were diluted to 25 ng / well with 1% Blocker A in PBS-T, and washed four times with 1×PBS-T between each incubation step. After the final wash, 2× Read Buffer T (#R92TC, MSD) was added, and the plate was read using an MSD SECTOR imaging system. The concentration of the test construct in the sample was evaluated using the 4PL curve fitting algorithm and a curve weighting of 1 / Y2 for the curve of the corresponding test construct calibration material, using MSD Discovery Workbench software.

[0159] The results are shown in Figure 36. As shown in Figure 36A, during the study period, mAb000-Gen2D-h26D3-HC6 showed higher peak brain concentrations and higher brain exposure (indicated by the area under the curve) compared with mAb000. As shown in Figure 36B, plasma exposure of mAb000-Gen2D-h26D3-HC6 was lower than that of mAb000, indicating hTfR1 binding and clearance of the test construct from plasma to hTfR1-expressing tissue. In summary, these data support the conclusion that the test construct mAb000-Gen2D-h26D3-HC6 undergoes blood-brain barrier transport via hTfR1.

[0160] Example 22 In vivo immunohistochemistry of Gen2D constructs The in vivo binding of both hTfR1 and amyloid beta by the test construct mAb000-Gen2D-h26D3-HC6 and the control antibody mAb000 (see Example 21) was further investigated using qualitative immunohistochemistry (IHC) analysis. Briefly, the right hemisphere of animals euthanized in Example 21 was embedded in sucrose in OCT compound (LAMB / OCT, Thermo Fisher Scientific, USA) and rapidly frozen in dry ice. The embedded right hemisphere was sectioned, and 20 μm sagittal slides were collected on Superfrost cryoslides (J1800AMNZ, Thermo Fisher Scientific), air-dried, and then subjected to IHC. Brain sections were pre-treated with 4% PFA (HL96753.1000, HistoLab, Sweden) for 20 minutes, washed with purified water for 5 minutes, and incubated with 70% FA for 5 minutes for antigen recovery. After washing with 1×PBS for 10 minutes twice, the slides were blocked with MOM mouse IgG blocking reagent (MKB-2213-1, Vector Laboratories) at room temperature for 1 hour. The primary antibody was diluted with 0.1% Triton X-100 in 1×PBS and incubated overnight at 4°C, and the secondary antibody was diluted with 1×PBS and incubated at room temperature for 1.5 hours.

[0161] Amyloid beta was visualized using mouse antibodies 6E10 (1 μg / ml) (803002, Biolegend) and 4G8 (1 μg / ml) (800702, Biolegend), as well as Alexa555 anti-mouse IgGH+L (1:500) (A21424, Invitrogen). The test compounds were visualized using Alexa647 anti-human IgG H+L (1:500) (A21206, Invitrogen). All incubations were performed in a PBS humidified chamber. After incubation, slides were washed in a cuvette with 1×PBS (usually 5 times for 5 minutes each). Sections were mounted on Fluoromount-G (00-4958-02, Invitrogen), and images were acquired using a Leica Stellaris 5 confocal system with an HC PL APO 40x / 1.25 GLYC motCORR CS2 objective lens (Leica, #11506423).

[0162] The obtained images are shown in Figure 37. Co-localization of hIgG antibodies and amyloid-beta antibodies was observed, and mAb000-Gen2D-h26D3-HC6 was shown to be involved in broad amyloid-beta targeting in 7-month-old 5×FAD / hTfR-KI brains 72 hours after administration (Figure 37A). Major core plaques were mainly positive for mAb000-Gen2D-h26D3-HC6, but low co-localization of amyloid-beta plaques was observed with mAb000 antibodies lacking the hTfR1 binding module (Figure 37B).

[0163] Example 23 Competition between the Gen2D construct and the antibody M-A712 for hTfR1 binding. As a complementary experiment to the test described in Example 3, the competition of test constructs for hTfR1 binding was investigated using a different method. The test constructs examined were "mAb158-Gen2D-h26D3VH-first" and "mAb158-Gen2D-h26D3VL-first" from Example 18, and "mAb000-Gen2D-h26D3-HC6" from Example 21, all of which have "VH-first" and "VL-first" configurations, and are here referred to as "mAb000-Gen2D-h26D3-HC6V H-first" and "mAb000-Gen2D-h26D3-HC6V L-first," respectively.

[0164] The method of this example uses the anti-CD71 (anti-hTfR1) antibody M-A712 as a marker for a specific epitope on the apical domain of hTfR1. The M-A712 antibody has been reported to bind to residues 208-212 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 described binding site for human ferritin on human TfR1 (Montemiglio et al (2019), Nat Commun. 10(1):1121). The binding of the binders of this disclosure to hTfR1, i.e., the binding of the binders identified as described in Example 1 to the protease-like domain of hTfR1, was evaluated in competition with the M-A712 antibody. Furthermore, M-A712 was also examined in competition with recombinant human ferritin heavy chain 1 (FTH1).

[0165] K562 lymphoblastic cells (Sigma) were used for the competition experiment. To evaluate the competition between M-A712 and the disclosed binder and to confirm the binding of the labeled M-A712 antibody, cells were first incubated with human Fc receptor block (BD Pharmingen, 564220) at 4°C for 30 minutes to block antibody binding via nonspecific Fc receptors. Next, cells were incubated with serially diluted test constructs and PE-conjugated M-A712 antibody (monoclonal, BD Pharmingen, 555537) and incubated at 4°C for 1 hour. After incubation, cells were washed three times with staining buffer (1% BSA and 0.1% sodium azide in 1× DPBS). M-A712 antibody bound to hTfR1 on the cell surface was analyzed by flow cytometry, and the mean fluorescence intensity (MFI(PE)) was plotted. Figure 38 shows no direct competition between the h26D3 conjugates and M-A712 in the different constructs described. When unlabeled (unbound) M-A712 antibody was used as a positive control for competition, binding of labeled (PE) M-A712 signal decreased in a concentration-dependent manner. This experiment demonstrates that conjugates targeting the protease-like domain of TfR1 do not directly compete for the same epitope as M-A712 antibody.

[0166] Similar experiments were performed using human ferritin heavy chain 1 (FTH1; Sino Biologicals, #13217-HNAE, lot number LC15NO0415) labeled with Alexa647. As shown in Figure 39, competition was observed at high concentrations compared to the competitive positive control (M-A712). This data clearly demonstrates that the hTfR1 conjugate of this disclosure, which binds to the protease-like domain of hTfR1, does not compete with the described epitope on the apical domain corresponding to the binding site used by the ferritin protein.

[0167] List of embodiments by category 1. A TfR1-binding molecule capable of selectively binding to the epitope located in the protease-like domain of transferrin receptor 1 (TfR1), as defined by amino acid residues 121-183 and 384-605 of SEQ ID NO: 85.

[0168] 2. The binding molecule according to item 1, wherein the epitope comprises or consists of amino acid residues 150, 151, 154, 158, 159, 161, 163, and 385 of SEQ ID NO: 85.

[0169] 3. A binding molecule according to any one of items 1 to 2, comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the VH region and the VL region form a VH / VL pair including an antigen-binding surface, and wherein the antigen-binding surface confers on the binding molecule the ability to selectively bind to the epitope.

[0170] 4. The binding molecule according to item 3, wherein the antigen-binding surface is composed of three complementarity-determining regions (CDRs) derived from the VH region and three CDRs derived from the 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: X4INPX5X6X7TTSX8X9X10KFKG (SEQ ID NO: 2), where X4 is selected from D and A, X5 is selected from D, N, and A, X6 is selected from Y and A, X7 is selected from D and A, X8 is selected from Y and A, X9 is selected from N and S, and X10 is selected from E and Q, VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID NO: 4) where X11 is selected from Y and A, X12 is selected from T and S, X13 is selected from Q and R, and X14 is selected from Y and A. VLCDR2:X15ASTRES(Sequence ID 5) Here X15 can be selected from W and A, VLCDR3:QQX16X17X18X19PX20T (Sequence ID 6) Here, X16 is selected from Y and A. X17 is selected from F and Y. X18 is selected from I and N. X19 is selected from Y and A, and X20 is selected from R and Y.

[0171] 5. The binding molecule relating to item 4, wherein the CDR further includes the following: VHCDR3:GGX21SGSSX22X23HPMX24X25 (Sequence ID 3) Here, X21 is selected from Y and A. X22 is selected from Y and A. X23 is selected from Y and A. X24 is selected from D and A, and X25 is selected from Y and A.

[0172] 6. The binding molecule relating to any one of items 4 to 5, wherein the VHCDR2 is as follows: VHCDR2:X4INPX5X6X7TTSX8NEKFKG (Sequence ID 7) Here, 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.

[0173] 7. The binding molecule relating to any one of items 4 to 6, wherein the VLCDR1 is as follows: VLCDR1: KSSQSLLX11STNQKNX14LA (SEQ ID NO: 8) Here X11 is selected from Y and A, and X14 is selected from Y and A.

[0174] 8. The binding molecule according to any one of items 4 to 7, wherein the VLCDR3 is as follows:: VLCDR3: QQX16FIX19PRT (SEQ ID NO: 9) Here X16 is selected from Y and A, and X19 is selected from Y and A.

[0175] 9. The binding molecule according to any one of items 4 to 8, wherein the amino acid sequence of the VHCDR1 is selected from the group consisting of SEQ ID NOs: 10 and 16 to 18.

[0176] 10. The binding molecule according to any one of items 4 to 9, wherein the amino acid sequence of the VHCDR2 is selected from the group consisting of SEQ ID NOs: 11, 19 to 23, and 34, for example, selected from the group consisting of SEQ ID NOs: 11 and 19 to 23.

[0177] 11. The binding molecule according to any one of items 4 to 10, wherein the amino acid sequence of the VHCDR3 is selected from the group consisting of SEQ ID NOs: 12, 24 to 28, and 35, for example, selected from the group consisting of SEQ ID NOs: 12 and 24 to 28.

[0178] 12. The binding molecule according to any one of items 4 to 11, wherein the amino acid sequence of the VLCDR1 is selected from the group consisting of SEQ ID NOs: 13, 29, 30, and 36, for example, selected from the group consisting of SEQ ID NOs: 13, 29, and 30.

[0179] 13. The binding molecule according to any one of items 4 to 12, wherein the amino acid sequence of the VLCDR2 is selected from the group consisting of SEQ ID NOs: 14 and 31.

[0180] 14. A binding molecule relating to any one of items 4 to 13, wherein the amino acid sequence of VLCDR3 is selected from the group consisting of SEQ ID NOs: 15, 32, 33, and 37, for example, selected from the group consisting of SEQ ID NOs: 15, 32, and 33.

[0181] 15. The amino acid sequences of the six CDRs mentioned above are as follows: Binding molecule relating to any one of items 4-14: VHCDR1:DYNMD (Sequence ID 10), VHCDR2:DINPDYDTTSYNEKFKG (Sequence ID 11), VHCDR3:GGYSGSSYYHPMDY (Sequence ID 12), VLCDR1:KSSQSLLYSTNQKNYLA (Sequence ID 13), VLCDR2:WASTRES (SEQ ID NO: 14), VLCDR3:QQYFIYPRT(Sequence ID 15)

[0182] 16. The binding molecule relating to any one of items 4 to 14, wherein the amino acid sequences of the six CDRs are as follows: VHCDR1:DYNMD (Sequence ID 10), VHCDR2:DINPDADTTSYNEKFKG (Sequence ID 21), VHCDR3:GGYSGSSYYHPMDY (Sequence ID 12), VLCDR1:KSSQSLLYSTNQKNYLA (Sequence ID 13), VLCDR2:WASTRES (SEQ ID NO: 14), VLCDR3:QQYFIYPRT(Sequence ID 15)

[0183] 17. The amino acid sequences of the six CDRs mentioned above are as follows: Binding molecule related to item 4: VHCDR1:DYNMD (Sequence ID 10), VHCDR2:DINPNYDTTSYSQKFKG (Sequence ID 34), VHCDR3:SEAGNYYWYFDV (Sequence ID 35), VLCDR1:KSSQSLLYSSNRKNYLA (Sequence ID 36), VLCDR2:WASTRES (SEQ ID NO: 14), VLCDR3:QQYYNYPYT(Sequence ID 37)

[0184] 18. A binding molecule relating to any one of items 3 to 17, wherein the VH region contains or consists of an amino acid sequence selected from the following: (i) The group consisting of sequence numbers 44-57, 65, and 67, for example the group consisting of sequence numbers 44-57, for example the group consisting of sequence numbers 44 and 50, and (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 the sequence defined in (i) (provided that the sequence of the CDR region is 100% identical with the sequence defined in (i)).

[0185] 19. A binding molecule relating to any one of items 3 to 18, wherein the VL region contains or consists of an amino acid sequence selected from the following: (i) A group consisting of sequence numbers 58-64, 66, and 68, for example, a group consisting of sequence numbers 58-64, and (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 the sequence defined in (i) (provided that the sequence of the CDR region is 100% identical with the sequence defined in (i)).

[0186] 20. A binding molecule relating to any one of items 18 to 19, wherein the VH region is as defined in item 18 and the VL region is as defined in item 19.

[0187] 21. A binding molecule relating to item 20, wherein the VH region includes sequence number 44, and the VL region includes a sequence selected from sequence numbers 58 to 64.

[0188] 22. A binding molecule relating to item 20, wherein the VH region includes a sequence selected from sequence numbers 44 to 57, and the VL region includes sequence number 58.

[0189] 23. A binding molecule relating to any one of items 21 to 22, wherein the VH region includes SEQ ID NO: 44 and the VL region includes SEQ ID NO: 58.

[0190] 24. A binding molecule relating to any one of items 21 to 22, wherein the VH region includes SEQ ID NO: 50 and the VL region includes SEQ ID NO: 58.

[0191] 25. A binding molecule relating to item 3, where, The antigen-binding surface is composed of three complementarity-determining regions (CDRs) derived from the VH region and three CDRs derived from the VL region, and here, The CDR contains the following sequence of binding molecules: VHCDR1:NYWLG (Sequence ID 38), VHCDR2:DIFPGSDNTYYNEKFKG (Sequence ID 39), VHCDR3:SGNFYAMDY (Sequence ID 40), VLCDR1:SASSSVNYMN (Sequence ID 41), VLCDR2:DTSKLAS (SEQ ID NO: 42), VLCDR3:FQGSGYPFT (Sequence ID 43).

[0192] 26. A binding molecule relating to item 25, wherein the VH region comprises or consists of an amino acid sequence selected from SEQ ID NO: 69 and 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 SEQ ID NO: 69, wherein the sequence of the CDR region is 100% identical to that of SEQ ID NO: 69.

[0193] 27. A binding molecule relating to any one of items 25 to 26, wherein the VL region comprises or consists of an amino acid sequence selected from SEQ ID NO: 70 and 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 SEQ ID NO: 70, provided that the sequence of the CDR region is 100% identical to that of SEQ ID NO: 70.

[0194] 28. A binding molecule relating to any one of items 26 to 27, wherein the VH region is as defined in item 26 and the VL region is as defined in item 27.

[0195] 29. A binding molecule relating to any one of items 3 to 28, wherein the VH / VL pair forms part of the antibody construct.

[0196] 30. A binding molecule relating to item 29, wherein the antibody construct has two or more binding specificities.

[0197] 31. A binding molecule relating to item 30, wherein the antibody construct is bispecific.

[0198] 32. A binding molecule relating to item 31, wherein a VH / VL pair, as defined in any one of items 3 to 28, is present in an antibody fragment selected from the group consisting of a Fab fragment, a single-stranded Fab (scFab) fragment, an Fv fragment, and a single-stranded (scFv) fragment.

[0199] 33. The binding molecule relating to item 32, wherein the antibody fragment is scFv.

[0200] 34. A binding molecule relating to any one of items 30 to 33, further comprising an antibody or an antigen-binding fragment thereof that can selectively bind to a target present in the brain of a mammal.

[0201] 35. A binding molecule relating to item 34, wherein the target is selected from the group consisting of amyloid-beta peptide or its derivatives or fragments, alpha-synuclein or its derivatives or fragments, TAR DNA-binding protein 43 (TDP-43) or its derivatives or fragments, induction receptor 2 (TREM2) expressed in bone marrow cells, beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, tau, phosphorylated tau or its fragments, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma-secretase, death receptor 6, amyloid-beta precursor protein, p75 neurotrophic factor receptor, neuregulin, and caspase 6.

[0202] 36. A binding molecule relating to item 35, wherein the target is selected from the group consisting of amyloid-beta peptide or its derivatives or fragments, alpha-synuclein or its derivatives or fragments, TAR DNA-binding protein 43 (TDP-43) or its derivatives or fragments, induction receptor 2 (TREM2) expressed in bone marrow cells, tau, phosphorylated tau or its fragments, and apolipoprotein E4.

[0203] 37. A binding molecule relating to item 36, wherein the target is selected from the group consisting of amyloid-beta peptide or its derivatives or fragments, alpha-synuclein or its derivatives or fragments, and TAR DNA-binding protein 43 (TDP-43) or its derivatives or fragments.

[0204] 38. A binding molecule relating to any one of items 34 to 37, wherein the antibody or antigen-binding fragment thereof, which is selectively capable of binding to a target present in the brain of a mammal, is an antibody selected from the group consisting of anti-Aβ antibodies, such as lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06, and KHK6640.

[0205] 39. A binding molecule relating to any one of items 34 to 37, wherein the antibody or antigen-binding fragment thereof, which is selectively capable of binding to a target present in the brain of a mammal, is an antibody selected from the group consisting of anti-alpha-synuclein antibodies, such as pracinezumab, UCB7853, LuAF82422, TAK-341, and BAN0805.

[0206] 40. A pharmaceutical composition comprising a binding molecule relating to any of the above items and a pharmaceutically acceptable carrier or excipient.

[0207] 41. A binding molecule relating to any one of items 1 to 39, or a composition relating to item 40, for use in treatments such as therapeutic or prophylactic procedures.

[0208] 42. A binding molecule relating to any one of items 1 to 39, or a composition relating to item 40, for use in in vivo diagnosis or in vivo prognosis determination.

[0209] 43. A conjugate molecule or composition for use relating to any one of items 41 to 42, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is relating to a neurodegenerative disease, such as Alzheimer's disease and other diseases 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-associated dementia (PDD), Lewy body Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy.

[0210] 44. A conjugating molecule or composition for use relating to item 43, wherein the treatment, prevention, in vivo diagnosis, or in vivo prognosis relates to a disease selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, Parkinson's disease (PD), Parkinsonian dementia (PDD), and Lewy body Alzheimer's disease.

[0211] 45. A conjugating molecule or composition for use relating to item 44, wherein the treatment, prevention, in vivo diagnosis, or in vivo prognosis relates to a disease selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD).

[0212] 46. ​​A conjugating molecule or composition for use in accordance with item 45, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is related to Alzheimer's disease.

[0213] 47. A conjugated molecule or composition for use in accordance with any one of items 41 to 42, wherein the treatment, prevention, in vivo diagnosis, or in vivo prognosis is related to a disease selected from brain cancer, multiple sclerosis, and lysosomal storage disorders.

[0214] 48. A method for therapeutic or prophylactic treatment of a mammal having a disability or being at risk of developing a disability, comprising administering to the mammal a therapeutically effective amount of a binding molecule relating to any one of items 1 to 39 or a composition relating to item 40.

[0215] 49. A method relating to item 48, wherein the disorder is a neurodegenerative disease, for example, a neurodegenerative disease as defined in any one of items 43 to 46.

[0216] 50. A method relating to item 48, wherein the obstruction is as defined in item 47.

Claims

1. A TfR1-binding molecule capable of selectively binding to an epitope located in the protease-like domain of transferrin receptor 1 (TfR1) as defined by amino acid residues 121-183 and 384-605 of SEQ ID NO: 85, wherein the epitope optionally contains or consists of amino acid residues 150, 151, 154, 158, 159, 161, 163, and 385 of SEQ ID NO:

85.

2. A binding molecule according to claim 1, comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the VH region and the VL region form a VH / VL pair including an antigen-binding surface, and the antigen-binding surface provides the binding molecule with the ability to selectively bind to the epitope.

3. A binding molecule according to claim 2, wherein the antigen-binding surface is composed of three complementarity-determining regions (CDRs) derived from the VH region and three CDRs derived from the VL region, and wherein the CDRs include the following: VHCDR1:X1X2NMX3 (Sequence ID 1), Here, X1 is selected from D and A, X2 is selected from Y and A, and X3 is selected from D and A. VHCDR2:X4INPX5X6X7TTSX8X9X10KFKG (Sequence No. 2), Here X4 is selected from D and A. X5 is selected from D, N, and A. X6 is selected from Y and A, X7 is selected from D and A. X8 is selected from Y and A. X9 is selected from N and S, and X10 is selected from E and Q. VLCDR1:KSSQSLLX11SX12NX13KNX14LA (Sequence ID 4) Here X11 is selected from Y and A, X12 is selected from T and S, X13 is selected from Q and R, and X14 is selected from Y and A. VLCDR2:X15ASTRES (Sequence ID 5) Here X15 is selected from W and A, and VLCDR3: QQX16X17X18X19PX20T (Sequence ID 6) Here, X16 is selected from Y and A, X17 is selected from F and Y. X18 is selected from I and N, X19 is selected from Y and A, and X20 is selected from R and Y. Optionally, further VHCDR3: GGX21SGSSX22X23HPMX24X25 (Sequence ID 3) Here, X21 is selected from Y and A, X22 is selected from Y and A, X23 is selected from Y and A, X24 is selected from D and A, and X25 is selected from Y and A.

4. The binding molecule according to claim 3, wherein the amino acid sequences of the six CDRs are as follows: VHCDR1:DYNMD (SEQ ID NO: 10) VHCDR2:DINPDDYDTTSYNEKFKG (Sequence ID 11) VHCDR3:GGYSGSSYYHPMDY (Sequence ID 12) VLCDR1:KSSQSLLYSTNQKNYLA (Sequence ID 13) VLCDR2:WASTRES (Sequence ID 14) VLCDR3:QQYFIYPRT (Sequence ID 15)

5. The binding molecule according to claim 3, wherein the amino acid sequences of the six CDRs are as follows: VHCDR1:DYNMD (SEQ ID NO: 10) VHCDR2:DINPDADTTSYNEKFKG (Sequence ID: SEQ ID NO: 21), VHCDR3:GGYSGSSYYHPMDY (Sequence ID 12), VLCDR1:KSSQSLLYSTNQKNYLA (Sequence ID 13), VLCDR2:WASTRES (Sequence ID 14), VLCDR3:QQYFIYPRT (Sequence ID 15)

6. A binding molecule according to any one of claims 2 to 5, wherein the VH region comprises or consists of an amino acid sequence selected from the following: (i) The group consisting of sequence numbers 44-57, 65, and 67, for example the group consisting of sequence numbers 44-57, for example the group consisting of sequence numbers 44 and 50, and (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 the sequence defined in (i) (provided that the sequence of the CDR region is 100% identical to the sequence defined in (i)).

7. A binding molecule according to any one of claims 2 to 6, wherein the VL region comprises or consists of an amino acid sequence selected from the following: (i) A group consisting of sequence numbers 58-64, 66, and 68, for example, a group consisting of sequence numbers 58-64, and (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 the sequence defined in (i) (provided that the sequence of the CDR region is 100% identical with the sequence defined in (i)).

8. A binding molecule according to any one of claims 6 to 7, wherein the VH region is as defined in claim 6, and the VL region is as defined in claim 7.

9. The binding molecule according to any one of claims 2 to 8, wherein the VH / VL pair forms part of an antibody construct, for example, a bispecific antibody construct.

10. A binding molecule according to claim 9, wherein the VH / VL pair, as defined in any one of claims 2 to 8, is present in an antibody fragment selected from the group consisting of a Fab fragment, a single-stranded Fab (scFab) fragment, an Fv fragment, and a single-stranded (scFv) fragment, for example, scFv.

11. A binding molecule according to any one of claims 9 to 10, further comprising an antibody or antigen-binding fragment thereof that can selectively bind to a target present in the brain of a mammal, wherein the target is selected from the group consisting of amyloid-beta peptide or its derivative or fragment, alpha-synuclein or its derivative or fragment, TAR DNA-binding protein 43 (TDP-43) or its derivative or fragment, bone marrow-expressing induction 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 its fragment, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma-secretase, death receptor 6, amyloid-beta precursor protein, p75 neurotrophic factor receptor, neuregulin, and caspase 6.

12. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier or excipient.

13. A binding molecule according to any one of claims 1 to 11 or a composition according to claim 12, for use in treatments such as therapeutic or preventive measures.

14. A conjugating molecule or composition for use according to claim 13, wherein the treatment or prevention relates to a neurodegenerative disease, such as Alzheimer's disease and other diseases 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-associated dementia (PDD), Lewy body Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy.

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