Bispecific binding molecule

Abstract

The present disclosure provides a bispecific binding molecule, which binds to protofibrils of amyloid beta and to the protease-like domain of human transferrin receptor 1 (hTfR1), as well as therapeutic and diagnostic uses thereof.

Description

[0001] BISPECIFIC BINDING MOLECULE

[0002] Field

[0003] The present disclosure relates to a bispecific binding molecule, which binds to amyloid beta (AP) protofibrils and to the protease-like domain of human transferrin receptor 1 (hTfRl), as well as therapeutic and diagnostic uses thereof.

[0004] Alzheimer's disease (AD) is a progressive neurodegenerative dementia disorder which exists in a more common late-onset form and an early-onset familial form. AD is characterized by progressive loss of memory and cognitive function. It is estimated that about 33 million people worldwide are presently suffering from AD, and the number of people suffering from AD is expected to increase due to the aging population. The prevalence of AD doubles approximately every 5 years from the age of 60, from 10% of individuals at the age of 65 to 50% of individuals at the age of 85 or more (Gustavsson et al (2023), Alzheimer's Dement 19:658-670).

[0005] Accumulation of AP peptide in the brain is thought to play an important role in the neuropathology of AD. A is generated from the amyloid precursor protein (APP) by sequential proteolysis and secreted via major regulated as well as minor constitutive secretory pathways. AP is a normal product of cell metabolism, which is present in the plasma and cerebrospinal fluid in healthy individuals. However, abnormal and excessive accumulation of AP in the brain leads to the formation of toxic AP aggregates that induce synaptic dysfunction and neuronal loss.

[0006] AP exists as various species, including monomers, soluble aggregates of varying size (e.g., oligomers and protofibrils), and insoluble fibrils in plaques. Studies of AP with the Arctic mutation showed that the peptide had an increased propensity to form soluble AP protofibrils and data indicated that these protofibrils were neurotoxic and contributed to the disease process. Furthermore, it has been shown that soluble AP aggregates are more neurotoxic than monomers and insoluble fibrils. It is therefore hypothesized that removal of these soluble AP aggregates represents an effective approach for the treatment of AD.

[0007] Antibodies against AP with the desirable high selectivity for the protofibril form have been described. One example is lecanemab, approved on the US market for treatment of AD and described in W02007 / 108756. Variants of lecanemab, in which single point mutations were introduced in order to influence properties such as the isoelectric point and the immunogenicity of the protein, are described in W02016 / 005466.

[0008] Treatment modalities for brain and neurological diseases are furthermore limited by the impermeability of the blood vessels of the brain to most substances carried in the bloodstream (Freskgard and Urich (2017), Neuropharmacology 120:38- 55; Stanimirovic et al (2018), BioDrugs 32:547-559). The small blood vessels (capillaries) of the brain, referred to collectively as the blood-brain barrier (BBB), are unique when compared to the blood vessels found in the periphery of the body. Tight apposition of BBB endothelial cells (EC) to neural cells, such as astrocytes, pericytes and neurons, induces phenotypic features that contribute to the observed impermeability. Tight junctions between ECs in the BBB limit paracellular transport, while the lack of passive pinocytotic vesicles and fenestrae limit non-specific transcellular transport. These factors combine to restrict molecular flux from the blood to the brain in general to molecules that are less than 500 Da in size and lipophilic. Thus, the otherwise promising prospect of using the large mass transfer surface area (over 20 m2from 600 km of capillaries in a human brain) of the blood stream as a delivery vehicle is made largely infeasible, except in those circumstances where a drug with the desired pharmacological properties fortuitously possesses size and lipophilicity attributes which allow it to pass through the BBB. Because of such restrictions, it has been estimated that more than 98 % of all small molecule pharmaceuticals and nearly 100 % of the emerging class of protein and gene therapeutics do not cross the BBB.

[0009] WO91 / 03259 proposes a principle for transporting a neuropharmaceutical agent across the BBB, which involves conjugating the agent to an antibody which is reactive with the transferrin receptor. According to this disclosure, binding of the conjugate to the transferrin receptor leads to active transport of the conjugate across the BBB. Later work has developed this concept further, for example as described in W02012 / 075037, W02014 / 033074, W02018 / 011353 and WO2022 / 258841, all describing different formats for achieving transport of a biopharmaceutical agent across the BBB by utilizing the transferrin receptor. There exist two forms of the human transferrin receptor (TfR), namely TfRl and TfR2. TfRl is one of the targets for the bispecific binding molecule of the present disclosure. TfRl is an iron transporter protein, which maintains cellular iron levels by recognizing and internalizing through specific binding of the iron carrier proteins transferrin (Tf) and ferritin (Ft) into cells through endocytosis mediated by clathrin- coated vesicles. TfRl is expressed in numerous cells and organs, but expression levels vary and, importantly, TfRl is expressed to a higher degree on BBB endothelial cells than on other endothelial cells, making the receptor a target for neuropharmaceutical delivery. Structurally, TfRl is a dimeric transmembrane glycoprotein comprising the amino acid sequence SEQ ID NO:98, which has a large ectodomain (residues 89-760), an intramembrane region (residues 62-88) and a cytoplasmic domain (residues 1-61). The ectodomain in turn has three distinct domains held separate from the cell surface by a 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).

[0010] In the context of BBB transport via the TfRl, antibodies and fragments thereof which have affinity for TfRl have been described. By way of example, a number of TfRl-binding antibodies are disclosed in WO2014 / 189973, in which antibodies are grouped according to epitope specificity in classes I - IV (see e.g. Figure 3 and the associated figure description on page 30 lines 11-15). Classes I - III of WO2014 / 189973 are denoted "apical binders" whereas the antibody of class IV is denoted a "non-apical binder". Other TfRl-binding antibodies are disclosed in EP3088518, EP3315606 and EP3560958, however without any information about the epitope specificity of these disclosed antibodies.

[0011] Thus, most work on using TfRl as a target for binding and BBB transport has focused on apical binders. This is thought to be because the apical domain is the structure within TfRl that seems to provoke a strong immune response and thus to trigger antibody generation in animals when used as an immunogen. Thus, most known antibody binders against TfRl have epitopes that are located within the apical domain. Another indication that the apical domain contains structures prone to engage with various ligands is that viruses have been described to utilize epitopes within the apical domain to enter cells (Cohen-Dvashi et al (2020), Nat Commun 11:67).

[0012] Furthermore, the detailed structure of the TfRl and ferritin complex was recently determined (Montemiglio et al (2019), Nat Commun 10:1121), showing that the interface between TfRl and ferritin is located within the apical domain. This suggests that TfRl apical binders could potentially interfere with the binding of ferritin to TfRl if used for BBB transport and in this way influence the normal function of ferritin in iron transport. Also, the binding and uptake of H-ferritin have been shown to be mediated by TfRl (Li et al (2010), Proc Natl Acad Sci USA 107(8):3505-10). Thus, there are reasons to conclude that binders directed against the apical domain of TfRl, and especially binding to the binding site used by ferritin, may negatively influence the important function of ferritin in transporting iron via the binding to TfRl.

[0013] It has been reported that TfRl apical binders can induce both acute clinical signs and decreased in circulating reticulocytes (Couch et al (2013), Sci Transl Med 5:183ra57). The TfRl has also been described in relation to anemia and iron deficiency (Braga et al (2014), Clin Chim Acta 431:143-147). Anemia due to autoantibodies to TfRl has also been described (Hyman et al (1984), N Engl J Med 311:214-218). Taken together, the data suggest that TfRl-binding and interfering with iron transporters such as transferrin and / or ferritin could lead to safety issues such as reduction in reticulocyte levels and anemia.

[0014] To date, the focus within the field has been to avoid interfering with one of the described TfRl ligands, namely transferrin. This has guided the field to utilize binding sites in the apical domain of TfRl, distant from the binding site of transferrin. However, such apical binders may still interfere with the other important TfRl ligand, ferritin, leading to interference in iron transport and function.

[0015] Despite the existence of candidate antibodies within the field, there remains a need in the art for novel therapeutic, prophylactic, diagnostic and prognostic tools for detecting and treating AD and other neurodegenerative diseases. There also remains a need in the field for antibodies and other binding molecules which have a binding affinity for TfRl, but which do not exhibit the drawbacks and risks associated with hitherto known binding molecules. Disclosure of the invention

[0016] One object of the disclosure is to provide binding molecules having one or more novel and useful binding specificity / specificities.

[0017] Another object of the disclosure is to provide novel candidate molecules for the treatment of neurodegenerative diseases via targeting of soluble aggregates of AP, for example AP protofibrils, with a beneficial and unique binding profile.

[0018] Another object of the disclosure is to enable the diagnosis of AD and other neurodegenerative disorders via detection of soluble aggregates of AP, for example AP protofibrils, implicated in disease formation and / or progression.

[0019] Another object of the disclosure is to provide molecules that bind to soluble aggregates of AP, for example A protofibrils, with high affinity.

[0020] Another object of the disclosure is to provide molecules that bind to soluble aggregates of AP, for example AP protofibrils, with high specificity.

[0021] Another object of the disclosure is to provide molecules that bind to soluble aggregates of AP, for example AP protofibrils, with high selectivity with respect to other AP peptide variants, for example AP monomers or AP fibrils.

[0022] Another object of the disclosure is to provide Ap-binding molecules that combine desirable properties for development into a biopharmaceutical product.

[0023] Another object of the disclosure is to provide Ap-binding molecules that exhibit little or no immunogenicity upon administration in human subjects.

[0024] Another object of the disclosure is to provide binding molecules that show a beneficial pharmacokinetic profile upon administration in human subjects, for example evidenced by one or more of a long half-life, a high total exposure, a low immunogenicity and a low clearance.

[0025] Another object of the disclosure is to provide a TfRl-binding molecule which utilizes a different binding site on TfRl than the naturally occurring ligands.

[0026] One such object is to provide a TfRl-binding molecule which utilizes a different binding site on TfRl than transferrin.

[0027] Another such object is to provide a TfRl-binding molecule which utilizes a different binding site on TfRl than ferritin.

[0028] Yet another such object is to provide a TfRl-binding molecule which utilizes a different binding site on TfRl than HFE (homeostatic iron regulator). A related object of the disclosure is to provide a TfRl-binding molecule which interacts with TfRl in a way which minimizes the interference with TfRl itself and / or its normal function.

[0029] A related object of the disclosure is to provide a TfRl-binding molecule which exhibits an improved stability, e.g. in the form of storage stability and / or resistance against multimerization, as compared to other TfRl-binding molecules.

[0030] Another object of the disclosure is to provide a TfRl-binding molecule suitable for use as a fusion partner in constructs arranged for transport through the BBB.

[0031] It is also an object of the disclosure to combine beneficial properties of different moieties into a bispecific binding molecule in which a therapeutic target in the brain is engaged more effectively through the provision of a moiety which enables transport through the blood-brain barrier.

[0032] One or more of these objects, and other objects that are evident to the skilled person from the teachings herein, are met by the various aspects of the disclosure.

[0033] Thus, in a first aspect, the present disclosure provides a bispecific binding molecule, comprising

[0034] - a first moiety Ml, which is an A|3 protofibril binding moiety comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), said VH and VL domains forming a VH / VL pair comprising an antigen-binding surface, said VH domain in Ml comprising the amino acid sequence SEQ ID NO:1:

[0035] EVQLVESGGG LVQPGGSLRL SCSASGFTFS SFGMHWVRQXaPGKGLEWVAY I SSGSSTIYY GDTVKGRFTI SRDNAKNSLF LQMSSLRAED TAVYYCAREG GYYYGRSYYT MDYWGQGTTV TVS , wherein

[0036] Xais selected from the group consisting of A, N and T; said VL domain in Ml comprising the amino acid sequence SEQ ID NO:5:

[0037] DWMTQSPLS LPXbTPGDPAS XcSCRSSQSIV HSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLXdI XeXfVXgAEDVGI YYCFQGSHVP PTFGPGTKLE IK, wherein

[0038] Xb is selected from the group consisting of V and A;

[0039] Xcis selected from the group consisting of I and V;

[0040] Xd is selected from the group consisting of R and T;

[0041] Xeis selected from the group consisting of S and Q;

[0042] Xf is selected from the group consisting of R and S; and

[0043] Xgis selected from the group consisting of E and D; and

[0044] - a second moiety M2, which is a human transferrin receptor 1 (hTfRl) binding moiety comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), said VH and VL domains forming a VH / VL pair comprising an antigen-binding surface, in which said antigenbinding surface provides the binding protein with the capacity to bind selectively to an epitope located in the protease-like domain of hTfRl defined by amino acid residues 121-183 and 384-605 in SEQ ID NO:98.

[0045] In a second aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific binding molecule in accordance with the first aspect of the invention and a pharmaceutically acceptable excipient or carrier.

[0046] In further aspects, the present disclosure provides bispecific binding molecules and / or pharmaceutical compositions comprising the same for use in methods of treatment or for use in methods of detection or diagnosis as described herein.

[0047] A6 protofibril binding moiety Ml

[0048] As described above, in a first aspect, the disclosure provides a bispecific binding molecule comprising a first moiety Ml, which has affinity for A|3 protofibrils, and in which the sequences of the VH and VL domains are as defined above with reference to SEQ ID NO:1 and SEQ ID NO:5.

[0049] AP protofibril binding moiety Ml is derived from the lecanemab variants disclosed in WQ2016 / 005466, hereby incorporated in its entirety into the present disclosure. As disclosed therein, it was identified that the half-life as well as in vivo exposure of lecanemab could be enhanced by introducing one or more mutations in certain positions, namely 17, 79 and / or 82, of the variable light chain of lecanemab. Also, it was found that low immunogenicity could be achieved by combining the mutations above, providing increased half-life, with mutations at one or more neighboring positions, namely 13, 21, 81 and 84, of the variable light chain and / or with mutations at position 40 of the variable heavy chain.

[0050] Without wishing to be bound by theory, it is contemplated that the binding molecules of the disclosure are useful in the diagnosis, prognosis and / or treatment of neurodegenerative diseases such as AD, through specific and selective binding to soluble AP aggregates, such as A protofibrils, by way of the AP protofibril-binding moiety Ml.

[0051] As defined herein, embodiments of the bispecific binding molecule of the first aspect of the disclosure are characterized by specific amino acid sequences in the regions determining its binding capability, such as the CDRs of the heavy and / or light chain variable domains of Ml and M2, or indeed the entire VL and / or VH domains or regions of Ml and M2. It is contemplated that the specific sequence information provided for the molecules generated as described in the Examples enables the skilled person to define combinations and variations of these sequences within the scope of the disclosure.

[0052] With respect to the VH domain in the AP protofibril binding moiety Ml, it comprises SEQ ID NO:1, with the variable amino acid Xaat position 40 being defined as being selected from A, N and T.

[0053] In one embodiment of the first aspect, said VH domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 3. This VH sequence is shared by the antibodies denoted A17D / R79T_DI 1 - 8 in WQ2016 / 005466 (see pages 12-13 thereof).

[0054] In one embodiment, said VH domain in Ml comprises the amino acid sequence SEQ ID NO:2, as exhibited by the antibodies denoted A17D / R79T_DI 5 - 8 in WQ2016 / 005466.

[0055] In another embodiment, said VH domain in Ml comprises the amino acid sequence SEQ ID NO:3, as exhibited by the antibodies denoted A17D / R79T_DI 1 - 4 in WQ2016 / 005466.

[0056] In another embodiment, said VH domain in Ml comprises the amino acid sequence SEQ ID NO:4, as exhibited by lecanemab. With respect to the VL domain in the A|3 protofibril binding moiety Ml, it comprises SEQ ID NO:5, with the variable amino acids Xb-Xgbeing defined as listed above.

[0057] In one embodiment, one of the following alternatives applies to the variable amino acids Xb-Xgin SEQ ID NO:5 of said VL domain in Ml:

[0058] Xd is R and Xf is R;

[0059] Xd is T and Xf is R;

[0060] Xd is R and Xf is S; and

[0061] Xd is T and Xf is S.

[0062] When Xd is R and Xf is R in SEQ ID NO:5, the VL domain comprises the A17D mutation, in comparison to the original lecanemab VL sequence.

[0063] When Xd is T and Xf is R in SEQ ID NO:5, the VL domain comprises the A17D and R79T mutations, in comparison to the original lecanemab VL sequence.

[0064] When Xd is R and Xf is S in SEQ ID NO:5, the VL domain comprises the A17D and R82S mutations, in comparison to the original lecanemab VL sequence.

[0065] When Xd is T and Xf is S in SEQ ID NO:5, the VL domain comprises the A17D, R79T and R82S mutations, in comparison to the original lecanemab VL sequence.

[0066] In one such embodiment, said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6-13.

[0067] In one embodiment, Xd is T and Xf is R in SEQ ID NO:5 of said VL domain in Ml, i.e. the VL domain comprises the A17D and R79T mutations, in comparison to the original lecanemab VL sequence.

[0068] In a more specific such embodiment, said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 and 10-13. This VL sequence is shared by the antibodies denoted A17D / R79T and A17D / R79T_DI 1 - 8 in WQ2016 / 005466 (see pages 12-13 thereof).

[0069] In one embodiment, Xb is A in SEQ ID NO:5 of said VL domain in Ml, i.e. the VL domain comprises the V13A mutation, in comparison to the original lecanemab VL sequence.

[0070] In a more specific such embodiment, said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NQ:10-ll, as exhibited by the antibodies denoted A17D / R79T_DI 3, 4, 7 and 8 in W02016 / 005466.

[0071] In one embodiment, Xgis D in SEQ ID NO:5 of said VL domain in Ml, i.e. the VL domain comprises the E84D mutation, in comparison to the original lecanemab VL sequence.

[0072] In a more specific such embodiment, said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10 and 12, as exhibited by the antibodies denoted A17D / R79T_DI 2, 4, 6 and 8 in WQ2016 / 005466.

[0073] In an even more specific embodiment, said VL domain in Ml comprises the amino acid sequence SEQ ID NQ:10, as exhibited by the antibody denoted A17D / R79T_DI 8 in WQ2016 / 005466.

[0074] In one embodiment, Xeis Q in SEQ ID NO:5 of said VL domain in Ml, i.e. the VL domain comprises the S81Q mutation, in comparison to the original lecanemab VL sequence.

[0075] In a more specific such embodiment, said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11 and 13, as exhibited by the antibodies denoted A17D / R79T_DI 1, 3, 5 and 7 in WQ2016 / 005466.

[0076] In one embodiment, Xcis V in SEQ ID NO:5 of said VL domain in Ml, i.e. the VL domain comprises the 121V mutation, in comparison to the original lecanemab VL sequence.

[0077] In a more specific such embodiment, said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12-13, as exhibited by the antibodies denoted A17D / R79T_DI 1, 2, 5 and 6 in WQ2016 / 005466.

[0078] In one embodiment of the binding molecule of the disclosure, said VH domain in Ml is as defined in any one of the above embodiments and said VL domain in Ml is as defined in any one of the above embodiments.

[0079] For example, moiety Ml in the bispecific binding molecule of the first aspect comprises a heavy chain variable domain and a light chain variable domain selected from the group consisting of the following VH / VL combinations: a) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10; b) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:11; c) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:12; d) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:13; e) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NQ:10; f) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:11; g) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:12; h) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:13; and i) a VH domain comprising SEQ ID NO:4 and a VL domain comprising SEQ ID NO:7.

[0080] In another embodiment, the Ml VH domain and the Ml VL domain are represented by one of the following VH / VL combinations: a) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10; b) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:11; c) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:12; d) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:13; e) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NQ:10; f) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:11; g) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:12; and h) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:13.

[0081] In another embodiment, the Ml VH domain and the Ml VL domain are represented by one of the following VH / VL combinations: a) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10; b) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NQ:10; and c) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:11.

[0082] In another embodiment, the Ml VH domain and the Ml VL domain are represented by a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10.

[0083] As a person of skill in the art is aware, A|3 peptides may exist in various forms along the progressive aggregation thereof from monomers to insoluble plaques. Of particular relevance to the present disclosure, soluble forms of A|3 peptides may be present in monomer form, or in various oligomeric or further aggregated forms. Soluble forms of polymerized or aggregated A|3 peptides are collectively referred to as "protofibrils" in the present disclosure. For clarity, the term "protofibril" is intended to encompass oligomers and higher order aggregates, but excludes insoluble fibrils or amyloid plaques. The bispecific binding molecule of the first aspect has an affinity for A|3 protofibrils.

[0084] The bispecific binding molecule may exhibit a preference or selectivity for one form of A|3 over another. In one such embodiment, the bispecific binding molecule has a higher affinity for A|3 protofibrils than for A|3 monomers. Without wishing to be bound by theory, such higher affinity for protofibrils in embodiments of the bispecific binding molecule may be due to avidity effects, insofar as the protofibril form of A|3 is thought to present a plurality of epitopes to bind, in comparison to the monomeric form. As such, the affinity of a binding molecule for protofibrils may be measured and reported herein as an "apparent affinity" in a manner known to the skilled person. In certain embodiments, the bispecific binding molecule of the first aspect binds selectively to A|3 protofibrils. As used herein, the term "bind selectively" refers to the preferential binding to the A|3 protofibril target. In certain embodiments, the bispecific binding molecule of the first aspect does not bind to any appreciable extent to A|3 monomers. In one embodiment, the bispecific binding molecule has at least 2x higher affinity for A|3 protofibrils than for A|3 monomers, such as at least lOx higher, such as at least 40x higher, such as at least lOOx higher, such as at least 200x higher, such as at least lOOOx higher, such as at least 2000x higher, such as at least 3000x higher binding affinity.

[0085] In one embodiment of the bispecific binding molecule, it has a binding affinity (or an apparent binding affinity) for A|3 protofibrils that corresponds to a KD value of no more than 5 nM, such as no more than 2 nM, such as no more than 1 nM, such as between 10 and 500 pM, such as between 200 and 400 pM, such as between 250 and 350 pM, as determined by surface plasmon resonance. hTfRl-binding moiety M2

[0086] As described above, in the first aspect, the present disclosure provides a bispecific binding protein in which moiety M2 is a human transferrin receptor 1 (hTfRl) binding moiety, capable of selective binding to an epitope located in the protease-like domain of hTfRl defined by amino acid residues 121-183 and 384-605 in SEQ ID NO:98. Without wishing to be bound by theory, the binding by moiety M2 to hTfRl to an epitope, or binding site, within the protease-like domain is contemplated to offer advantages in terms of avoiding the drawbacks associated with known binders to hTfRl, in particular those known binders which have affinity for epitopes or binding sites located in the apical domain of TfRl.

[0087] In a specific embodiment, the epitope or binding site for the hTfRl-binding moiety M2 comprises the amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO:98. In another embodiment, the epitope or binding site for the hTfRl binding moiety M2 consists of the amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO:98. In an alternative specific embodiment, the epitope or binding site for the hTfRl binding moiety M2 comprises or consists of at least one, at least two, at least three, at least four, at least five, at least six, at least seven or all eight of the amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO:98. As shown in the examples which follow, for example with reference to Figure 7, this embodiment of the epitope for the hTfRl binding moiety disclosed herein ensures binding that does not interfere with the natural hTfRl ligands transferrin and ferritin.

[0088] As known to a person skilled in the art, an epitope (or "antigenic determinant") is a group of amino acids or other chemical groups exposed on the surface of a molecule, frequently a protein, here hTfRl, which can generate an antigenic response and bind antibody. An epitope is a localized region on the surface of an antigen that is recognized by the immune system, specifically by antibodies. A conformational epitope is composed of neighboring amino acid residues located on an antigenic protein surface structure. Conformational epitopes bind their complementary paratopes in B-cell receptors and / or antibodies. In one embodiment of the disclosure, the epitope bound by the hTfRl-binding moiety M2 of the bispecific binding molecule is a conformational epitope.

[0089] In one embodiment, the binding to hTfRl by the M2 binding moiety is monovalent.

[0090] As described above, the hTfRl-binding moiety M2 comprises a VH / VL pair with an antigen-binding surface. For clarity with regard to both Ml and M2, the designation of "VH / VL" as used in relation to a VH / VL pair does not limit the construct to any particular order of the VH and VL domains in the polypeptide chain, but is only used to convey that both the VH and VL domains are present, and that they are capable of pairwise association to form an immunoglobulin domain with an antigen-binding surface. As non-limiting alternatives, the term "VH / VL pair" encompasses, for example, constructs in which the VL domain precedes the VH domain in a single chain Fv, constructs in which the VH domain precedes the VL domain in a single chain Fv, and constructs in which the VH and VL domains are non- covalently associated with each other. In a specific embodiment of the binding protein, the VH / VL pair in M2 is arranged such that the VL domain precedes the VH domain in a single chain Fv construct.

[0091] The VH / VL pair comprised in moiety M2 comprises an antigen-binding surface. In one embodiment, said antigen-binding surface is composed of three complementarity-determining regions (CDRs) from each of the VH and VL domains.

[0092] In one embodiment, said CDRs comprise the following amino acid sequences:

[0093] VHCDR1: X1X2NMX3 (SEQ ID NO:14), wherein

[0094] XI is selected from D and A;

[0095] X2 is selected from Y and A; and X3 is selected from D and A;

[0096] VHCDR2: X4INPX5X6X7TTSX8X9X10KFKG (SEQ ID NO:15), wherein 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;

[0097] X9 is selected from N and S; and X10 is selected from E and Q;

[0098] VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID NO:17), wherein

[0099] XII 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;

[0100] VLCDR2: X15ASTRES (SEQ ID NO:18) wherein X15 is selected from W and A; and

[0101] VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:19) wherein X16 is selected from Y and A; X17 is selected from

[0102] X18 is selected from X19 is selected from X20 is selected from

[0103] In one embodiment, the CDRs of moiety M2 further comprise:

[0104] VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:16) wherein X21 is selected from Y and A;

[0105] X22 is selected from Y and A;

[0106] X23 is selected from Y and A; X24 is selected from D and A; and X25 is selected from Y and A.

[0107] In an alternative embodiment, the said antigen-binding surface of moiety M2 further comprise:

[0108] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:48)

[0109] As defined herein, embodiments of the hTfRl-binding moiety M2 in the bispecific binding molecule of the first aspect of the disclosure have specific amino acid sequences in the regions determining its binding capability, such as the CDRs of the heavy and / or light chain variable domain, or indeed the entire VL and / or VH domains or regions. Non-limiting examples of such specific amino acid sequences are provided herein for the specific antibodies and fragments thereof generated and characterized as described in Examples 1-15. Further development of these antibodies into bispecific binding molecules of the present disclosure is detailed in Examples 16-23.

[0110] It is contemplated that the specific sequence information provided for the generated binding molecules enables the skilled person to define combinations and variations of these sequences within the scope of the invention, such as including the combinations and variations afforded by the variation in the general CDR sequences provided herein.

[0111] In one embodiment, said VHCDR2 of moiety M2 is:

[0112] VHCDR2: X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO:20), wherein

[0113] X4 is selected from D and A;

[0114] X5 is selected from D and A;

[0115] X6 is selected from Y and A;

[0116] X7 is selected from D and A; and

[0117] X8 is selected from Y and A. In one embodiment, said VLCDR1 of moiety M2 is:

[0118] VLCDR1: KSSQSLLX11STNQKNX14LA (SEQ ID NO:21), wherein Xll is selected from Y and A; and X14 is selected from Y and A.

[0119] In one embodiment, said VLCDR3 of moiety M2 is:

[0120] VLCDR3: QQX16FIX19PRT (SEQ ID NO:22) wherein X16 is selected from Y and A; X19 is selected from Y and A.

[0121] In one embodiment, the amino acid sequence of said VHCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:23 and 29-31.

[0122] In one embodiment, the amino acid sequence of said VHCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:24, 32-36 and 47, for example selected from the group consisting of SEQ ID NO:24 and 32-36.

[0123] In one embodiment, the amino acid sequence of said VHCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:25, 37-41 and 48, for example selected from the group consisting of SEQ ID NO:25 and 37-41.

[0124] In one embodiment, the amino acid sequence of said VLCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:26, 42, 43 and 49, for example selected from the group consisting of SEQ ID NO:26, 42 and 43.

[0125] In one embodiment, the amino acid sequence of said VLCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:27 and 44.

[0126] In one embodiment, the amino acid sequence of said VLCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:28, 45, 46 and 50, for example selected from the group consisting of SEQ ID NO:28, 45 and 46.

[0127] In some embodiments, the CDR sequences can be freely combined among the options listed above. Such embodiments for example include, but are not limited to, those combinations exemplified in Example 9 for alanine substituted variants of the representative M2 moiety h26D3. In a specific embodiment of a bispecific binding molecule of the disclosure, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0128] VHCDR1: DYNMD (SEQ ID NO:23),

[0129] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:24),

[0130] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:25)

[0131] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:26),

[0132] VLCDR2: WASTRES (SEQ ID NO:27), and

[0133] VLCDR3: QQYFIYPRT (SEQ ID NO:28).

[0134] In another specific embodiment of a bispecific binding molecule of the disclosure, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0135] VHCDR1: DYNMD (SEQ ID NO:23),

[0136] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:34),

[0137] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:25),

[0138] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:26),

[0139] VLCDR2: WASTRES (SEQ ID NO:27), and

[0140] VLCDR3: QQYFIYPRT (SEQ ID NO:28).

[0141] In another specific embodiment of a bispecific binding molecule of the disclosure, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0142] VHCDR1: DYNMD (SEQ ID NO:23),

[0143] VHCDR2: DINPNYDTTSYSQKFKG (SEQ ID NO:47),

[0144] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:48),

[0145] VLCDR1: KSSQSLLYSSNRKNYLA (SEQ ID NO:49),

[0146] VLCDR2: WASTRES (SEQ ID NO:27), and

[0147] VLCDR3: QQYYNYPYT (SEQ ID NQ:50).

[0148] In another specific embodiment of a bispecific binding molecule of the disclosure, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0149] VHCDR1: NYWLG (SEQ ID NO:51),

[0150] VHCDR2: DIFPGSDNTYYNEKFKG (SEQ ID NO:52),

[0151] VHCDR3: SGNFYAMDY (SEQ ID NO:53),

[0152] VLCDR1: SASSSVNYMN (SEQ ID NO:54),

[0153] VLCDR2: DTSKLAS (SEQ ID NO:55), and

[0154] VLCDR3: FQGSGYPFT (SEQ ID NO:56).

[0155] In one embodiment of the bispecific binding molecule of the disclosure, the VH domain in moiety M2 comprises or consists of an amino acid sequence selected from (i) the group consisting of SEQ ID NO:57-70, 78 and 80, for example the group consisting of SEQ ID NO:57-70, for example the group consisting of SEQ ID NO:57 and 63; and

[0156] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i).

[0157] In one embodiment of the bispecific binding molecule of the disclosure, the VL domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0158] (i) the group consisting of SEQ ID NO:71-77, 79 and 81, for example the group consisting of SEQ ID NO:71-77; and

[0159] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions a sequence defined in (i).

[0160] In a particular such embodiment, the VH domain and VL domain in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NQ:57-70 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NO:71-77 and sequences having at least 80 % sequence identity thereto.

[0161] In another embodiment of the bispecific binding molecule of the disclosure, said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:82 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 to SEQ ID NO:82, provided that the sequences of the CDR regions are 100% identical to those of SEQ ID NO:82.

[0162] In another embodiment of the bispecific binding molecule of the disclosure, said VL domain of moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:83 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 to SEQ ID NO:83, provided that the sequences of the CDR regions are 100% identical to those of SEQ ID NO:83.

[0163] In a particular such embodiment, the M2 VH domain and VL domain are both as defined immediately above, i.e. a VH comprising or consisting of the sequence SEQ ID NO:82 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of the sequence SEQ ID NO:83 and sequences having at least 80 % sequence identity thereto.

[0164] In one embodiment of the bispecific binding molecule of the disclosure, said VH domain in M2 comprises SEQ ID NO:57 and said VL domain in M2 comprises a sequence selected from SEQ ID NO:71-77.

[0165] In one embodiment of the bispecific binding molecule of the disclosure, said VH domain in M2 comprises a sequence selected from SEQ ID NQ:57-70 and said VL domain in M2 comprises SEQ ID NO:71.

[0166] In one embodiment of the bispecific binding molecule of the disclosure, said VH domain in M2 comprises SEQ ID NO:57 and said VL domain in M2 comprises SEQ ID NO:71.

[0167] In one embodiment of the bispecific binding molecule of the disclosure, said VH domain in M2 comprises SEQ ID NO:63 and said VL domain in M2 comprises SEQ ID NO:71.

[0168] In some embodiments of the bispecific binding molecule of the disclosure, the hTfRl-binding moiety M2 comprises one first cysteine residue in the VH domain thereof, and one second cysteine residue in the VL domain thereof, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL domains.

[0169] Without wishing to be bound by theory, the provision of the first and second cysteine residues in the VH and VL domains of moiety M2, respectively, and the resultant disulfide bridge between VH and VL, is contemplated to allow for the formation of a more stable VH / VL pairing. Non-limiting examples of advantages with such increased stability include an improved storage stability and an increased resistance towards multimerization. The introduction of cysteine residues and the resulting formation of a disulfide bridge in moiety M2 in the bispecific binding molecule of the disclosure is contemplated to increase the stability of the bispecific binding molecule. As realized by a person of skill in the art from the context herein, such increased stability may for example be measured as an increase in the monomeric content of the bispecific binding molecule in a sample after storage, compared to the monomeric content of a binding molecule having an identical sequence except for the cysteine residues. Evaluating monomeric content may for example be done using size exclusion liquid chromatography (SEC) after simulated stress conditions and / or long-term storage. This will provide measures of the monomer content and presence of aggregates. In one embodiment, the bispecific binding molecule of the disclosure is defined as stable if it exhibits a monomer content of 90 % or more after storage for two weeks at 40 °C as determined by SEC. In another embodiment, the bispecific binding molecule of the disclosure is defined as stable if it exhibits a monomer content of 95 % or more after storage for two weeks at 40 °C as determined by SEC. In yet another embodiment, the bispecific binding molecule of the disclosure is defined as stable if it exhibits a monomer content of 98 % or more after storage for two weeks at 40 °C as determined by SEC.

[0170] Importantly, the increased size and avidity of dimers, or further multimers, of binding molecules comprising pairs of VH and VL domains may cause undesirable cross-linking of targets in vivo and altered pharmacodynamic properties. This is especially important when binding to the transferrin receptor to cross the blood brain barrier, because it is crucial to avoid multimerization, as this leads to downregulation of the transferrin receptor. Such down-regulation, in turn, reduces the transport capacity over the blood brain barrier and can potentially cause safety problems for a biopharmaceutical product, due to a lower abundance of transferrin receptors on the cell surface. In addition, dimerization and further oligomerization is contemplated to pose considerable challenges with respect to the production, analysis, formulation and storage of biologies in connection with commercial or clinical applications.

[0171] Thus, in this embodiment of the bispecific binding molecule of the disclosure, the hTfRl-binding moiety M2 is engineered to comprise a disulfide bridge in order to stabilize the VL / VH or VH / VL forms. This is shown to be beneficial for producing antibody constructs that are stable and only bind in a monomeric form to hTfRl. Data show that the presence of only small amounts of dimeric forms of binding molecules leads to avidity binding to the transferrin receptor. Also, without the stabilizing disulfide, the disclosed constructs may be produced in a dimeric or oligomeric form and be unstable over time under various conditions. Thus, by introducing a disulfide bond between the VL / VH or VH / VL domains, the bispecific binding molecules of the disclosure are contemplated to be both more stably produced and in addition prevent avidity binding by moiety M2 to hTfRl.

[0172] In one embodiment, said first cysteine (in the M2 VH domain) is located at an amino acid position selected from VH position 39-49 as determined by reference to the Kabat numbering scheme. In a more specific embodiment, said first cysteine is located at an amino acid position selected from VH position 41-47, such as selected from VH position 43-45, all as determined by reference to the Kabat numbering scheme. In a yet more specific embodiment, the first cysteine is located at VH position 44 per Kabat numbering.

[0173] In one embodiment, said second cysteine (in the M2 VL domain) is located at an amino acid position selected from VL position 95-105 as determined by reference to the Kabat numbering scheme. In a more specific embodiment, said first cysteine is located at an amino acid position selected from VL position 97-103, such as selected from VL position 99-101, all as determined by reference to the Kabat numbering scheme. In a yet more specific embodiment, the first cysteine is located at VL position 100 per Kabat numbering.

[0174] In one exemplary embodiment, said first cysteine residue is located at M2 VH position 44 and said second cysteine residue is located at M2 VL position 100, as determined by reference to the Kabat numbering scheme.

[0175] In one embodiment of the bispecific binding molecule of the disclosure, the VH domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0176] (i) the group consisting of SEQ ID NO:101-116, for example the group consisting of SEQ ID NO:101-114, for example the group consisting of SEQ ID NQ:101 and 107; and

[0177] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 44 (Kabat position 44). In one embodiment of the bispecific binding molecule of the disclosure, the VL domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0178] (i) the group consisting of SEQ ID NO:118-126, for example the group consisting of SEQ ID NO:118-124; and

[0179] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 106 (Kabat position 100).

[0180] In a particular such embodiment, the VH domain and VL domain in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NQ:101-116 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NO:118-126 and sequences having at least 80 % sequence identity thereto, subject to the defined provisos.

[0181] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises SEQ ID NQ:101 and the M2 VL domain comprises a sequence selected from SEQ ID NO:118-124.

[0182] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises a sequence selected from SEQ ID NQ:101-114 and the M2 VL domain comprises SEQ ID NO:118.

[0183] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises SEQ ID NQ:101 and the M2 VL domain comprises SEQ ID NO:118.

[0184] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises SEQ ID NQ:107 and the M2 VL domain comprises SEQ ID NO:118.

[0185] In one embodiment of the bispecific binding molecule of the disclosure, the

[0186] VH domain in moiety M2 comprises or consists of an amino acid sequence selected from (i) the group consisting of SEQ ID NO:142-152, for example the group consisting of SEQ ID NO:142-147, for example the group consisting of SEQ ID NO:142-143; and

[0187] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 44 (Kabat position 44).

[0188] In one such embodiment of the bispecific binding molecule of the disclosure, the VL domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0189] (i) the group consisting of SEQ ID NO:118-126, for example the group consisting of SEQ ID NO:118-124; and

[0190] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 106 (Kabat position 100).

[0191] In a particular such embodiment, the VH domain and VL domain in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NO:142-152 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NO:118-126 and sequences having at least 80 % sequence identity thereto, subject to the defined provisos.

[0192] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises a sequence selected from SEQ ID NO:142-143 and the M2 VL domain comprises SEQ ID NO:118.

[0193] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises SEQ ID NO:142 and the M2 VL domain comprises SEQ ID NO:118.

[0194] In one embodiment of a bispecific binding molecule of the disclosure, the M2

[0195] VH domain comprises SEQ ID NO:143 and the M2 VL domain comprises SEQ ID NO:118. In another embodiment of the bispecific binding molecule of the disclosure, the VH domain in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:117 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 to SEQ ID NO:117, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NO:117, and provided that the sequence comprises a cysteine residue at position 44 (Kabat position 44).

[0196] In another of the bispecific binding molecule of the disclosure, the VL domain in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:127 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 to SEQ ID NO:127, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NO:127, and provided that the sequence comprises a cysteine residue at position 99 (Kabat position 100).

[0197] In a particular such embodiment, the M2 VH domain and M2 VL domain are both as defined immediately above, i.e. a VH comprising or consisting of the sequence SEQ ID NO:117 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of the sequence SEQ ID NO:127 and sequences having at least 80 % sequence identity thereto, subject to the defined provisos.

[0198] Linkage of Ml and M2

[0199] In one embodiment of a bispecific binding molecule of the disclosure, the VH / VL pair of M2 forms part of an antibody construct. In one such embodiment, the VH / VL pair of M2 is present in an antibody fragment selected from the group consisting of a Fab fragment, a single chain Fab (scFab) fragment, an Fv fragment and a single chain (scFv) fragment. In a particular embodiment, said antibody fragment is an scFv.

[0200] Thus, in one embodiment of the bispecific binding molecule of the disclosure, the M2 moiety comprises an scFv. In other words, the VH / VL pair in M2 forms part of an scFv, in which the VH and VL domains are coupled together by a peptide scFv linker. In one such embodiment, the scFv linker may either be attached to the C- terminal amino acid residue of the VH domain and to the N-terminal amino acid residue of the VL domain, or to the N-terminal amino acid residue of the VH domain and to the C-terminal amino acid residue of the VL domain. In the first configuration, the VH domain precedes the VL domain in the polypeptide chain constituting the scFv, while in the second configuration, the VL domain precedes the VH domain. The two different configurations are sometimes denoted "VH first" and "VL first" in the present disclosure.

[0201] The design and selection of suitable peptide linkers for use within and between domains and moieties of fusion proteins, antibody constructs and other such engineered polypeptides is within the capacity of a person of skill in the art. In some embodiments where the hTfRl-binding moiety M2 comprises or consists of an scFv, the scFv linker is a flexible peptide linker, consisting of from 5 to 40 amino acid residues, for example from 10 to 30 amino acid residues, for example from 15 to 25 amino acid residues, for example about 15 amino acid residues, for example 15 amino acid residues, for example comprising or consisting of the sequence (648)3 (SEQ ID NO:137).

[0202] The same or similar design considerations apply to linkers used to attach the AP protofibril-binding moiety Ml to the hTfRl-binding moiety M2. In one embodiment, Ml and M2 are connected to each other by at least one peptide linker between Ml and M2. In one embodiment, said at least one peptide linker between Ml and M2 is attached, on the M2 side, to the C-terminal amino acid residue of the VH domain of M2 or to the N-terminal amino acid residue of the VL domain of M2.

[0203] As described above, the design and selection of suitable peptide linkers for use within and between domains and moieties of fusion proteins, antibody constructs and other such engineered polypeptides is within the capacity of a person of skill in the art. In one embodiment of the binding protein of the disclosure, Ml and M2 are linked by at least one flexible peptide linker. In one embodiment, the at least one flexible peptide linker comprises glycine, serine, alanine and / or threonine residues. In a more specific embodiment, said linker(s) has a general formula selected from (GnSm)p and (SnGm)p, wherein, independently, n = 1-7, m = 0-7, n + m < 8 and p = 1-10. In some embodiments, at least one linker is between 10 and 50 amino acid residues long, such as between 10 and 30 amino acid residues long, such as between 15 and 25 amino acid residues long or between 10 and 20 amino acids long. In case Ml and M2 are linked via two or more linkers, all of the disclosed, optional linker designs apply individually to each linker present independently of the other linkers. Thus, for example, if there are two linkers, they may be of the same or different length, and have the same amino acid sequence or different amino acid sequences.

[0204] In one embodiment of the bispecific binding molecule of the disclosure, moiety Ml is provided as a knob-into-hole antibody comprising two identical antibody light chains; one antibody hole heavy chain; and one antibody knob heavy chain; and M2 is provided as an scFv linked to the C-terminal amino acid residue of the knob heavy chain of Ml.

[0205] In one such embodiment of the bispecific binding molecule, the amino acid sequence of the antibody light chain of Ml comprises or consists of SEQ ID NO:138; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:139, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NQ:140.

[0206] In another such embodiment of the bispecific binding molecule, the amino acid sequence of the antibody light chain of Ml comprises or consists of SEQ ID NO:138; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:139, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:164.

[0207] In another such embodiment of the bispecific binding molecule, the amino acid sequence of the antibody light chain of Ml comprises or consists of SEQ ID NO:138; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:139, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:165.

[0208] Amino acid sequences

[0209] In various embodiments of the bispecific binding molecule of the disclosure, the VH and VL sequences, when present in either moiety Ml or moiety M2 of the binding molecule, may be individually selected from any one of the listed sequences and 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. For embodiments wherein variable domains of the binding molecules of the disclosure are defined by such a particular percentage sequence identity to a reference sequence, the VH and / or VL domains may retain the identical CDR sequences of those present in the reference sequence such that the allowed percentage variation is present only within the framework regions.

[0210] In various embodiments of the bispecific binding molecule of the disclosure, sequences of complementarity determining regions (CDR regions) and general amino acid position numbering within antibody sequences may suitably be defined using the Kabat convention, which is well known to a person of skill in the art of antibody technology (see e.g. Kabat (1991), Sequences of Proteins of Immunological Interest, 5thedition, NIH Publication no 91-3242 from the US Department of Health and Human Services).

[0211] Affinity for a target

[0212] As used herein, the terms "specific binding to X", "selective binding to X" and "affinity for X", wherein X is a target (e.g. an antigen or an epitope, such as the A|3 protofibril bound by moiety Ml or the hTfRl bound by moiety M2 of the bispecific binding molecule of the disclosure), refer to a property of a binding molecule which may be tested for example by ELISA, by surface plasmon resonance (SPR), by Kinetic Exclusion Assay (KinExA®) or by bio-layer interferometry (BLI). The skilled person is aware of these methods and others.

[0213] For example, binding affinity for antigen or epitope X may be tested in an experiment in which a binding molecule to be tested is captured on ELISA plates coated with target or antigen X, or an antigen exhibiting the epitope X, and a biotinylated detector antibody is added, followed by streptavidin-conjugated horse radish peroxidase (HRP). Alternatively, said detector antibody may be directly conjugated with HRP. Tetramethylbenzidine (TMB) substrate is added and the absorbance at 450 nm is measured using an ELISA multi-well plate reader. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the binding molecule for X. If a quantitative measure is desired, for example to determine the EC50 value (the half maximal effective concentration) for the interaction, ELISA may also be used. The response of the binding molecule against a dilution series of X may be measured using ELISA as described above. The skilled person may then interpret the results obtained by such experiments and EC50 values may be calculated from the results, using for example GraphPad Prism v.9 and non-linear regression.

[0214] As used herein, the term "EC50" refers to the half maximal effective concentration of a binding molecule which induces a response halfway between the baseline and maximum after a specified exposure time.

[0215] Additionally, inhibition ELISA may be used to obtain a quantitative measure of interaction by determination of the "IC50" (the half maximal inhibitory concentration). In an inhibition ELISA, the concentration of a target or an antigen or epitope X in a fluid sample is measured by detecting interference in an expected signal output. In principle, a known antigen or epitope-bearing substance is used to coat a multi-well plate. In parallel, a binding molecule with putative affinity for the target, antigen or epitope is added and incubated with a solution containing antigen at varied concentrations. Following standard blocking and washing steps, samples containing the mixture of the binding molecule with the antigen or epitope are added to the well. Labeled detection antibody with affinity for the antigen- or epitope-binding molecule is then applied for detection using relevant substrates (for example TMB). In principle, if there is a high concentration of antigen or epitope in the fluid sample, a significant reduction in signal output will be observed. In contrast, if there is very little antigen or epitope in the fluid sample, there will be very little reduction in the expected signal output. The skilled person appreciates that the signal output is also dependent on the affinity of the binding molecule for said antigen or epitope.

[0216] As used herein, the term "IC50" refers to the half maximal inhibitory concentration of a binding molecule which induces a response halfway between the baseline and maximum inhibition after a specified exposure time. Herein, a lower IC50 value indicates that a lower concentration of antigen or epitope is required to interfere with the binding of the detection antibody to the known antigen or epitope coated on the plate, as compared to a higher IC50 value. Thus, a lower IC50 value typically corresponds to a higher affinity of the binding molecule.

[0217] The binding affinity of a binding molecule may also be tested by SPR. For example, said binding affinity may be tested in an experiment in which antigen or epitope X is immobilized on a sensor chip of the instrument, and the sample containing the binding molecule to be tested is passed over the chip. Alternatively, the binding molecule to be tested may be immobilized on a sensor chip of the instrument, and a sample containing X is passed over the chip. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the moiety for X. If a quantitative measure is desired, for example to determine a KD value for the interaction, SPR may also be used. Binding values may for example be defined in a Biacore (Cytiva) or ProteOn XPR 36 (Bio-Rad) instrument. The antigen or epitope is suitably immobilized on a sensor chip of the instrument, and samples of the binding molecule whose affinity is to be determined are prepared by serial dilution and injected. KD values may then be calculated from the results using for example the 1:1 Langmuir binding model of the Biacore Insight Evaluation Software 2.0 or other suitable software, typically provided by the instrument manufacturer.

[0218] Another method for determining binding affinity of a binding molecule to antigen or epitope X is the Kinetic Exclusion Assay (KinExA; Sapidyne Instruments Inc; Darling and Brault, Assay and Drug Dev Tech (2004) 2(6) :647-657) for measurements of the equilibrium binding affinity and kinetics between unmodified molecules in solution. A KinExA KD analysis requires immobilization of one interaction partner (e.g. the titrated binding partner) to a solid phase, which is then used as a probe to capture the other interaction partner (e.g. the constant binding partner) free in solution once an equilibrium is reached.

[0219] The binding affinity may also be measured by bio-layer interferometry (BLI), a label-free technology for measuring biomolecular interactions within the interactome. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. The binding between a ligand (antigen or epitope X) immobilized on the biosensor tip surface and an analyte (such as a binding molecule with affinity for X) in solution produces an increase in optical thickness at the biosensor tip resulting in a wavelength shift, AX, which is a direct measure of the change in thickness of the biological layer. Interactions are measured in real time, providing the ability to monitor binding specificity, rates of association and dissociation, or concentration, with precision and accuracy. The skilled person is aware of the above-mentioned and other methods for measuring the affinity of a binding molecule for target, antigen or epitope X, either qualitatively or quantitatively or both.

[0220] Polynucleotides, vectors and cells

[0221] In another one of its aspects, the present disclosure provides a nucleotide sequence encoding a bispecific binding molecule disclosed herein. In certain embodiments, provided herein is a set of nucleotide sequences wherein the set encodes a bispecific binding molecule disclosed herein. In a specific embodiment, each nucleotide sequence of such a set encodes one polypeptide of a bispecific binding molecule disclosed herein. In one specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:138; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:139; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NQ:140. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:138; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:139; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:164. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:138; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:139; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:165.

[0222] This aspect of the disclosure provides polynucleotides, or sets of polynucleotides, encoding a bispecific binding molecule disclosed herein, as well as polynucleotides or sets of polynucleotides comprising one or more portion(s) thereof, and / or being complementary thereto. A set of polynucleotides encoding the bispecific binding molecule disclosed herein may encompass two or more polynucleotides, each encoding a portion of the bispecific binding molecule. Polynucleotides disclosed herein can be RNA or DNA (e.g., cDNA, genomic DNA, or synthetic DNA), and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

[0223] The present disclosure further provides a vector or a set of vectors comprising a polynucleotide, or a set of polynucleotides as disclosed herein. Such vectors are useful, for example, for amplifying the polynucleotide or set of polynucleotides in host cells to create useful quantities thereof, and for expressing the bispecific binding molecule as disclosed herein. Any suitable vectors can be used to introduce one or more polynucleotides disclosed herein into a cell. Exemplary vectors include, but not limited to, lentivirus vectors, adeno-associated viral (AAV) vectors, adenoviral (AV) and liposomal vectors.

[0224] The present disclosure further provides a cell (e.g., a host cell) comprising any one or more of: a bispecific binding molecule as disclosed herein, a polynucleotide or a set of polynucleotides as disclosed herein, or a vector or a set of vectors as disclosed herein. In certain embodiments, the cell replicates the polynucleotide or set of polynucleotides as disclosed herein or the vector or set of vectors as disclosed herein. Non-limiting examples of cells as disclosed herein include Escherichia coli, mammalian cells (e.g. myeloma cells, Chinese Hamster Ovary (CHO) cells, or hybridoma cells), yeast cells, insect cells and plant cells. Mammalian cells may provide translational modifications (e.g. glycosylation, truncation, lipidation or phosphorylation) that may confer beneficial biological properties on recombinant expression products.

[0225] The present disclosure further provides a method of making a bispecific binding molecule disclosed herein, comprising culturing said cell under conditions that result in the expression of the bispecific binding molecule, and isolating the bispecific binding molecule.

[0226] Pharmaceutical compositions

[0227] In a second aspect, there is provided a pharmaceutical composition comprising a bispecific binding molecule as described herein and at least one pharmaceutically acceptable excipient or carrier.

[0228] Techniques for formulating antibodies, fragments thereof and other related binding molecules for human therapeutic use are well known in the art and are reviewed, for example, in Wang et al. (2007), J Pharm Sci, 96:1-26, the contents of which are incorporated herein in their entirety.

[0229] Pharmaceutically acceptable excipients that may be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylenepolyoxypropylene block polymers, polyethylene glycol and wool fat.

[0230] In certain embodiments, the pharmaceutical compositions are formulated for administration to a subject via any suitable route of administration including but not limited to intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalational, buccal (e.g., sublingual) and transdermal administration. In preferred embodiments, the composition is formulated for intravenous or subcutaneous administration.

[0231] Methods of prevention, treatment, diagnosis, prognosis and detection

[0232] The bispecific binding molecules according to the present disclosure may be useful as therapeutic and / or diagnostic agents.

[0233] Hence, in a further aspect of the disclosure, there is provided a bispecific binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a medicament.

[0234] In yet another aspect of the disclosure, there is provided a bispecific binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a diagnostic agent.

[0235] In yet another aspect of the disclosure, there is provided a bispecific binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a prognostic agent.

[0236] Also provided are methods of preventing disease, treating disease or diagnosing disease or assessing disease prognosis, wherein a bispecific binding molecule as disclosed herein is administered to a subject, typically a human subject.

[0237] Also provided is the use of the disclosed bispecific binding molecule for the manufacture of compositions (such as medicaments) for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of any one of the listed diseases. Also provided are methods of detecting and / or diagnosing a disease in a subject, wherein the methods comprise contacting a sample obtained from the subject with a bispecific binding molecule as described herein. These methods are typically in vitro methods.

[0238] Thus, said bispecific binding molecule, or pharmaceutical composition comprising it, is useful in the treatment, prevention and / or diagnosis of a condition selected from neurological disorders or conditions characterized by accumulation and / or aggregation of AP, such as formation of amyloid plaques. Such diseases or conditions include but are not limited to Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), Lewy body dementia (including Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB)), neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type); as well as other diseases which are based on or associated with amylogenic proteins, such as progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataract due to AP deposition, traumatic brain injury with an accumulation of A , adult onset diabetes, senile cardiac amyloidosis and macular degeneration.

[0239] Thus, in one embodiment, there is provided a bispecific binding molecule, or pharmaceutical composition comprising it, for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of an AP peptide-associated condition. In one embodiment, there is provided a bispecific binding molecule, or pharmaceutical composition comprising it, for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of an A peptide-associated condition, selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), Lewy body dementia, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataract due to AP deposition, traumatic brain injury with an accumulation of AP, adult onset diabetes, senile cardiac amyloidosis and macular degeneration. In one specific embodiment, said bispecific binding molecule, or pharmaceutical composition comprising it, is provided for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of Alzheimer's disease.

[0240] In another aspect, there is provided a method of the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of an A|3 peptide-associated condition in a mammal having, or being at risk of developing, said disorder, comprising administering to said mammal an amount, such as a therapeutically effective amount, of a bispecific binding molecule, or pharmaceutical composition comprising it.

[0241] In one embodiment, said A|3 peptide-associated condition is, for example, selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), Lewy body dementia (including Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB)), neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataract due to A|3 deposition, traumatic brain injury with an accumulation of A|3, adult onset diabetes, senile cardiac amyloidosis and macular degeneration. In a more specific embodiment, said A|3 peptide-associated condition is Alzheimer's disease.

[0242] With regard to therapeutic and / or preventive use of the disclosed bispecific binding molecule for the treatment of neurodegenerative diseases, there are several putative mechanisms of action. Without wishing to be bound by theory, non-limiting and independently possible mechanisms of action are for example binding to A|3 protofibrils to prevent their neurotoxic action, or binding to and removal of soluble neurotoxic A|3 protofibrils and / or amyloid plaques, all with the end purpose of alleviating amyloidosis and improving cognitive function.

[0243] With regard to diagnostic and / or prognostic use of the disclosed bispecific binding molecule in neurodegenerative diseases, the harmful A|3 protofibril species can be detected and measured in patients at risk of disease or showing signs of incipient disease. One such method is PET scan using a radio-labelled antibody of the disclosure. Another method for diagnosis and prognosis is biochemical analysis analyzing the levels of A|3 protofibrils in blood, plasma, CSF and other fluids, using such methods as ELISA, Mesoscale Discovery (MSD), SMCxPro or Simoa.

[0244] Kit

[0245] The present disclosure also provides, in a further aspect, a kit comprising a bispecific binding molecule disclosed herein, or a composition provided herein, packaged into suitable packaging material. A kit optionally includes a label or packaging insert comprising a description of the components and / or instructions for use of the components therein in vitro, in vivo or ex vivo. A kit according to this aspect of the disclosure can additionally include other components. Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. reference

[0246] Various publications are cited in the present application, each of which is incorporated by reference herein in its entirety.

[0247] Brief description of the figures

[0248] Figure 1 shows the results of a binding screen of the indicated IgG antibodies from the immunization described in Example 1 towards human (hTfRl), cynomolgus (cTfRl) and mouse (mTfRl) TfRl in crude hybridoma supernatants by biolayer interferometry (BLI).

[0249] Figure 2 shows the result of the BLI binding analysis described in Example 2 for the indicated Fab fragments of mouse antibodies 24B4, 26D3 and 37D10 as well as for a Fab fragment of control antibody 8D3.

[0250] Figure 3 shows mapping of antibody binding epitopes to the protease-like domain of hTfRl as described in Example 2, by selective antibody binding to ELISA plates coated with either human, mouse or one of three different chimeric human / mouse TfRl receptors. Antibodies 24B4, 26D3 and 37D10 bind to hTfRl (A) but not to mTfRl (B). In addition, 24B4, 26D3 and 37D10 also bind to h / m protease like domain chimera (D), but not to any of the plates coated with the other chimeric receptors (C and E). Figure 4 illustrates the epitope binning assay described in Example 2, with the following main four steps: Step 1 - immobilization of bio-TfRl on sensor chip; Step 2 - wash of non-binding material; Step 3 - binding of competing binder to TfRl; Step 4 - association of binders to the TfRl:binder complex formed in Step 3. The data in Step 4 determines whether the two investigated binders compete in binding to hTfRl.

[0251] Figure 5 shows the result of carrying out the epitope binning assay as described in Example 2, showing the degree of competition between antibodies for simultaneous binding to hTfRl. Binding of (A) antibody 26D3, (B) antibody 24B4 and (C) control antibody 15G11-1 to preformed complexes of hTfRl and either of the indicated antibodies. Binding responses for all antibodies are normalized to the binding response measured to free hTfRl (no competing antibody).

[0252] Figure 6 shows binding by the indicated binders to hTfRl on the surface of cells, studied as described in Example 2. The Y axes of both diagrams show the mean fluorescence intensity when staining cells with (A) whole antibodies and (B) Fab fragments of the indicated binders. No background staining is detected with the negative isotype control IgG (A) or the non-related Fab fragment, Lyl28 (B).

[0253] Figure 7 shows the result of competition analysis of indicated binders with ferritin and transferrin as described in Example 3. The diagrams show (A) MFI of the indicated binders binding to TfRl expressed on THP-1 cell surfaces, (B) MFI of ferritin on THP-1 cell surface when exposed to the indicated binders, with the positive control antibody MA-712 competing with ferritin, and (C) MFI of transferrin on K562 cell surfaces when exposed to the indicated binders.

[0254] Figure 8 is a collection of sensorgrams showing the result of SPR analysis of original 26D3 and 26D3 humanized as described in Example 4 (h26D3) in Fab formats when binding to hTfRl and cTfRl as indicated.

[0255] Figure 9 shows the result of BLI and ELISA binding studies carried out on mouse and humanized versions of 26D3 in an scFv format as described in Example 4. (A) Sensorgrams obtained by BLI measurement of binding of the indicated constructs to hTfRl. (B) Binding responses from ELISA measurement of binding of the indicated constructs to coated TfRl.

[0256] Figure 10 are depictions of the x-ray structure of the complex of h26D3-Fab and hTfRl, determined as described in Example 5. The chain names as used in the coordinate files are indicated. (A) Refined structure showing overall folds of three independent complexes in the asymmetric unit. (B) Example of electron density (2m|Fo|-D|Fc|) contoured at the 1 G level. The protein chains are drawn in cartoon representation while sugar moieties are shown in stick representation.

[0257] Figure 11 is a ribbon representation of the h26D3-Fab human TfRl complex determined with x-ray crystallography as described in Example 5. h26D3-Fab is depicted in dark gray and hTfRl in white. The binding interface (epitope / paratope) is encircled.

[0258] Figure 12 is a surface area representation of hTfRl with the binding sites for the natural ligands ferritin and transferrin indicated, as well as the epitope for the binder 26D3 of the present disclosure. The different binding sites and epitope are depicted with a circle around each specific site.

[0259] Figure 13 illustrates the work on generating and characterizing an hTfRl-KI mouse model as described in Example 6. (A) Schematic illustration of the transgenic hTfRl-KI mouse construct. The extracellular domain of human TFRC was inserted in the murine Tfrc gene by homologous recombination. (B) Quantitative reverse transcription PCR (RT-qPCR) analysis of mouse Tfrc and human TFRC gene expression in brain (N=3 / genotype). hTfRl-KI mice (grey circles) express human TFRC and mouse Tfrc in total brain homogenate, WT littermates only express mouse Tfrc (white). (C) Western blot analysis for hTfRl, total TfRl, and |3 -actin control in brain. hTfRl-KI animals at 6-8 months (N=5) and 15 months (N=4) express comparable levels of hTfRl protein. Total TfRl levels are comparable between hTfRl-KI transgenic and WT littermates (N=3).

[0260] Figure 14 shows the results of in vivo brain and plasma exposure analysis of various indicated hTfRl-binding molecules in hTfRl-KI transgenic mice as described in Example 7. (A) Brain exposure 24 h after i.v. administration of the indicated hTfRl binders. (B) Plasma exposure 24 h after i.v. administration of the indicated hTfRl binders. (C) Brai Plasma ratio 24 h after i.v. administration of the indicated hTfRl binders. The negative control is denoted "158", and the positive control "15G11-1".

[0261] Figure 15 shows the results of in vivo brain exposure analysis of various indicated hTfRl-binding molecules in hTfRl-KI mice by immunohistochemistry as described in Example 8. Cortical brain capillary staining observed for several binding molecules, including h26D3. Reference hTfRl-binder "15G11-1" and non-TfRl binder "158" were used as positive and negative control, respectively.

[0262] Figure 16 shows BLI sensorgrams for the indicated alanine variants of h26D3 as described in Example 9. Each variant showed a different kinetic profile, illustrating the possibility to generate variants with different affinities against human TfRl with specific mutations in the CDR regions of the heavy or light chain.

[0263] Figure 17 shows representative SPR sensorgrams of the interaction between the indicated alanine variants of h26D3 with hTfRl and cTfRl, measured as described in Example 9.

[0264] Figure 18 shows the results of indirect ELISA analysis of the binding of the indicated alanine variants of h26D3 with hTfRl and cTfRl, measured as described in Example 9.

[0265] Figure 19 shows SPR sensorgrams of the interaction between the indicated alanine variants of h26D3, studied as scFv building blocks within a bispecific protein format as described in Example 9.

[0266] Figure 20 shows chromatograms from preparative SEC of (A) h26D3-HC6_DS and (B) h26D3-HC6, carried out as described in Example 10.

[0267] Figure 21 shows chromatograms from analytical SEC of the indicated scFv proteins after formulation and short-term storage at -80 °C, as described in Example 11.

[0268] Figure 22 shows chromatograms from analytical SEC analysis of the indicated scFv proteins kept at -80 °C (TO) and then at 40 °C for 1, 2 and 4 weeks as indicated, carried out as described in Example 12.

[0269] Figure 23 is a series of bar diagrams showing the percentage of monomeric scFv, as measured by analytical SEC, in samples of the respective indicated scFv molecule subjected to the thermal stability evaluation described in Example 12.

[0270] Figure 24 shows chromatograms from analytical SEC analysis of the indicated scFv proteins kept at -80 °C (TO) and then at 40 °C for 1, 2 and 4 weeks as indicated, carried out as described in Example 12. The asterisk (*) in Figure 24C highlights a shift in retention time for h26D3-LCl_DS which occurred due to drift in the chromatography equipment. The shift was also seen for a standard size control (not shown) injected on the same column, and is unrelated to the analyzed sample. Figure 25 demonstrates the results of the ELISA experiment described in Example 13, showing (A) hTfRl binding of a stabilized binding molecule of the disclosure after 48 h incubation in mouse serum at 37 °C and 4 °C in three separate experiments, (B) binding curves obtained from the binding molecule incubated in serum, in comparison to incubation in PBS, and (C) the ratio of binding activity at 37 °C to the binding activity at 4 °C in serum or PBS as indicated.

[0271] Figure 26 shows representative SPR sensorgrams of the interaction between the indicated variants of h26D3 with hTfRl, measured as described in Example 15.

[0272] Figure 27 shows the binding interaction for the bispecific binding molecule BA101 against human TfR-1, measured by SPR as described in Example 17. Black line = fit, Grey line = experimental data, y-axis = response units, x-axis = time in seconds.

[0273] Figure 28 shows the binding interactions for binding molecules BA101 (A) and BA102 (B) against A 1-42 protofibrils, measured by SPR as described in Example 17. Black line = fit. Grey line = experimental data, y-axis = response units, x-axis = time in seconds.

[0274] Figure 29 shows the binding interactions for binding molecules BA101 (A) and BA102 (B) against Api-28 monomers, measured by SPR as described in Example 17. Black line = fit. Grey line = experimental data, y-axis = response units, x-axis = time in seconds.

[0275] Figure 30 shows the binding and selectivity for A|3 protofibrils compared to AP monomers for the indicated binding molecules using inhibition ELISA, as described in Example 17. Binding inhibition-response curves by A 1-42 protofibrils in solution are shown by open circles and open triangles, and inhibition-response curves by A 1-28 monomers in solution are shown by closed circles and closed triangles.

[0276] Figure 31 shows the binding of binding molecules BA101 (A) and BA102 (B) to AP plaques in AD brain sections, as described in Example 18.

[0277] Figure 32 is a diagram showing the immunoprecipitation of target A 42 in a TBS soluble AD brain extract pool by the indicated binding molecules, as described in Example 18. Bead Control (-) values were below LLOQ.

[0278] Figure 33 shows the concentration-response effects of binding molecules BA101 and BA102 on uptake of HiLyte Flour 488-labelled Api-42 protofibrils in THP- 1 cells, as described in Example 19. Figure 34 shows the concentration-response of the indicated binding molecules on A|3 plaque clearance in AD brain sections, as described in Example 19.

[0279] Figure 35 shows a diagram of the antibody-dependent cellular cytotoxicity assay with the indicated binding molecules, as described in Example 20.

[0280] Figure 36 shows a diagram of the complement-dependent cytotoxicity assay with the indicated binding molecules, as described in Example 20.

[0281] Figure 37 shows brain and plasma exposure 48 h after intravenous administration of binding molecules BA101 and BA102 in Tg-ArcSwe x hTfRl-KI mice, as described in Example 21. Brain exposure (A), Plasma exposure (B), Brai Plasma ratio (C).

[0282] Figure 38 shows cortex lOx images of sagittal brain sections from Tg-ArcSwe x hTfRl-KI mice 48 h after intravenous injection with binding molecules BA101 (A) or BA102 (B) as described in Example 22. Total A|3 and hlgG representative images.

[0283] Figure 39 shows a diagram of BA101 competition with transferrin in K562 cells, as described in Example 23.

[0284] Figure 40 shows example sensorgrams for binding of BA101 (A) and BA102 (B) to FcyRI, measured by SPR as described in Example 24. Black line = fit. Grey line = experimental data, y-axis = response units, x-axis = time in seconds.

[0285] Figure 41 shows the mean binding interactions for BA101 (black) and BA102 (white) to hFcRn, measured by the Lumit® FcRn Binding Immunoassay kit as described in Example 25. y-axis = the % relative light unit, x-axis = concentration of binding molecule (nM). Error bars represent standard deviation.

[0286] Figure 42 shows example sensorgrams of BA101 serum stability samples binding to A 1-42 protofibrils (A) and hTfRl (B) measured by SPR as described in Example 26. y-axis = normalized response units, x-axis = time in seconds.

[0287] Figure 43 shows mean dose-normalized plasma concentration-time profiles in scid hFcRn mice after intravenous administration of BA101 at doses of 5.7, 17, and 57 nmol / kg (A) and of BA102 at a dose of 57 nmol / kg (B) as described in Example 27. Error bars represent standard deviation.

[0288] Figure 44 shows mean concentration-time profiles in plasma and brain homogenates following intravenous administration of 57 nmol / kg BA101 in hTfRl-KI and 5xFAD x hTfRl-KI littermate female mice as described in Example 28. Error bars represent standard deviation. Brain homogenate concentrations at 504 h in hTfRl-KI mice were below LLOQ and hence not reported.

[0289] Figure 45 shows plasma and brain exposure up to 168 h after intravenous administration of 57 nmol / kg BA101 or BA102 in female 5xFAD x hTfRl-KI mice, as described in Example 28. (A) Mean plasma concentration-time profile; (B) Brain concentration; (C) Brai Plasma ratio. Error bars represent standard deviation, and in (B) and (C) bars indicate mean, and circles represent individual values.

[0290] Figure 46 shows mean quantitative amyloid-p target engagement in cortex (A-B) and hippocampus (C-D) after intravenous administration of 57 nmol / kg BA101 or BA102 in female 5xFAD x hTfRl-KI mice, as described in Example 28. Colocalization of test substances (IgG) on amyloid-p plaques (LOC) is reported as immunoreactive area (A, C) and object density (B, D). Data presented as mean ± standard deviation, and circles represent individual values.

[0291] Figure 47 is a diagram showing the brain concentrations of amyloid-p protofibrils 7 days after intravenous administration of 57 nmol / kg BA101 in 5xFAD x hTfRl-KI mice or BA102 in 5xFAD littermates compared to PBS-treated controls as described in Example 29. Concentrations were determined in TBS 16k brain extracts. Data presented as mean ± standard deviation, circles represent individual values, (x) denotes the hTfRl-KI genotype, ** represents an adjusted p-value < 0.01 and ns = not significant.

[0292] Figure 48 shows reticulocyte subfractions from hTfRl-KI mice 24 h after single intravenous administration of BA101, BA102 or PBS as described in Example 30. % change denotes the 24 h post-dose sample alteration compared to the individual pre-dose sample for (A) Low fluorescence reticulocytes (LFR); (B) Medium fluorescence reticulocytes (MFR); (C) High fluorescence reticulocytes (HFR). Data presented as mean ± standard deviation, and circles represent individual values. The relative levels of TfRl expression and reticulocyte maturation are indicated in the gray bars under the graphs.

[0293] Figure 49 shows (A) mean reticulocyte counts over time after single intravenous administrations of BA101 at doses of 3 or 10 mg / kg, or BA102 at a dose of 20 mg / kg in non-human primates, with pre-samples collected on Day 1 just before dosing, and post-dose samples collected at 24 h (Day 2), 72 h (Day 4), and 240 h (Day 11); and (B) median post-dose reticulocyte counts normalized against pre-dose reticulocyte counts, as described in Example 31. (A) Error bars represent standard deviation. (B) Circles represent individual values.

[0294] Figure 50 shows mean dose-normalized plasma concentration-time profiles from serial sampling up to 240 h in non-human primates after single intravenous administration of BA101 at doses of 3 and 10 mg / kg, or of BA102 at a dose of 20 mg / kg as described in Example 31. Error bars represent standard deviation.

[0295] Figure 51 shows mean dose-normalized brain concentration-time profiles from sparse, composite sampling of parietal cortex (A), hippocampus (B), and striatum (C) up to 240 h in non-human primates after single intravenous administration of BA101 at a dose of 10 mg / kg (grey circles) or BA102 at a dose of 20 mg / kg (white circles) as described in Example 31. Error bars represent standard deviation.

[0296] Figure 52 shows stacked chromatograms from analytical SEC of the indicated scFv samples collected as TO or after temperature hold at 40 °C for 1, 2 or 4 weeks respectively, as described in Example 33. The relative absorbance mAU (220 nm) is shown on the X-axis of respective graph (A-F).

[0297] Figure 53 shows representative SPR sensorgrams of the interaction between the indicated scFv variants and hTfRl, as described in Example 35.

[0298] Figure 54 shows representative SPR sensorgrams of the interaction between the indicated scFv variants and cTfRl, as described in Example 35.

[0299] Figure 55 shows the results from screening and competition ELISA with serum from 107 donors against h26D3-HC6 scFv variants, as described in Example 36: (A) PE-ADA screening ELISA with 21 serum samples against TfRl binding scFv variants, as indicated; (B) Screening ELISA with serum from 107 donors against six selected variants show a reduced response frequency for all six variants compared to "h26D3-HC6_DS, VL-first"; (C) ELISA with 107 serum samples against h26D3- HC6_DS, VL-first, with competition (HC6 competition) och without competition (HC6 screen) (dotted line represents the assay cut-off point in the competition setting); (D) Among the serum samples from 107 donors, a majority of the samples render a response level above the assay cut-off point (horisontal line) in the ELISA setting without competition.

[0300] Figure 56 shows brain exposure after repeated intravenous administrations of indicated doses of BA101 or BA102 in female 5xFAD x hTfRl-KI mice, as described in Example 38. Boxes represent the interquartile range with the median as a straight line, the whiskers indicate min and max, and filled circles represent individual values.

[0301] Figure 57 shows levels of amyloid-p protofibrils in TBS 16k brain extracts after repeated intravenous administrations of indicated doses of BA101 or BA102 in female 5xFAD x hTfRl-KI mice, as described in Example 38. Data is presented as mean ± standard deviation, circles represent individual values, the dotted line refers to mean in baseline group, and % decrease refers to mean decrease in indicated group compared to mean of PBS-treated group. Adjusted p-values < 0.05, < 0.01, < 0.001, < 0.0001 represented by one, two, three or four symbols, respectively, where (#) indicates significant difference compared to PBS-treated group and (*) refers to difference between indicated groups.

[0302] Figure 58 shows cortical diffuse amyloid-p plaques in female 5xFAD x hTfRl- KI mice after repeated intravenous administrations of indicated doses of BA101 or BA102, as described in Example 38. Data presented as mean ± standard deviation, circles represent individual values, and the dotted line refers to mean in baseline group for (A) immunoreactive area; (B) object density; (C) mean object size. Adjusted p-values < 0.05, < 0.01, < 0.001, < 0.0001 represented by one, two, three or four symbols, respectively, where (#) indicates significant difference compared to PBS-treated group and (*) refers to difference between indicated groups.

[0303] Examples

[0304] While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention is not limited to any particular embodiment, but that the invention will include all embodiments falling within the scope of the appended claims.

[0305] The invention will be further illustrated by the following non-limiting Examples. They are offered for illustrative purposes only and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperatures, etc.), but some experimental errors and deviations may be present. Unless otherwise indicated, the practice of the invention employs conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the existing literature. Additionally, it will be apparent to one of skill in the art that the methods for protein engineering applied herein can also be applied to other constructs described herein and contemplated by the present inventors to fall within the scope of the disclosure.

[0306] EXAMPLE 1

[0307] Identification of binders of human TfRl by immunization and screening Immunization and hybridoma screening

[0308] To identify monoclonal antibodies that bind human transferrin receptor 1 (hTfRl), four 6-10 weeks old Balb / c or C57BL / 6 mice were immunized subcutaneously with immunogen together with adjuvant. The hTfRl immunogen was designed to contain the ectodomain of the human TfRl protein, N-terminally fused to a T-cell epitope from tetanus toxin, P2 (Kovacs-Nolan and Mine (2006), Biochim Biophys Acta 1760:1884-1893) via a GSS linker, and an N-terminal 10x histidine tag (Hisio-P2-hTfRl; SEQ ID NO:84). Following gene construction, recombinant Hisio-P2-hTfRl protein was generated by transient transfection in Hek293 cells using the Expi293™ Expression system (Gibco), purified on a nickel column (HisTrap FF, cat. no. 17-5255-01, GE Healthcare), buffer exchanged to PBS and concentrated to 1 mg / ml. Expressed TfRl immunogen was aliquoted and stored at -80°C until use. Quil-A adjuvant (vac-quil, InvivoGen) was used for all immunizations except for the final booster injection in which no adjuvant was included. For use, Quil-A was resuspended in ddH2O at a concentration of 1 mg / ml, sterile filtered and aliquoted in 0.1 ml aliquots stored at -80°C. Quil-A was administered at a dose of 10 pg / mouse.

[0309] Animals were immunized every month with the recombinantly produced immunogen, Hisw-P2-hTfRl, mixed and co-administered with Quil-A. Three weeks after each immunization, blood samples were collected, and the plasma was analyzed for presence of antibodies reactive towards recombinantly produced human TfRl and mouse TfRl. Titers were considered high enough when the ELISA response at 1 / 100,000 dilution exceeded the average of the blanks (i.e. background) plus 3 standard deviations of the blanks. The four mice used in this study received between 4 and 6 immunizations each.

[0310] Three days before fusion, the final intraperitoneal booster injection was given to the mice in absence of adjuvant. At sacrifice, mice were anesthetized with isoflurane. Intact spleens were collected by opening the abdominal cavity and dissected. Briefly, a single cell suspension of the spleen from an immunized mouse was prepared and mixed with Sp2 / 0 cells at a 3:1 ratio. The cells were fused using PEG and the cells were added to a bottle of ClonaCell™-HY Medium D (STEMCELL Technologies). 60-70 pl per well was then dispensed into 96-well plates. After 6-7 days, 150 pl HAT-medium was added to each well in the semi solid 96-well plates. The day after, 120 pl of supernatant was discarded from each well and 100 pl fresh HAT-medium was added. The next day, 100 pl of the supernatant of each well was taken and transferred to a storage plate and tested for presence of antibodies against mouse TfRl using indirect ELISA on nickel-coated plates according to the protocol below. A repeated screen of the hybridoma plates was performed by adding 120 pl HAT-medium on day 12 and by 3 days later transferring 25 pl supernatant to ELISA plates to screen for reactivity against mouse TfRl (both screens referred to as "primary screen"). Clones that were positive towards mouse TfRl with OD>0.2 were transferred to 24-well plates, cultured for at least 3 days, and subjected to a secondary screen for reactivity towards murine, human and cynomolgus TfRl in solution using biolayer interferometry (BLI) (referred to as "secondary screen"). Whereas binding of both hTfRl and cynomolgus TfRl was indicated, only very weak or no binding was detected for mTfRl in the secondary screen. Supernatants from 24-well plates were also screened for binding towards His-tagged hTfRl as well as lack of binding towards His-tagged amyloid-p precursor protein (APP; negative control) using both direct coated TfRl plates and nickel- coated plates as described below. Binding towards cynomolgus TfRl (cTfRl) was also analyzed using direct TfRl coat. Notably, ELISA responses (OD450 values) were very low for mTfRl compared to hTfRl and cTfRl, indicating weaker binding to mTfRl compared to the binding to hTfRl and cTfRl for all positive clones. Selected clones were diluted using limiting dilution assays (LDA) to reach monoclonality. Reactivity against mouse TfRl and human TfRl were re-tested by ELISA on monoclonal cultures following LDA and expansion.

[0311] Indirect ELISA screening

[0312] ELISA assays were performed according to standard ELISA protocols in order to screen plasma samples for reactivity towards the target antigens after immunizations, or to identify hybridoma clones producing antibodies with reactivity against the TfRl target protein. Briefly, 96-well half area plates (Corning) were coated with 1 pg / ml Hisio-mTfRl (SEQ. ID NO:85) or Hisw-hTfRl (SEQ ID NO:86). Hisio-mTfRl and Hisw-hTfRl were recombinantly produced and purified using the procedure described above for the Hisio-P2-hTfRl immunogen. The plates were blocked with 150 pl / well of protein free blocking solution (Pierce) for 1 h at room temperature 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 a starting dilution of 1 / 450 or hybridoma supernatants diluted 1 / 2 were added to the plates (50 pl / well; dilution buffer: PBS with 0.1 % BSA and 0.05 % TWEEN®-20) and incubated for 2 h at room temperature and then the plates were washed four times. Detection antibody (HRP-conjugated anti-mouse IgG, Southern Biotech, cat. no. 1030-05, diluted 1 / 5000 in dilution buffer) was added at 50 pl / well, and the plates were incubated for 1 h at room temperature. After another wash (as above), 50 pl / well TMB substrate (K-Blue® Aqueous, Neogen) was added, and the reaction was stopped after 10-15 min with 50 pl / well of 0.5 M H2SO4. The optical density at 450 nm was read using a plate reader (Tecan). The endpoint titers were defined as the dilution above the average of the blank wells (background) plus 3 standard deviations of the blank wells.

[0313] The primary screen of hybridoma clones producing antibodies with reactivity against the target protein was performed using nickel-coated ELISA plates. Briefly, 96-well Ni-coated plates (PIERCE) supplied pre-blocked with BSA were incubated with 3 pg / ml (100 pl) Hisio-mTfRl without shake overnight at 4°C. The plates were washed four times with PBS containing 0.1 % TWEEN®-20 and Kathon™. Hybridoma supernatants diluted 1 / 4 were added to the plates (dilution buffer: PBS with 0.1 % BSA and 0.05 % TWEEN®-20) and incubated for 2 h at room temperature and then the plates were washed four times. Detection antibody (HRP-conjugated anti-mouse IgG, Southern Biotech, cat. no. 1030-05, diluted 1 / 5000 in dilution buffer) was added at 100 pl / well, and the plates were incubated for 1 h at room temperature. After another wash (as above), 100 pl / well of K-Blue® Aqueous substrate (Neogen) was added, and the reaction was stopped after 10-15 min with 100 pl / well of 0.5 M H2SO4. The optical density at 450 nm was read using an ELISA plate reader (Tecan).

[0314] Examples of clones considered to be positive in binding mouse TfRl and human TfRl are shown in Table 1. These clones were also confirmed to bind both His-tagged hTfR and cTfR by ELISA, and to lack binding to His-tagged APP (negative control). Selected clones were further characterized in various assays.

[0315] Table 1: Examples of identified clones from hybridoma screening

[0316] Biolayer interferometry measurements

[0317] Selected clones were investigated using biolayer interferometry (BLI) on an Octet instrument (Octet Red384, ForteBio). In the setup used, the adopted method involves capture of IgG from the respective clone on the individual sensor tips to allow for detection of antibodies that bind to target in solution. In addition to providing a measure of binding, BLI measurements provide more details about the overall binding properties, because they include estimates of the on-rate and off- rate.

[0318] Figure 1 shows the results of BLI measurements for three selected clones provided as examples, with binding measured directly in the crude hybridoma supernatant. Briefly, mouse IgG antibody clones in hybridoma supernatants, diluted 1:1 in running buffer (PBS, 0.02% TWEEN®-20 and 0.01% BSA), were captured on anti-mouse capture biosensors (anti-mouse capture, AMC, Molecular devices, Cat. 18-5580). Next, sensors with immobilized IgGs were briefly washed for 10 s before incubation in running buffer to establish a baseline signal. Association to target antigens were measured by incubating sensors for 120 s in wells of the assay plate containing the following concentrations of respective target antigen: 500 nM mTfRl, 250 nM hTfRl and 250 nM cTfRl. All proteins were diluted in running buffer. Target dissociation was measured by incubating the biosensors in running buffer for 90 s. All tested clones, i.e. 24B4, 26D3 and 37D10, bind to both human and cynomolgus TfRl but very weakly to mouse TfRl. Overall, most clones showed more crossreactivity towards human and cynomolgus TfRl than against mouse TfRl.

[0319] Sequencing of selected clones Clones of interest were cryopreserved and sequenced by whole transcriptome shotgun sequencing. Among the sequenced hybridoma clones were clones denoted 26D3, 24B4 and 37D10. The amino acid sequences obtained for the respective heavy chain variable (VH) and light chain variable (VL) domains of these antibodies are given in Table 2 below:

[0320] Table 2: Variable domain amino acid sequences for selected primary antibodies

[0321] The complementarity determining regions (CDRs) of these antibodies were identified using the Kabat definition, and are given in Table 3 below.

[0322] Table 3: CDR sequences of primary antibodies The VH and VL domains of the identified antibodies are mutated to introduce cysteine residues for the provision of a disulfide bridge between the VH and VL domains. The resulting sequences, variously denoted "disulfide stabilized variants", "DS stabilized variants", "DS versions" or similar herein, are given in Table 4.

[0323] Table 4: Disulfide stabilized variants of primary antibodies

[0324] EXAMPLE 2

[0325] In vitro binding to human and cynomolgus TfRl and epitope screen A more detailed binding analysis by BLI was performed on purified, selected antibodies. Binding of Fab fragments from the murine antibodies 26D3, 24B4 and 37D10 to human TfRl and cynomolgus TfRl was investigated. For example, the BLI instrument Octet Red384 was used to measure binding between immobilized TfRl and the tested Fab fragments in solution. Antibody binding to TfRl was measured with TfRl complexed to the human transferrin ligand (Tf). Tf / TfRl-complexes were formed on streptavidin biosensors by first loading sensors with biotinylated human holo-transferrin followed by a complex-formation step by capturing either hTfRl or cTfRl on the sensors. Final complex density on the sensors was similar for both hTfRl and cTfRl. Antibody binding to TfRl was measured during an association phase of 120 s and a dissociation phase of 300 s. Figure 2 shows sensorgrams for 15 nM of each of 24B4-Fab, 26D3-Fab and 37D10-Fab, as well as for a Fab derived from the known TfRl-binding antibody 8D3 (Boado et al (2009), Biotechnol Bioeng 102:1251-1258). The data indicate a similar binding profile against human and cTfRl for both 24B4-Fab and 26D3-Fab, and cross-reactive binding to both species is also detected for 37D10-Fab, while no significant binding of 8D3-Fab against human or cynomolgus TfRl was detected. Importantly, the experiment shows that 24B4-Fab, 26D3-Fab and 37D10-Fab all bind to TfRl when the natural ligand transferrin is in complex with TfRl.

[0326] Next, an ELISA experiment showed that antibodies 26D3, 24B4 and 37D10 bind to the protease-like domain of TfRl. In the ELISA experiment, human, mouse or three different chimeric TfRl receptors were used to coat ELISA plates (Figure 3). The ELISA protocol was slightly modified as follows from the indirect ELISA described in Example 1. Briefly, ELISA plates were coated with the following His-tagged antigens at 1 pg / ml: ectodomain of human TfRl (HislO-hTfRl; SEQ ID NO:87), ectodomain of mouse TfRl (HislO-mTfRl; SEQ ID NO:88), chimeric TfRl consisting of human apical domain grafted on mouse TfRl ectodomain (h / m apical domain chimera, mhHD_TFRl; SEQ ID NO:89), chimeric TfRl consisting of human helical domain grafted on mouse TfRl ectodomain (h / m helical domain chimera, mhHD_TfRl; SEQ ID NQ:90) or chimeric TfRl consisting of human protease like domain grafted on mouse TfRl ectodomain (h / m protease-like domain chimera, mhPLD_TfRl; SEQ ID NO:91). The coated plates were then blocked. Dilution series of mouse IgG of the analyzed antibodies were prepared in PBS and incubated on the ELISA plates. Unbound antibodies were then washed off before incubating wells with a HRP-conjugated secondary, anti-mouse-IgG for 1 h. Plates were then washed again before addition of HRP substrate TMB for development and detection of antibody binding to the wells. TMB development was stopped by adding 0.5 M H2SO4 to the wells and ELISA responses measured as the OD at 450 nm in an ELISA plate reader. As illustrated in Figure 3, 26D3, 24B4 and 37D10 only bind hTfRl (A) and not mTfRl (B). There is no binding of 26D3, 24B4 or 37D10 to the construct with the human apical domain grafted onto the rest of the mTfRl ectodomain (C). The control antibody 15G11-1 (Yu et al (2014), Sci Transl Med 6:261ral54) known to bind to the human apical domain shows binding to the h / m apical domain chimera as expected (C). In addition, 26D3, 24B4 and 37D10 bind to the h / m protease-like domain chimera (D), but not to any of the plates coated with the other chimeric receptors (C and E). Further, the control antibody 8D3, with an epitope in the apical domain of mTfRl, binds to all plates coated with TfRl antigens including this domain, namely mTfRl (B), h / m protease-like domain chimera (D) and h / m helical domain chimera (E), In summary, the experiment demonstrates that the epitope or epitopes for 26D3, 24B4 and 37D10 lie(s) predominantly within the protease-like domain of hTfRl, and that this is in contrast to the control antibodies 15G11-1 and 8D3.

[0327] In a further BLI experiment carried out for the purpose of epitope binning (binding competition), it was then shown that binding by both 26D3 and 24B4 is targeted to the same or overlapping regions of hTfRl, with an epitope located outside the apical domain (Figure 4). The epitope binning experiment by BLI was conducted on an Octet Red384 instrument (ForteBio) by first (Step 1) immobilizing biotinylated hTfRl to streptavidin biosensors (High precision biosensors, ForteBio). Next (Step 2), a washing step was carried out. Then (Step 3), hTfRl loaded sensors were incubated in either buffer (non-competitive reference) or 200 nM of the respective antibody (Ab) to form hTfRl:Ab complexes on the sensors. Finally (Step 4), sensors with free hTfRl (reference) or respective preformed hTfRl:Ab complex was incubated in 200 nM of respective antibody to measure binding to hTfRl in complex with the competing antibody. Figure 4 shows representative BLI sensorgrams obtained during the indicated main assay steps. The signal in Step 4 is indicative of the degree of competition between the two analyzed antibodies. If the antibodies compete for binding to the same or overlapping epitope, there is no increase in the signal of the sensorgram in Step 4. Conversely, if the two tested antibodies bind to distinct and different epitopes, there will be an increased signal from Step 4.

[0328] The results of competitive screening of antibody binding to epitopes on hTfRl by epitope binning as described above is illustrated in Figure 5. Antibodies 26D3 (dark grey bars) and 24B4 (light grey bars) were shown to bind to an overlapping epitope, which is distinct from the hTfRl apical domain epitope of control antibody 15G11-1 (black bars). Figure 5A shows that the binding response for 26D3 is reduced by over 70% when hTfRl is in complex with 24B4. As expected, binding of 26D3 to pre-formed hTfRl:26D3-complex is nearly fully inhibited, illustrating that it blocks itself. Similarly, Figure 5B shows that the binding response for 24B4 is 70% lower when hTfRl is in complex with 26D3 and nearly fully inhibited by itself. Both 24B4 and 26D3 retain the full binding response to hTfRl when hTfRl is in complex with the control antibody 15G11-1, which has its binding epitope within the apical domain of hTfRl (Figures 5A and 5B, black bars). As shown in Figure 5C, the control antibody 15G11-1 has similar binding responses to the apical domain of hTfRl, regardless of whether it is tested against hTfRl without competition antibody or when the receptor is in complex with 24B4 or 26D3. In Figure 5, all responses were normalized to the respective antibody's maximal binding response to free hTfRl.

[0329] Furthermore, antibody binding to endogenous hTfRl on brain endothelial cells was studied. Binding to endogenous hTfRl on cell surfaces was monitored using flow cytometry and human hCMEC / D3 cells (Weksler et al (2013), Fluids Barriers CNS 10:16), which are known to express significant levels of hTfRl on their surface. Cells that stained positively were plotted and the mean fluorescence intensity (MFI) is shown in Figure 6. Both Figure 6A (IgGl antibodies) and 6B (Fab fragments) show that cells were positively stained for hTfRl with 24B4 and 26D3 to a similar degree (MFI) compared to the positive control antibody 15G11-1 having a high hTfRl affinity and to a higher degree than the low affinity control antibody 15G11-2 (Yu et al (2014), supra). No background staining was detected with the negative isotype control (Figure 6A) or the non-related Fab fragment Lyl28 (Figure 6B). This data illustrates that both 24B4 and 26D3 bind to hTfRl expressed on a cell surface.

[0330] EXAMPLE 3

[0331] Competition for hTfRl-binding with ferritin and transferrin The unique binding to hTfRl of the binders according to the disclosure, binding to the protease-like domain of hTfRl and identified as described in Example 1, was evaluated for competition with natural TfRl ligands ferritin (Ft) and transferrin (Tf). In order to test ferritin competition with antibody, the human monocytic cell line THP-1 (Sigma / ECACC) was used. Binding of the scFv-Fc format (see Example 4 below) and control antibody (M-A712) to hTfRl on the THP-1 cell surface was confirmed, as shown in Figure 7A. For evaluating the competition between ferritin and the disclosed binders, cells were incubated with serially diluted test binders along with ferritin from human liver (BioRad, 4420-4804) for 1 h at 4 °C. After incubation, ferritin that had bound to hTfRl on the cell surface was captured using a primary sheep antibody against human liver ferritin (BioRad, AHP2179G) and analyzed using flow cytometry. The results are displayed in Figure 7B, and show that the 26D3 scFv-Fc does not compete with ferritin on the cell surface, whereas the control antibody anti-CD71, clone M-A712, known to bind to the same epitope on hTfRl as Ft (Maier et al (2016), Mol Ther Nucleic Acids 5:e321) clearly competes with Ft binding. Also for the identified 26D3 hTfRl binder, the impact on Ft binding is much less, illustrating that 26D3 has a different epitope on hTfRl than the binding site for Ft (Figure 7B).

[0332] For transferrin competition, hTfRl-expressing K562 lymphoblast cells (Sigma / ECACC) were used. Cells were incubated with serially diluted test constructs along with Alexa Fluor 488 conjugated, human holo-transferrin (Thermo Fisher; T13342) and incubated for 1 h at 4 °C. Transferrin bound to hTfRl on cell surfaces was captured using flow cytometry, and the mean fluorescence intensity was plotted. Figure 7C shows that there is no competition between the 26D3 binder and transferrin. When non-labeled (unconjugated) Tf was used as positive control for competition, the binding of labeled (AF488) Tf signal was reduced in a concentration dependent way. The experiment illustrates that a binder directed against the protease-like domain of TfRl does not compete directly for the same epitope as transferrin.

[0333] Overall, this example shows that binding of 26D3 to hTfRl does not negatively affect the ability of the two endogenous ligands ferritin and transferrin to bind to the receptor.

[0334] EXAMPLE 4

[0335] Humanization of hTfRl binder 26D3

[0336] The Fab sequence of mouse antibody 26D3, identified and characterized as described in Examples 1-3, was analyzed and an in silica model of the 26D3 Fab 3D structure was generated using Bioluminate Software (Schrodinger). This murine Fab model was used as input for humanization. In this process, the CDRs of the VH and VL domains of 26D3 (see Table 3; SEQ ID NO:23-28) were grafted in silica into various human variable domains and some residues were back mutated to murine framework at some positions. Three variants having the fewest back mutations and otherwise desirable characteristics were generated and extracted from the software. One such humanized variant was selected for expression and denoted h26D3. h26D3 has the VH domain sequence defined in SEQ ID NO:57 and the VL domain sequence defined in SEQ ID NO:71. A DS version of h26D3 has the VH and VL amino acid sequences SEQ ID NQ:101 and 118, respectively.

[0337] The humanized version h26D3 and the murine original sequence 26D3 were both expressed as His-tagged Fabs by transient transfection of Chinese Hamster Ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. The harvested supernatant was purified using HiTrap IMAC Sepharose FF (Cytiva) followed by a size exclusion chromatography on HiLoad Superdex 200pg 26 / 600 (Cytiva). The following buffers were used: Ni-NTA wash buffer: 20 mM Tris pH 8.0, 10 mM imidazole and 200 mM NaCI; Ni-NTA elution buffer: 20 mM Tris pH 8.0, 200 mM NaCI and 500 mM imidazole; size-exclusion buffer (SEC): lxdPBS (Thermo Fisher).

[0338] Binding of the purified Fabs to human and cynomolgus TfRl was evaluated using surface plasmon resonance (SPR) on a Biacore 8K instrument (Cytiva) and the results are shown in Figure 8. 1 pg / ml of human TfRl (truncated hTfRl of SEQ ID NO:99) or cynomolgus TfRl (truncated cTfRl of SEQ ID NQ:100) was immobilized on a Cm5 sensor chip (Cytiva, WBR100399) using the amine coupling kit type 2 (Cytiva, WBR100633) according to the manufacturer's instruction. The h26D3 and 26D3 Fabs were injected over the chip using a 2-fold dilution series in five steps starting at 25 nM. The interaction was measured using the single cycle kinetics method with a contact time of 120 s at a flow rate of 30 pl / ml followed by a dissociation time of 600 s. Regeneration of the surface between cycles was done by injecting 3M MgCL The binding data were fitted to a 1:1 interaction model. The Fabs were diluted in HBS-EP+ (Cytiva, WBR100669). Experiments were performed at 25°C. The data confirm that the humanized variant of 26D3, i.e. h26D3, retained binding capacity for human and cynomolgus TfRl (Figure 8). The kinetic parameters obtained in the experiment are given in Table 5 below. Table 5: SPR analysis of murine and humanized 26D3 Fobs vs. hTfRl and cTfRl

[0339] Both murine 26D3 and the humanized variant h26D3 were converted to the scFv format and confirmed to have maintained target binding as scFv (Figure 9). Murine and humanized 26D3 were reformatted to scFv (SEQ ID NO:92 and SEQ ID NO:93 respectively) and produced as monovalent Fc-fused scFv antibody fragments by employing the knob-into-hole (Ki H) technology. In this format, one scFv fragment is fused only to the knob half of the Fc (SEQ ID NO:94), while the hole half of Fc (SEQ ID NO:95) is left unfused. The resulting antibody format is a one-armed scFv-Fc. The 26D3 scFv fused to the knob half of the Fc has the complete amino acid sequence SEQ ID NO:96, whereas the h26D3 scFv fused to the knob half of the Fc has the complete amino acid sequence SEQ ID NO:97. The binding profiles for murine and humanized 26D3 in this scFv format are similar and confirm binding activity in the scFv format. Binding responses agree with those of the antibody in Fab format. This was confirmed by several methods, including a kinetic experiment using BLI (results shown in Figure 9A) and an ELISA (results shown in Figure 9B). Binding kinetics for murine and humanized 26D3-scFv-Fc were measured by BLI by first immobilizing biotinylated hTfRl to streptavidin biosensors (Fortebio). Sensors were then washed in buffer (Kinetics buffer, Fortebio) before measuring association of 26D3-scFv-Fc (murine) and h26D3-scFv-Fc (humanized) at 25 nM concentrations followed by a 500 s dissociation phase. In the ELISA experiment, hTfRl was used to coat the plates for standard binding ELISA experiments using the protocol for indirect ELISA described in Example 1.

[0340] EXAMPLE 5 Crystallization and structure determination of h26D3-Fab in complex with hTfRl This example describes crystallization of a complex between h26D3-Fab and hTfRl and determination of the binding interface. Ectodomain of human TfRl (SEQ ID NO:87) was expressed by transient transfection of human embryonic kidney cells (Expi297; Thermo Fisher Scientific) according to the manufacturer's instructions. The harvested supernatant was purified using HiTrap IMAC Sepharose FF (Cytiva) followed by size exclusion chromatography on HiLoad Superdex 200pg 26 / 600 (Cytiva). The buffers used and purification of the humanized Fab were as described in Example 4.

[0341] The formation of a complex between humanized h26D3-Fab and hTfRl was done by mixing of the two components at a molar ratio of 1:1 in lx dPBS and incubation at room temperature for 1 h. Subsequently, the complex was purified using size exclusion chromatography on HiLoad Superdex 200pg 26 / 600 (Cytiva) as described in Example 4.

[0342] Crystallization was performed using a stock solution of hTfRl-h26D3 at 15 mg / ml in PBS which was diluted to 4 mg / ml in PBS supplemented with 4 mM |3- mercaptoethanol. A 100+100 nl drop was set up using the additive screen in reservoir: 0.1 M sodium potassium phosphate pH 6.5, 10% PEG 3000, 0.05% dichloromethane and 2 mM |3-mercaptoethanol. The crystal was flash-frozen in reservoir solution supplemented by 8% glycerol and 16% PEG 400.

[0343] X-ray data collection and refinement were performed as follows. Data was collected to 3.87 A at Diamond Light Source beamline 104. The beamline was equipped with a DECTRIS Eiger2 XE 16M detector. The data set was integrated using XDS (Kabsch (2010), Acta Crystallogr D Biol Crystallogr 66:125-132) with STARANISO anisotropic scaling (Tickle et al (2018), Global Phasing Ltd) and diffracted to 3.87 A along the c* direction of the reciprocal lattice, and to 4.82 A in the a* / b* plane. Three complexes were found in the asymmetric unit. The structure was refined using the Buster refinement software and model building was carried out in Coot. Data collection and refinement parameters and statistics are given in Table 6 below.

[0344] The final, refined structure of the complexes showing the overall folds is depicted in Figure 10. As shown in Figure 10A, there were three independent complexes in the asymmetric unit. The chain names as used in the coordinate files are indicated. Figure 10B shows an example of the electron density contoured at the interface between hTfRl and heavy / light chain of h26D3-Fab. The protein chains are drawn in cartoon representation while sugar moieties are shown in stick representation. The binding interface interaction between h26D3 and human TfRl was extracted from the X-ray structure and described in the following to provide information about the precise binding of h26D3 to human TfRl. The binding interface between hTfRl and h26D3-Fab is depicted in Figures 10 and 11, and interaction was observed between the amino acid residues indicated in Table ?.

[0345] Table 7: Amino acid residues involved in interaction between h26D3 and hTfRl

[0346] Table 7 describes the key residues from 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 between h26D3 and hTfRl. In addition, as described in Example 9 below, several positions outside the observed binding interaction show important participation in binding of h26D3 to hTfRl.

[0347] In Table 8 below, the amino acids of human TfRl that are involved in the respective interactions with h26D3, Ft and Tf are listed. Notably, no amino acids involved in the binding of h26D3 form part of any of the binding interfaces for the endogenous ligands. This illustrates that the binders of the present disclosure, as exemplified by h26D3, bind to hTfRl outside the binding sites used by Ft and Tf. Table 8: Amino acid residues in hTfRl which interact with the respective ligand

[0348] * Montemiglio et al (2019), Nat Commun 10:1121

[0349] # Eckenroth et al (2011), Proc Natl Acad Sci USA 108:13089 The different epitopes on the hTfRl structure (pdb: 1SUV) are illustrated further in Figure 12. As shown in Figure 12, the Ft binding site is located on the apical domain of hTfRl, the Tf binding site is mainly located on the helical domain of hTfRl and the h26D3 epitope is located on the protease-like domain of hTfRl. The structure illustrates that the different ligands and binder use distinct, specific surface areas on the hTfRl structure. hTfRl is a homodimer having two identical chains, and the epitopes are only indicated on one of these chains.

[0350] EXAMPLE 6

[0351] Generation and characterization of hTFRl knock-in mice Human TfRl knock-in (hTfRl-KI; TFRIC-Kl) mice were generated by homologous recombination (experimental work performed at Cyagen US). A cDNA vector carrying the TFR1C (NCBI Reference Sequence: NM_001128148.3) ectodomain and murine Tfrc transmembrane and intracellular domain were introduced by pronuclear microinjection in C57BL / 6N ES cells Tfrc. The coding region of Tfrc exon 2 plus partial intron 2 were replaced with the TFR1C chimeric cassette (Figure 13A). Correct insertion of hTfRl cDNA was verified by Southern blot and PCR. Transgene expression in hTfRl-KI mice was confirmed in brain tissue by qRT-PCR (Figure 13B) and western blot (Figure 13C), indicating endogenous expression levels. hTfRl-KI mice were maintained on a C57BL / 6N background and only heterozygous hTfRl-KI mice were used for experiments.

[0352] EXAMPLE 7

[0353] Brain uptake of hTfRl-binding constructs in vivo

[0354] To evaluate hTfRl-mediated brain uptake in vivo, monovalent Fc-scFv constructs (see Example 4) were produced for four different binding proteins. A known binder to hTfRl, 15G11-1, was used as a control (Yu et al (2014), supra). This hTfRl binder has been described to be active in vivo and is used as a positive reference control for brain uptake. In addition, a construct containing a non-hTfRl scFv binder based on the anti-amyloid 0 antibody mAbl58 was designed and included as a negative control in the form of an Fc fusion construct (Fc-scFvl58, also referred to as simply "158" here and in the figures). The different Fc-scFv constructs were injected intravenously (i.v.) into hTfRl knock-in (hTfRl-KI) mice produced as described in Example 6 (n=4 per construct) at equimolar doses of 30 nmol / kg (corresponding to approximately 2.3 mg / kg). Plasma and brain exposure was assessed 24 h after dose.

[0355] The animals were anaesthetized using isoflurane and terminal blood samples were collected from the orbital plexus into BD Microtainer K2EDTA tubes. The samples were inverted and centrifuged at 2400 x g for 10 min at 4 °C. Plasma was extracted and transferred to Eppendorf tubes and frozen at -80 °C. Immediately following blood sampling, the abdomen of the animals was cut open and a cannula (21 G) was inserted into the left ventricle of the heart. A small cut was made in the right atrium and transcardial perfusion was performed with a minimum of 50 ml of cold PBS. Following perfusion, brains were extracted and the olfactory bulbs removed. The brains were separated into left and right hemispheres and cerebellum was removed from the left hemisphere, after which the left hemisphere was weighed and snap frozen on dry ice and stored at -80 °C until further preparation and analysis of the concentrations of injected constructs using a Meso Scale Discovery (MSD) based assay. The right hemispheres were placed in 4 % formaldehyde and stored at 4 °C for 24 h, after which they were rinsed in cold PBS, transferred to cold 30 % sucrose solution prepared in PBS and stored at 4 °C for further immunohistochemistry (IHC) processing (see Example 8 below).

[0356] For brain concentration measurements, frozen left hemispheres were thawed on ice and homogenized in TBS by automated bead homogenization. Triton was added to the homogenate to a final Triton concentration of 0.5% before centrifugation at 16 000 x g, after which supernatants were collected.

[0357] Brain and plasma concentrations of anti-hTfRl Fc-scFv were determined using a custom build MSD assay detecting the human Fc. A standard 96-well MSD plate (MSD, #L15XA-3) was coated with 0.5 pg / ml goat anti-human IgG, Fey fragment specific antibody (Jackson Immuno Research Europe Ltd, #109-005-098) diluted in lxPBS (Medicago AB, #09-9400-100). After incubation at 4 °C overnight, the plate was washed 4x in lxPBS-TWEEN (Fisher Scientific, #09-9410-100) and blocked with 150 pl 1% BlockerA in PBS-TWEEN (MSD, #R93BA-4) per well. Samples and corresponding standards, ranging from 400 pM to 0.1 pM in 1:4 dilution steps, were added and incubated for 2 h and 900 rpm at room temperature. A 1 h incubation step with mouse anti-human IgG (Mabtech, 3850-1-1000, MT145) diluted to 0.5 pg / ml was included, followed by 1 h incubation of SULFO-TAG conjugated anti-mouse antibody (MSD, R32AC-1) diluted to 0.5 pg / ml when the plate was incubated for another hour at room temperature and 900 rpm. 150 pl MSD read buffer (MSD, #R92TC) per well was added before reading the plates in an MSD SECTOR Imager. Between each incubation step, a 4x wash in lxPBS-TWEEN was performed. All antibodies and samples, except the coating antibody, were diluted in 1% Blocker A in PBS-TWEEN and added in a volume of 50 pl / well. The concentration of the analytes in the samples were evaluated with the MSD workbench software, using a 4PL curve fitting algorithm and curve weighting 1 / Y2 for the standard curve. Statistical analysis was performed in GraphPad Prism (v. 9.0.0) using one way ANOVA with Tukey's post hoc test.

[0358] The results are shown in Figure 14. As shown in Figure 14A, substantially higher brain concentrations were observed for the two test constructs and the positive control 15G11-1, compared to the negative control (158) at 24 h after dose. As shown in Figure 14B, the plasma concentrations of the two test constructs and the positive control 15G11-1 were lower at 24 h compared to that of 158, indicating that hTfRl engagement leads to a faster plasma clearance. The brain-to-plasma concentration ratios are shown in Figure 14C. The two test constructs and the positive control 15G11-1 showed a significantly enhanced brain exposure relative to plasma in comparison to the negative control. Taken together, the data supports hTfRl-mediated BBB transport in this experiment for the tested, novel hTfRl binders.

[0359] EXAMPLE 8

[0360] Immunohistochemistry data on brain exposure

[0361] In vivo engagement of hTfRl by the Fc-scFv construct was studied further using a qualitative immunohistochemistry (IHC) analysis. In brief, coronal brain sections at a thickness of 20 pm were obtained from PBS-perfused brain hemispheres of the mice described in Example 7 using a cryostat (Microm NX50 CryoStar, Epredia). The sections were collected on Superfrost plus slides (Menzel- Glaser, #J1800AMNZ) and air-dried prior to IHC. The brain sections were washed with PBS (pH 7.4) for 15 min and incubated in blocking buffer (5 % BSA, 0.25 % Triton-X in PBS) for 2 h at room temperature. To visualize i.v. dosed constructs, brain sections were incubated with a secondary goat anti-human IgG (heavy and light chain specific) conjugated to Alexa Fluor 488 (Invitrogen, #A11013) for 120 min at room temperature followed by 3x15 min wash in PBS. Slides were mounted with Fluoromount-G (Invitrogen, #00-4958-02) for imaging analysis. Confocal images from cerebral cortex were captured using a Leica Stellaris 5 confocal system equipped with a HC PL APO 40x / 1.25 GLYC motCORR CS2 objective (Leica, #11506423).

[0362] Distinct IHC immunofluorescence signals were observed in brain capillaries with positive reference module 15G11-1, while a minimal IHC signal was detected in brain sections from mice injected with negative control 158 (Figure 15). Brain capillary IHC signal was observed for the two test constructs h26D3 and 37D10, of which h26D3 showed the strongest immunofluorescence signal, comparable to the positive control 15G11-1. Taken together, the MSD (Example 7) and IHC (this Example) analyses demonstrate that the hTfRl binders of the disclosure in a scFv format exhibit an increased brain exposure in hTfRl-KI mice. EXAMPLE 9

[0363] Generation of affinity variants and affinity determinations

[0364] Several variants of the parental antibody h26D3 were generated by substituting tyrosine, tryptophan and aspartic acid residues in the CDRs one by one for alanine residues. The resulting variant VH domains were denoted HC1-HC13 and their amino acid sequences are provided in the sequence listing as SEQ ID NO:58-70, respectively. Disulfide stabilized versions of these variant VH domains have the amino acid sequences SEQ ID NO:102-114, respectively. Variant CDR sequences comprised in these variant VH domains are listed as SEQ ID NO:29-41, respectively. The resulting variant VL domains were denoted LC1-LC6 and their amino acid sequences are provided in the sequence listing as SEQ ID NO:72-77, respectively. Disulfide stabilized versions of these variant VL domains have the amino acid sequences SEQ ID NO:119-124, respectively. Variant CDR sequences comprised in these variant VL domains are listed as SEQ ID NO:42-46, respectively. Table 9 below provides a summary of the specific mutations in each of the alanine variants.

[0365] Table 9: Alanine substitution variants of VH and VL ofh26D3

[0366] The generated alanine variants were expressed as single mutant, His-tagged Fabs by transient transfection of Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. Clarified media, into which the Fabs had been secreted, was used to assess binding to hTfRl by BLI (Octet RED384, ForteBio). The expressed Fabs were loaded from the cell supernatants onto anti-Fab biosensors during 240 s. Thereafter, association of ectodomain of hTfRl (SEQ ID NQ:110), diluted to 3.75 pg / ml in lx Kinetics buffer (ForteBio), to the loaded sensors was measured for 300 s, followed by dissociation for 300 s. All variants were confirmed to bind hTfRl but were affected to different extents (Figure 16). Variants showing affected binding to hTfRl in the screen were selected for further characterization. In addition, double mutants were generated by combining heavy and light chains with alanine substitutions. Table 10 below provides a summary of the specific mutations in each of the alanine variants that were selected.

[0367] Table 10: Variants of VH and VL ofh26D3 selected for further characterization

[0368] The selected variants were expressed as His-tagged Fabs by transient transfection of Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. The Fabs were purified at small scale with HisPur™ Ni-NTA Magnetic Beads (Thermo Scientific) according to the manufacturer's instructions followed by buffer exchange into DPBS pH 7.4. Selected variants were also purified at a larger scale by application on a HisTrap Excel column (Cytiva), which was washed with 20 mM Tris, 200 mM NaCI and 5 mM imidazole. The proteins were eluted with 20 mM Tris, 200 mM NaCI 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 Ultra centrifugal concentrator (30 MWCO; Mil lipore). Selected variants were further polished by size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex 200; Cytiva) in DPBS pH 7.4. Analytical characterization of the protein was done by UV protein determination, SDS-PAGE and HPLC-SEC.

[0369] Binding of the purified Fabs to human and cynomolgus TfRl was evaluated using either SPR (Figure 17) or indirect ELISA (Figure 18). For SPR, a Biacore 8K instrument (Cytiva) was used. 1 pg / ml of hTfRl (SEQ ID NO:99) or cTfRl (SEQ ID NO:100) was immobilized on a Cm5 sensor chip (Cytiva, WBR100399) using the amine coupling kit type 2 (Cytiva, WBR100633) according to the manufacturer's instruction. The Fabs were injected over the chip using a 2-fold dilution series in four steps starting at 100 nM. The interaction was measured using the single cycle kinetics method with a contact time of 120 s at a flow rate of 30 pl / min followed by a dissociation time of 1000 s. Regeneration of the surface between cycles was done by injecting 3M MgCL The binding data was fitted to a 1:1 interaction model. The Fabs were diluted in HBS-EP+ (Cytiva, WBR100669). Experiments were performed at 25 °C. The results are shown in Figure 17, and the calculated KD values are given in Table 11 below.

[0370] Table 11: SPR analysis of variant h26D3 Fabs vs. hTfRl and cTfRl For the indirect ELISA, half area 96-well plates (Corning, #3690) were coated with 1 pg / ml recombinant ectodomain of hTfRl (SEQ ID NO:110) in PBS overnight at 4 °C. The coated plates were blocked using Pierce protein-free blocking solution (Thermo Fisher Scientific, #37572) for 1 h at room temperature with shaking and washed four times in PBS containing 0.1 % TWEEN-20. Serial dilutions (1:3) of various expressed constructs in incubation buffer (1 % BSA, 0.1 % TWEEN-20 in PBS) were incubated for 1 h at room temperature. Following the four wash steps, bound test constructs were detected by addition of anti-human-IgG F(ab')2-HRP antibody (Jackson Immuno Research, #109-036-003) at 1:5000 dilution in incubation buffer (1 h, room temperature). Following four wash steps, K-Blue® Aqueous TMB substrate (Neogen, #331177) was added to the wells for 15 min at room temperature before the reaction was stopped with 1:1 dilution of 0.5 M H2SO4. The optical density at 450 nm was recorded (Spark, Tecan) and background signal was subtracted before analysis. The obtained results are shown in Figure 18.

[0371] Based on the Biacore and ELISA measurements, several variants were identified within a wide range of affinities for human TfRl. Many variants exhibited a retained cross-reactivity to cynomolgus TfRl.

[0372] Finally, selected variants were reformatted to scFv and used in the context of the bispecific binding molecule format disclosed in WO2022 / 258841. Bispecific binding molecules comprising scFv modules constructed from h26D3 and selected alanine mutants were expressed in ExpiCHO cells as described above. Filtered supernatants were applied to a MabSelect SuRe column (Cytiva) which was subsequently washed with DPBS pH 7.4. Expressed binding molecules were eluted by application of 0.7 % HAc pH 2.5, followed by immediate neutralization of the sample to pH 7.5. Purified samples were polished further by subjecting them to size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex 200; Cytiva) in DPBS pH 7.4. The purified constructs were concentrated using centrifugal concentrators Amicon Ultra (30 MWCO, Millipore). Each purified expressed construct was characterized using SDS-PAGE, size-exclusion chromatography (Superdex 200 Increase 3.2 / 300; Cytiva) and UV protein determination. Binding to hTfRl was evaluated using SPR as described above with adjustments of the concentration interval depending on the variant. As shown in Figure 19 and in Table 12 below, the different tested variants exhibited a range of affinities for the hTfRl target. Table 12: SPR analysis of variant h26D3 scFv in bispecific format vs. hTfRl

[0373] EXAMPLE 10

[0374] Design, production and preparative SEC of disulfide-stabilized hTfRl-binding molecules

[0375] A panel of hTfRl-binding molecules in the scFv format were designed, produced and purified. The designed hTfRl-binding scFv molecules are listed in Table 13.

[0376] Table 13: hTfRl-binding scFv molecules and their amino acid sequences

[0377] The scFv variants whose respective designation includes the "_DS" suffix all comprise two mutations which introduce cysteine residues at position 44 of the VH domain and at position 106 of the VL domain of the respective starting sequences. It is contemplated that these introduced cysteine residues cause the formation of a stabilizing disulfide bond between the VH and VL domains.

[0378] The test items were produced as Hise tagged scFv constructs with the Hise tag spaced from the remainder of the scFv by a flexible (648)4 linker (combined tag sequence given by SEQ ID NO:135), by transient transfection of CHO cells in 400 ml culture volume per scFv. One of the test items, "h26D3-wt_DS, VL-first", was also produced with both an Hise tag and an Avi tag (combined tag sequence given by SEQ ID NO:136) for site directed in vivo biotinylation, and was expressed in 1 1 culture volume.

[0379] For purification, all scFv proteins were recovered by immobilized metal ion affinity chromatography (IMAC) purification from clarified cell supernatants. For IMAC, supernatants were loaded on a HisTrap excel 5 ml column (Cytiva) and unbound material washed out with wash buffer (PBS, 350 mM NaCI and 10 mM imidazole). Bound scFv was then eluted in elution buffer (PBS, 350 mM NaCI, 0.5 M imidazole, pH 7.5). Next, the eluted proteins were passed over a preparative SEC column (HiLoad 26 / 600 Superdex 200 pg; Cytiva) with PBS, pH 7.4 as running buffer. SEC fractions containing monomeric scFv were collected and brought to 1 mg / ml final concentration in PBS, pH 7.4. Representative chromatograms from this preparative SEC are shown in Figure 20 for h26D3-HC6_DS (A) and h26D3-HC6 (B), and show that the scFv molecules are recovered with different degrees of aggregated forms during the initial IMAC purification. For h26D3-HC6_DS (A), 45 % of the material elutes in the main peak and contain the monomeric, desired scFv. This is in contrast to h26D3-HC6 (B), for which the distribution between dimer and monomer is the opposite, showing scFv dimer in the main peak, and only 16 % of the material in the monomer peak. The size distribution of higher molecular weight (HMW) species is similar for both constructs.

[0380] EXAMPLE 11

[0381] Analytical SEC of disulfide-stabilized hTfRl-binding molecules

[0382] Following three freeze / thaw cycles between room temperature and -80 °C, 1 pg of each scFv variant produced in Example 10 was injected to a SEC column (Waters BioSuite 250 UHR SEC 4 pm, 4.6 x 300 mm). Analyses were done with a running buffer of 0.2 M potassium phosphate, 0.25 M KCI, pH 6.2 at a flow rate of 0.35 ml / min.

[0383] The results of the analytical SEC experiment are shown in Figure 21 and Table 14. The monomeric form of all scFv samples have a retention time of 11 min (Figure 21A-H). In scFv molecules lacking the DS mutations, additional peaks, corresponding to multimerized forms of scFv are detected (Figure 21A-D). In all samples with DS mutations, 100 % of the respective molecule migrate at 11 min as monomeric scFv (Figure 21E-H). The percentage distribution of integrated peak areas from the analytical SEC samples are listed in Table 14. Again, for the four samples with stabilizing DS mutations, 100 % of injected proteins are detected in the monomer peak, whereas additional peaks of multimeric forms are detected for corresponding samples without DS mutations.

[0384] Table 14: Distribution of peak areas from SEC chromatograms

[0385] EXAMPLE 12

[0386] Thermal stability of disulfide-stabilized hTfRl-binding molecules Monomer stability of scFv samples was evaluated by HPLC SEC analysis. The panel of scFv molecules produced and studied in Examples 10-11, purified and stored in PBS, were subjected to temperature hold for one, two or four weeks at temperatures 4 °C, 40 °C and frozen at -80 °C, except for h26D3-HC6_DS, VL-first and h26D3-LCl, VL-first, which were held frozen at -70 °C and -75 °C respectively. At each timepoint, samples of each variant from each temperature were analyzed by HPLC-SEC as described in Example 11. At the initiation of the study, frozen samples were thawed and analyzed, and are denoted TO.

[0387] The results for scFv molecules without DS mutations after storage at 40 °C for 1-4 weeks are shown in Figures 22 and 23. All samples were isolated as pure monomers in the preceding preparative SEC purification described in Example 10. However, analytical SEC revealed that all samples contain both monomer (retention at approximately 11 min) and dimer (retention at approximately 10 min) forms already at the initial time point TO (Figure 22A-D). The share of dimers is the most significant for h26D3-HC6 (Figure 22A) and h26D3-HC6, VL-first (Figure 22B), while the majority of scFv molecules are monomeric at TO for h26D3-LCl (Figure 22C) and h26D3wt (Figure 22D). The chromatograms show that the distribution between monomeric and dimeric forms shifts gradually during the study. In samples from 4 weeks, the monomer / dimer distributions are more similar between the different molecules as compared to the corresponding distributions at TO (Figure 22A-D). Multimers are observed for all scFv molecules as a minor peak with a retention time between 9-10 min (Figure 22A-D). The results indicate that, despite having been isolated in the pure monomeric form, scFv molecules without the stabilizing DS mutations form multimers during storage.

[0388] The same pattern is shown by the percentage proportions of monomeric scFv molecules exhibited in Figure 23. The molecules h26D3-HC6 (Figure 23A) and h26D3- HC6, VL-first (Figure 23B) have lower proportions of monomeric scFv at TO (reference sample kept at -80 °C). The degree of monomer increases for these molecules in samples kept at 40 °C for 1-4 weeks (Figure 23A and 23B). The opposite is seen for h26D3-LCl (Figure 23C) and h26D3wt (Figure 23D), where monomer content is high at TO and then decreases during storage at 40 °C for 1-4 weeks. The observation indicates that scFv molecules without DS mutations reach an equilibrium between monomeric and dimeric states during storage. For h26D3-HC6 and h26D3-HC6 VL-first, the monomer content increased over the course of the study, while for h26D3-LCl and h26D3wt, the monomer content decreased as compared to TO.

[0389] Corresponding chromatograms for scFv molecules with DS mutations after storage at 40 °C for 1-4 weeks are shown in Figure 24. As seen here, the molecules with introduced DS mutations are highly stable as monomers over the studied period, with uniform peaks of monomeric scFv detected at a retention time just above 11 min for all the variants (Figure 24A-C). Only in chromatograms from samples incubated for 4 weeks at 40 °C (Figure 24C), a very small peak is observed at 10 min retention time. The results demonstrate that the monomeric state of DS- stabilized scFv molecules is highly stable also at an extended storage time at 40 °C.

[0390] As can be seen in Table 15 below, the high stability of the scFv variants with DS mutations as compared to corresponding variants without DS mutations was also seen for samples kept at 4 °C or frozen. Table 15: Percentage of monomer forms of scFv samples EXAMPLE 13

[0391] Serum stability of disulfide-stabilized hTfRl binding molecules

[0392] Serum stability is a critical attribute for antibodies and different fragments such as scFv-containing biotherapeutics (Worn and Pluckthun (2001), J Mol Biol 305(5):989-1010; Austerberry et al (2017), Eur J Pharm Biopharm 115:18-30; Willuda et al (1999), Cancer Res 59:5758-67). In order to assess the stability in serum of scFv molecules with DS mutations, the variant h26D3wt_DS, VL-first expressed with Hise and Avi tags (see Example 10) was incubated in mouse serum (Capricon, MOU-1B) and lxPBS (#09-9400-100, Medicago AB) respectively at both 4 °C and 37 °C using a thermal mixer (Eppendorf ThermoMixer C, Eppendorf). After 48 h incubation, binding of the scFv to hTfRl was evaluated by ELISA. In brief, a half area 96-well plate (#3690, Corning) was coated overnight at 4 °C with hTfRl diluted in lxPBS, followed by blocking with Pierce Protein-Free Blocking Buffer (#37572, ThermoScientific) for 1 h at room temperature (RT) with shaking. h26D3wt_DS, VL- first was diluted or serially diluted in mouse serum or ELISA incubation buffer (EIB): lxPBS-0.05% Tween20, 0.1% BSA (PBS-T, #09-9410-100, Medicago AB, A7030-100G, Sigma-Aldrich), added to plate and incubated for 2 h at 4-8 °C with shaking. Bound biotinylated scFv was detected using streptavidin-horseradish-peroxidase (#3310-9- 100, Mabtech) in EIB for 1 h at RT with shaking, followed by TMB (#331177, Neogen). The reaction was stopped by 1:1 addition of 0.5 M sulfuric acid (#35354-lL, Honeywell). Optical density at 450 nm was obtained using a microplate reader (Spark, Tecan) and the collected data was plotted using GraphPad Prism software (GraphPad Software Inc). Serum stability of the scFv is displayed as % binding to hTfRl and determined using the following equation:

[0393] Serum stability = (ELISA OD450 at 37 °C) / (ELISA OD450 at 4 °C) x 100 %

[0394] The results are shown in Figure 25, and demonstrate stability and a highly retained hTfRl binding ability of the tested scFv variant after incubation in mouse serum for 48 h at the tested temperatures. The results can be compared to other published scFv stability data in mouse serum (Liu et al (2022), mAbs 14:1, 2073632). EXAMPLE 14

[0395] Dynamic light scattering analysis of disulfide-stabilized hTfRl binding molecules Dynamic light scattering (DLS) analysis of scFv variants h26D3-HC6_DS, VL- first; h26D3-HC6 and h26D3-HC6_DS (see Example 10) was performed at 25 °C or 20 °C using an Uncle instrument (Unchained Labs). Average hydrodynamic diameter and polydispersity index (PDI) were calculated from analyses run in triplicates h26D3-HC6_DS, VL-first) or duplicates (h26D3-HC6 and h26D3-HC6_DS). Samples were diluted to 1 mg / ml in PBS prior to analysis.

[0396] The results are shown in Table 16. The scFv variant without DS mutations (h26D3-HC6) exhibits a larger average hydrodynamic diameter than either of the two variants with DS mutations (h26D3-HC6_DS, VL-first and h26D3-HC6_DS). The results are expected from the high monomeric content for DS stabilized scFv as shown by analytical SEC (Figure 21 and Table 14), and agree with other reported DLS analyses of scFv (Morioka et al (2019), Molecules 24(14):2620). The observed PDI values are around 0.1 in all variants, indicating monodisperse (PDI < 0.1) or a low degree of polydispersity (PDI 0.1-0.2).

[0397] Table 16: Dynamic light scattering analysis ofscFv variants

[0398] EXAMPLE 15

[0399] Surface plasmon resonance analysis of disulfide-stabilized hTfRl-binding molecules

[0400] Binding to hTfRl of six different purified scFv variants from Example 10 was evaluated by surface plasmon resonance (SPR) using Biacore 8K (Cytiva). 30 pg / ml of DYKDDDDK Tag mAb FG4R (Thermo Fisher Scientific, MAI-91878) in 10 mM acetate buffer, pH 4.5 was immobilized on a CM5 sensor chip (Cytiva, WBR100399) using the amine coupling kit type 2 (Cytiva, WBR100633) according to the manufacturer's instruction, resulting in immobilization levels of approximately 10000 RU. hTfRl was captured via an N-terminal DYKDDDDK tag. To this end, the receptor was injected over the chip with a contact time of 60 s at a flow rate of 10 pl / min. Each of the six scFv variants h26D3-HC6; h26D3-HC6, VL-first; h26D3-LCl; h26D3-HC6_DS; h26D3- HC6_DS, VL-first and h26D3-LCl_DS, VL-first were injected over the chip using a 3- fold dilution series in five steps starting at 700 nM or 400 nM. Interaction was measured using the single cycle kinetics method with a contact time of 120 s at a flow rate of 30 pl / min followed by a dissociation time of 600 s. Regeneration of the surface between cycles was done by injecting 10 mM glycine-HCI pH 1.7 with a contact time of 30 s and a flow rate of 30 pl / min. The binding modules were diluted in HBS-EP+ (Cytiva, WBR100669). Experiments were performed at 25 °C.

[0401] The resulting binding curves are shown in Figure 26. As can be seen from the SPR diagrams, introduction of stabilizing DS mutations does not disrupt the binding of hTfRl by the scFv variants. It can also be seen that the scFv variants without DS mutations dissociate from the hTfRl antigen in a biphasic manner, which may be due to an element of avidity interaction observed for dimeric forms present in the samples.

[0402] EXAMPLE 16

[0403] Generation of bispecific binding molecule

[0404] This example describes the design and production of a bispecific binding molecule denoted BA101, which incorporates an A|3 protofibril-specific antibody described in W02016 / 005466 and a humanized, stabilized hTfRl-binding scFv variant as described in Example 10.

[0405] Materials and methods

[0406] Design of constructs: The bispecific binding molecule BA101 was designed as a knob-into-hole variant of the A|3 protofibril-specific antibody A17D / R79T_DI8 described in W02016 / 005466, with an hTfRl-binding scFv (h26D3-HC6_DS, VL first) linked to the C-terminal amino acid residue of the knob heavy chain of the antibody. As control, the A|3 protofibril-specific standard antibody A17D / R79T_DI8 was used and denoted BA102 herein. The amino acid sequences of the tested molecules are provided in the sequence listing as noted in Table 17. Table 17: Amino acid sequences of bispecific binder BA101 and con trol BA 102

[0407] Expression from transient transfection: The BA101 test molecule was expressed in CHO cells (ExpiCHO; ThermoFisher) and purified by affinity chromatography, cation exchange chromatography (CIEX), followed by preparative size-exclusion chromatography (SEC) and buffer exchange into phosphate buffered saline (PBS) solution. The purified molecules were characterized using SDS-PAGE, SEC, and UV protein determination. The reference antibody BA102 was expressed and purified as described in W02016 / 005466.

[0408] Results

[0409] The bispecific binding molecule was successfully produced and purified to a final concentration of 10 mg / ml. Protein purity, as defined by SEC-HPLC, was >98 % monomer.

[0410] EXAMPLE 17

[0411] Characterization of target binding

[0412] This example describes the binding of the bispecific binding molecule generated and produced in Example 16 to human TfRl and A|3 protofibrils and monomers by SPR and inhibition ELISA.

[0413] Materials and methods

[0414] Human TfRl: The human transferrin receptor 1 (hTfRl) was expressed in human embryonic kidney (HEK293) cells (Expi293; ThermoFisher) according to the manufacturer's instructions. The harvested supernatant was purified using HiTrap IMAC Sepharose FF (Cytiva) followed by SEC on HiLoad Superdex 200pg 26 / 600 (Cytiva). The following buffers were used: Ni-NTA wash buffer: 20 mM Tris pH 8.0, 10 mM imidazole and 200 mM NaCI; Ni-NTA elution buffer: 20 mM Tris pH 8.0, 200 mM NaCI and 500 mM imidazole; size-exclusion buffer (SEC): lx dPBS (ThermoFisher). Purified hTfRl (SEQ ID NO:99) was concentrated to 2 mg / ml and stored at -80 °C until analysis.

[0415] AI31-42 protofibrils: Protofibrils were prepared using an A 1-42 peptide (Bachem). Briefly, the A 1-42 peptide was dissolved in 10 mM NaOH, 0.005 % Tween-20, pH >11 at a concentration of 100 pM. Protofibrils were prepared by neutralizing the A 1-42 peptide to pH 7.4 by adding 1:1 volume of a 2x PBS buffer to a final concentration of 50 pM. The peptide was incubated for ~30 min at 37 °C for protofibril formation and purified from remaining monomers by HPLC on a Superdex 75 Increase 3.2 / 300 size-exclusion column using a mobile-phase of lxPBS, 0.1 % Tween-20, pH 7.4. Prior to injection, the protofibril reaction was centrifuged at 16000 x g for 10 min at 4 °C to remove insoluble fibrils. The void peak containing the A 1-42 protofibrils was collected and the concentration determined using SEC and a calibration curve of an A|3 protofibril standard with a known concentration.

[0416] A(3 1-28 monomers: A 1-28 monomers (Bachem) were dissolved in 10 mM NaOH, 0.005 % Tween-20, pH >11 at a concentration of 100 pM. Aliquots were made and stored at -80 °C until analysis. The monomers were neutralized prior to use in downstream assays.

[0417] Affinity evaluation and Kp determination by surface plasmon resonance: Binding interactions between antigens and binding molecules were evaluated by SPR using a Biacore 8K or 8K+ instrument (Cytiva) according to standard procedures.

[0418] Single cycle kinetics using capture was used to measure binding to hTfRl. For measurement, 30 pg / ml FLAG-Tag mAb FG4R (Invitrogen) was immobilized on a CM5 chip. 5 pg / ml hTfRl was captured for each cycle followed by injection of the binding molecules using a 3-fold dilution in five steps starting at 1500 nM for BA101, using a 2 min injection of every concentration and a 10 min dissociation time. Regeneration of the surface between each cycle was done by injecting 30 pl 10 mM Glycine-HCI, pH 1.7 (Cytiva, cat. No. 29215281). The binding data was fitted using a 1:1 interaction model.

[0419] Single cycle kinetics, with binding molecules as analytes, was used to measure binding to the A 1-42 protofibrils that were immobilized on a CM5 chip. For the measurements, 11.2 nM A 1-42 protofibrils were immobilized on the chip. The binding molecules were then injected over the chip using a 2-fold dilution in five steps starting at 10 nM, using 2 min injection of every concentration and a 20 min dissociation time. Regeneration of the surface between cycles was done by injecting 30 pl 3 M MgCl2 (Cytiva, cat. No. 29234600). The binding data was fitted to a bivalent analyte model.

[0420] Single cycle kinetics, with binding molecules immobilized on a CM5 chip, was used to measure binding to the AP 1-28 monomer. For the measurements, 45 pg / ml of binding molecule was immobilized on the chip. The monomer was then injected over the chip using a 2-fold dilution in five steps starting at 5000 nM, using 2 min injection of every monomer concentration and a 10 min dissociation time. Regeneration of the surface between cycles was done by injecting 30 pl 3 M MgCl2 (Cytiva, cat. No. 29234600). The binding data was fitted to a 1:1 interaction model.

[0421] In all SPR experiments, lxHBS-EP+ (Cytiva, cat. No. BR100669) was used as running buffer and to dilute analytes and capture ligands. Experiments were performed at 25 °C.

[0422] IC50 determination by inhibition ELISA: The binding of binding molecules BA101 and BA102 to A 1-42 protofibrils and A 1-28 monomers was evaluated by inhibition ELISA. The binding molecules were incubated at a fixed concentration (0.05 pg / ml) with titrating concentrations of the different AP antigens (Api-42 protofibrils: 125 nM - 0.0021 nM, Api-28 monomers: 12.5 pM - 0.21 nM). After incubation for 45 min at 900 rpm to reach equilibrium, the samples were added to a blocked and washed ELISA plate with an Api-40 coat (0.5 pM). The samples were incubated on the plate for 25 min without shaking followed by washing, incubation with alkaline phosphatase (ALP)-conjugated detection antibody, another wash step and finally incubation with alkaline phosphatase substrate. Optical density at 405 nm was read, and the collected data was analyzed using a four-parameter variable slope non-linear fit to determine IC50 values.

[0423] Results

[0424] Affinity evaluation and Kp determination by surface plasmon resonance: The binding of binding molecule BA101 to human TfRl was evaluated in SPR and the KD value was determined. The calculated ka, kd, and KD values are shown in Table 18. Representative sensorgrams are shown in Figure 27. Table 18: SPR analysis of binding to human TfRl

[0425] The binding of BA101 to A 1-42 protofibrils was evaluated in SPR and KD values were determined. BA101 demonstrated binding to A|3 protofibrils at a similar affinity to the comparator molecule BA102. The calculated ka, kd, and KD values are shown in Table 19. Representative sensorgrams are shown in Figure 28.

[0426] Table 19: SPR analysis of binding to A61-42 protofibrils

[0427] The binding of BA101 to A 1-28 monomers was evaluated in SPR and KD values were determined. BA101 demonstrated binding to A|3 monomers at a similar affinity to the comparator molecule BA102. The calculated ka, kd, and KD values are shown in Table 20. Representative sensorgrams are shown in Figure 29.

[0428] Table 20: SPR analysis of binding to A61-28 monomers

[0429] IC50 determination by inhibition ELISA: The binding of the bispecific binding molecule BA101 to A 1-42 protofibrils and A 1-28 monomers was evaluated using inhibition ELISA. BA101 demonstrated binding to A 1-42 protofibrils and A 1-28 monomers in solution (Figure 30), at similar IC50 values as the comparator molecule BA102 (Table 21). Table 21: Binding to A6 protofibrils and monomers using inhibition ELISA

[0430] EXAMPLE 18

[0431] Target binding in brain from human Alzheimer's disease patient

[0432] This example describes target binding of the bispecific binding molecule (BA101) and the comparator molecule (BA102), generated as described in Example 16 and tested by immunohistochemistry (IHC) and immunoprecipitation in a pool of human brain extract from AD patients.

[0433] Materials and methods

[0434] Brain tissue sample preparation: Fresh frozen human brain cortical tissue from subjects with clinical and neuropathological diagnosis of Alzheimer's disease (AD) were collected by the Netherlands Brain Bank (NBB), Netherlands Institute for Neuroscience, Amsterdam. For IHC analysis the temporal cortex tissues were sectioned (8-10 pm) and mounted onto Superfrost Plus slides (TermoFisher). The sections were air-dried at room temperature for 60 min and stored in a sealed box at - 80 °C until use. For immunoprecipitation experiments the brain tissues were homogenized in a Potter-Elvehjem homogenizer at 1:10 weight:volume in Trisbuffered saline (TBS) buffer followed by centrifugation at 16000 x g for 1 h. The resulting supernatants were frozen at -80 °C until analysis.

[0435] Target binding in Alzheimer's disease brain by immunohistochemistry: The frozen sections were transferred directly to ice-cold 50% acetone for 30 s, followed by 100% acetone for 5 min, and finally 1 x phosphate-buffered saline (PBS) for 5 min before wet-loading into a Ventana robotic platform. For the automated staining procedure, the binding molecules BA101 and BA102 were used at a working concentration of 6.8 nmol / L, followed by incubation with a secondary rabbit antihuman IgG and detection by DISCOVERY OmniMap anti-Rb HRP (Ventana, 760-4311) horseradish peroxidase (HRP) 3,3'-diaminobenzidine-based system. The sections were counterstained with hematoxylin before bright-field scanning using a Panoramic 250 FLASH III slide scanner. The resulting image files were uploaded into Slide Viewer software, version 2.5 (3DHISTECH) and adjusted for optimal brightness and contrast prior to manual assessment of staining.

[0436] Target binding in human Alzheimer's disease brain extracts by immu n Binding of BA101 and its comparator BA102 to their target (AP protofibrils) in human AD brain was analyzed by immunoprecipitation, which is a method for capturing of target protein in a sample using an antibody specific for the target molecule. Briefly, four concentrations (70, 700, 7000 and 70000 pM) of each of the tested antibodies were pre-incubated with soluble 16000 x g TBS brain extracts from a pool of 11 AD cases for 2 h. Magnetic Protein A Dynabeads were added to the sample and incubation continued for an additional 30 min. The beadbound target was separated by a magnet and eluted from the beads using 70% formic acid, which also monomerizes the immunoprecipitated AP target. After neutralization, the pellet (immunoprecipitation fraction) was analyzed using the V- PLEX A Peptide Panel 1 (4G8) Kit (Meso Scale Discovery) according to manufacturer's instructions. Obtained signal was correlated to the amount of immunoprecipitated AP peptides, of which AP42 was further reported.

[0437] Results

[0438] Target binding in AD brain by immunohistochemistry: The bispecific binding molecule BA101 and comparator BA102 were tested for their ability to bind to AP plaques in fresh frozen temporal cortex sections from an AD subject.

[0439] Immunohistochemistry demonstrates comparable binding of BA101 and BA102 to AP plaques (Figure 31).

[0440] Target binding in human Alzheimer's disease brain extracts by immu n The bispecific binding molecule BA101 and comparator BA102 were tested for their ability to bind to AP target in solution, in a pooled TBS brain extract from human AD patients. Immunoprecipitation of the brain extract using

[0441] BA101 and BA102 demonstrated a concentration-dependent immunoprecipitation of target AP42 by both binding molecules (Figure 32). EXAMPLE 19

[0442] Characterization of functional effects

[0443] This example describes the functional effects of the bispecific binding molecule generated and produced in Example 16. The potency of the bispecific binding molecule BA101 and comparator BA102 with regards to mediation of uptake of AP protofibrils into THP-1 cells and clearance of amyloid plaques ex vivo in AD brain sections was evaluated.

[0444] Materials and methods

[0445] Uptake of A protofibrils in THP-1 cells: An in vitro uptake assay was used to investigate whether the bispecific binding molecule BA101 and comparator binding molecule BA102 could induce uptake of AP protofibrils by human monocytic THP-1 cells. THP-1 cells were purchased from Sigma / ECACC and cultured in RPMI1640 (Gibco) supplemented with 10% FBS (Hyclone), lx GlutaMax (Gibco), lx Penicillin Streptomycin (Hyclone). Binding molecule and HiLyte Flour 488-labelled A protofibrils were incubated together for 30 min at RT (final concentrations 25-0.05 nM for binding molecule and 50 nM for AP protofibrils). HiLyte Flour 488-labelled Api-42 protofibrils were made from HiLyte 488-labelled Api-42 peptides (Bachem), using the protocol described for Api-42 protofibrils in Example 17. 200,000 THP-1 cells were added to the wells of a 96-well plate (Corning). Cells were pelleted by centrifugation at 300 x g 5 min at RT, resuspended with the binder / AP protofibril complexes and incubated at 37°C, 5% CO2 for 60 min. Cells were washed in PBS before data acquisition using a BD FACS Lyric flow cytometer. Data was evaluated using FlowJo software (BD Bioscience). EC50 values were calculated using non-linear regression with the sigmoidal 4PL equation in GraphPad Prism.

[0446] Ex vivo phagocytosis in AD brain: An ex vivo phagocytosis assay was used to investigate whether the bispecific binding molecule BA101 and comparator BA102 could induce plaque clearance by macrophages. Fresh frozen AD brain tissue was cryosectioned (20 pm) and tissue slices were collected onto poly-D-lysine (Gibco A38904-01, 50 pg / ml) coated 12 mm glass coverslips. Sections were then incubated with different concentrations of BA101 and BA102 (0.85-34 nM) or an IgGl isotype control antibody (CrownVivo, C-00012, 1 pg / ml, 34 nM) for 1 h at 37 °C 5% CO2 followed by incubation for 24 h with 5 x 105to 8.5 x 105primary human macrophages isolated from buffy coats. Plaque clearance was evaluated using immunohistochemistry with the mouse anti-human A|3 antibodies 6E10 (Covance WSIG-39320) and 4G8 (Covance #SIG-39200) by measuring the immunopositive area on selected ROIs analyzed in consecutive sections. Images were analyzed with Leica's LASX Software using Otsu or Inverted Triangle thresholding methods. Experiments were repeated 13 times with macrophages isolated from different buffy coats and in 3 different patients' temporal cortex with distinct ApoE genotypes (ApoE 3 / 3, ApoE 3 / 4 and ApoE 4 / 4). IC50 values were calculated assessing non-linear regression on GraphPad Prism and "Inhibitor vs. normalized response - Variable slope" analysis was performed (outliers removed based on ROUT method).

[0447] Results

[0448] Uptake of A(3 protofibrils in THP-1 cells: The ability of the bispecific binding molecule BA101 and comparator molecule BA102 to induce uptake of A|3 protofibrils by THP-1 human monocytic cells was evaluated. The results indicate that both binding molecules can induce uptake in a concentration-dependent manner (Figure 33). The calculated EC50 values are listed in Table 22.

[0449] Table 22: Uptake ofA61-42 protofibrils in THP-1 cells (Mean±SEM, n=4)

[0450] Ex vivo phagocytosis in human AD brain: The ability of the bispecific binding molecule BA101 and comparator molecule BA102 to induce clearance of A|3 plaques by macrophages in AD brain was evaluated. Compared to negative control samples pre-incubated with an isotype control IgGl antibody, A|3 plaques were reduced in a concentration-dependent manner after pre-incubation with BA101 and BA102. The results indicate that both BA101 and the comparator BA102 can induce plaque clearance by macrophages (Figure 34). The calculated IC50 values for % A|3- immunopositive area are listed in Table 23. Table 23: % A6-immunopositive area (Mean ± SEM, n=13)

[0451] EXAMPLE 20

[0452] Evaluation of immunotoxicity

[0453] This example describes the evaluation of immunotoxicity of the bispecific binding molecule generated and produced in Example 16 in cellular assays for antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and in a human blood loop system.

[0454] Materials and methods

[0455] ADCC measurements: To investigate the effector function of bispecific binding molecule BA101 and comparator molecule BA102, an ADCC reporter assay with Jurkat effector cells (Promega; #G7018) was used. The cells stably express the FcyRllla receptor, V158 (high affinity) variant, and an NFAT response element driving expression of firefly luciferase as a measurement of ADCC activity. Antibody coated target cells bind with their antibody Fc part to FcyR on the effector cells, which triggers luciferase activity in the engineered effector cells. Ramos cells (Sigma, cat: 85030802), which express high levels of hTfRl on the cell surface, were used as target cells. Effector and target cells were used in a ratio of 6:1, with and without serially diluted test constructs. Controls used were the BA102 antibody alone, i.e. lacking an hTfRl-binding scFv, as negative control and the monoclonal antibody rituximab (MabThera; Roche) as positive control. Target cells coated with test constructs were plated in a 96 well assay plate (Corning, #3917), mixed with effector cells and incubated for 18 h at 37°C with 5% CO2. After 18 h incubation, Bio-Gio luciferase reagent was added, and the luciferase signal was quantitated in a SPARK plate reader (Tecan). ADCC fold induction was calculated by dividing the signal obtained in the presence of indicated amount of test construct by the signal obtained in the absence of test construct.

[0456] CDC analysis: Complement activity is initiated by Clq binding to an Fc part of e.g. an antibody, which further leads to binding of other complement factors forming membrane attack complex (mac) that leads to cell death. To evaluate whether the bispecific binding molecule BA101 and comparator molecule BA102 trigger any CDC activity by enabling Clq binding to their respective Fc parts, Ramos cells (Sigma, cat: 85030802) were used as target cells for CDC analysis. To measure the cell death, Ramos cells were labeled with cell viability dye, Calcein-AM (Sigma, 17783). These labeled cells were then treated with serially diluted test molecules in the presence of pooled human complement serum (Innovative Research Inc, #39337) for 4 h at 37°C, 5% CO2. As positive control, rituximab was also tested. Treated cells were acquired using a BD Lyric flow cytometer (BD Biosciences). Samples were analyzed using FlowJo software (BD Biosciences). The frequency of cell death was determined on calcein AM quenched gated cells and plotted against concentration of the tested binding molecules or control.

[0457] Human blood loop: Blood from six healthy human volunteers (above 50 years of age) was used to investigate if and to what extent the bispecific binding molecule BA101 and comparator molecule BA102 induced cytokine release, complement activation and cell activation in freshly collected, circulating blood. The assessment was performed using an ex vivo blood loop test system (ID. Flow; Immuneed). BA101 and BA102 were evaluated at the five concentrations: 2 pM, 667 nM, 222 nM, 74.1 nM and 24.7 nM. Appropriate assay controls with known effects on the test parameters were included, i.e., lipopolysaccharide (LPS), alemtuzumab (anti-CD52), cetuximab (anti-EGFR) and ANC28.1 (anti-CD28). Alemtuzumab was included as a reference antibody with manageable cytokine release in the clinic by corticosteroid treatment prior to each administration. Blood parameters, including platelet (PLT), white blood cell (WBC) and red blood cell (RBC) counts, were also evaluated to assess any effects on blood cell viability.

[0458] Results

[0459] ADCC: The results are shown in Figure 35. Rituximab is known to be a strong inducer of ADCC, and this was verified in the assay setup. No ADCC activity was observed for the bispecific binding molecule BA101, which was similar to the negative control BA102. CPC: The results are shown in Figure 36. The antibody rituximab was used as a positive control to verify CDC activity. The bispecific binding molecule BA101 did not mediate any CDC activity, in similar to the negative control antibody BA102.

[0460] Human blood loop: At all concentrations of BA101 and BA102 tested in the blood loop system, cell counts were similar to those in the vehicle group. In addition, no hemolysis or macroscopic clots were observed in any of the test item samples. Addition of BA101 or BA102 did not result in any significant cytokine release (IFN-y, IL-2, IL-6, IL-8 and TNF) or complement activation (C3a and C5a) at any of the five concentrations tested. In addition, neither antibody had any effect on cell activation (i.e., frequency of CD69 positive T, B and NK cells, CD107a positive NK cells, CDllb positive granulocytes and CD83 positive monocytes), compared to the vehicle control. In conclusion, no effect was observed of BA101 or BA102 on cytokine release, complement activation or cell activation in circulating human blood at any of the concentrations tested.

[0461] EXAMPLE 21

[0462] Brain and plasma exposure in Tg-ArcSwe x hTfRl knock-in mice

[0463] This example describes the in vivo exposure of the bispecific binding molecule generated and produced in Example 16 in plasma and brain, following intravenous dosing in Tg-ArcSwe x hTfRl-KI mice.

[0464] Materials and methods

[0465] To evaluate the in vivo exposure of the bispecific binding molecule BA101 in comparison to the control antibody BA102, brain and plasma concentrations were investigated 48 h post intravenous (i.v.) dosing in Tg-ArcSwe x hTfRl-KI mice.

[0466] Tg-ArcSwe x hTfRl-KI mice were generated by crossing Tg-ArcSwe male mice on a C57BL / 6 BomTac background with hTfRl-KI females (Example 6). The Tg- ArcSwe mouse model is an Alzheimer's disease (AD) model with mice expressing human APP, including two AD-linked mutations: Arctic (E693G) and Swedish (KM670 / 671NL).

[0467] Tg-ArcSwe x hTfRl-KI mice at 20 months of age received a single i.v. injection of 40 nmol / kg of BA101 or BA102. The animals were then terminated at 48 h post dose by perfusion. Terminal blood samples were collected from the orbital plexus into BD microtainer K3EDTA tubes from anaesthetized mice. The samples were inverted and centrifuged at 2400 x g for 10 min at 4°C. Plasma was extracted and transferred to Eppendorf tubes and frozen at -80°C. Immediately following blood sampling, the abdomen of the animals was cut open and a cannula (21 G) was inserted into the left ventricle of the heart. A small cut was made in the right atrium and transcardial perfusion was performed with ice cold PBS. Following perfusion, brains were extracted and separated into left and right hemispheres. The olfactory bulb and cerebellum were removed from the left hemisphere, after which the left hemispheres were weighed and snap frozen on dry ice and stored at -80°C until further preparation and analysis of the concentrations of injected test constructs, using a Meso Scale Discovery (MSD) based assay.

[0468] For brain concentration measurements, frozen left hemispheres were thawed on ice and homogenized in Tris-buffered saline (TBS) containing complete protease inhibitor and PhosSTOP phosphatase inhibitor (#11836145001 and #04906837001, Roche) by automated bead homogenization using MP Biomedical's FastPrep-24 5G system with Lysing Matrix D for 5 s at 6 m / s. Triton X-100 (#X100, Merck) was added to the homogenate resulting in a final Triton X-100 concentration of 0.5% and a weight to volume ratio of 1:10. Homogenates were vortexed for 10 s and centrifuged at 16 000 x g for 1 h at 4°C, after which supernatants were collected and used for brain exposure measurements.

[0469] Brain and plasma concentrations of BA101 and BA102 were determined using a custom MSD assay detecting the human Fc domain. A 96-well MSD plate (#L15XA-3) was coated overnight at 4°C with 25 ng / well goat anti-human IgG, Fey fragment specific antibody (#109-005-098, Jackson Immuno Research Europe Ltd) diluted in lxPBS (#09-9400-100, Medicago AB). The coat was removed, and wells were blocked with 1% Blocker A (#R93BA-4, MSD) in PBS-0.05% Tween20 (PBS-T) (#09-9410-100, Medicago AB). Following 4x wash with lxPBS-T, the samples and test construct calibrators diluted in 1% Blocker A in PBS-T were added to plate and incubated in room temperature (RT) for 2 h at 900 rpm. Detection of bound molecules was done by sequential incubations for 1 h at 900 rpm at room temperature with the secondary antibody (mouse anti-human IgG #3850-1-1000, MT145, Mabtech), followed by 1 h at 900 rpm at room temperature incubation with the SULFO-TAG conjugated anti-mouse detection antibody (R32AC-1, MSD). Secondary and detection antibodies were both diluted to 25 ng / well in 1% Blocker A in PBS-T and 4x wash with lxPBS-T was performed between all incubation steps. Following the last wash, 2X Read Buffer T (#R92TC, MSD) was added prior reading the plates in an MSD SECTOR Imager. The test construct concentration in the samples was evaluated with the MSD Discovery Workbench software, using a 4PL curve fitting algorithm and curve weighting 1 / Y2 for the corresponding test construct calibrator curve.

[0470] Results

[0471] A higher brain exposure was observed 48 h post-dose for BA101 as compared to BA102 (Figure 37A). As shown in Figure 37B, the plasma exposure 48 h post-dose of BA101 was lower compared to BA102, indicating hTfRl engagement and clearance of test construct from the plasma to hTfRl expressing tissues. Consequently, the Brai Plasma ratio was higher for BA101 compared to BA102 (Figure 37C). Taken together, the data supports the conclusion that the bispecific binding molecule BA101 undergoes hTfRl-mediated BBB transport.

[0472] EXAMPLE 22

[0473] In vivo target engagement

[0474] This example describes the target engagement of the bispecific binding molecule generated and produced in Example 16 in brain, following intravenous dosing in Tg-ArcSwe x hTfRl-KI mice.

[0475] Materials and methods

[0476] In vivo target engagement with brain amyloid |3 pathology was investigated at 48 h post-dose in crossed Tg-ArcSwe x hTfRl-KI mice. The mice were treated and terminated as described in Example 21. Following termination with perfusion and brain extraction, the brains were separated into left and right hemispheres, and the right hemispheres were placed in 4% formaldehyde and stored at 4°C for 24 h, after which they were rinsed in cold PBS, transferred to cold 30% sucrose solution prepared in PBS and stored at 4°C for further immunohistochemistry (IHC) processing. In vivo target engagement of amyloid |3 by the bispecific binding molecule BA101 and control molecule BA102 was studied using a qualitative immunohistochemistry (IHC) analysis. In brief, the right hemispheres in sucrose were embedded in O.C.T. compound (LAMB / OCT, ThermoFisher) and fast-frozen in dry ice. Embedded right hemispheres were sectioned and sagittal 20 pm slides were collected onto Superfrost cryoslides (J 1800AM NZ, ThermoFisher) and air-dried prior to IHC. The brain sections were pre-treated for 20 min with 4% PFA (HL96753.1000, HistoLab, Sweden) followed by 5 min wash with dH2O and 5 min incubation with 70% FA for antigen retrieval. After washing 2x 10 min with 1XPBS, the slides were blocked with M.O.M. Mouse IgG Blocking Reagent (MKB-2213-1, Vector Laboratories) for 1 h at room temperature. Primary antibodies were diluted in 1XPBS 0.1% Triton X-100 and incubated over night at 4°C, and secondary antibodies were diluted in 1XPBS and incubated for 1.5 h at room temperature.

[0477] Amyloid |3 was visualized with rabbit antibodies anti-|3-amyloid 1-40 (2 pg / ml) (in house) and anti-|3-amyloid 1-42 (1 pg / ml) (805504, BioLegend) and Alexa488 donkey anti-rabbit IgG H+L (1:500) (A21206, Invitrogen). The tested compounds were visualized with Alexa647 donkey anti-human IgG H+L (1:500) (709-605-149, Jackson ImmunoResearch). All incubations were conducted in a PBS humidified chamber. Slides were washed in lxPBS in a cuvette (5x 5 min) after incubation. Sections were mounted with Fluoromount-G (00-4958-02, Invitrogen) and images were captured using a Leica DM6 B slide scanner microscope equipped with a HC PL APO lOx / O.75 CS2 objective (Leica, #11506517).

[0478] Results

[0479] The resulting images are shown in Figure 38. Co-localization of hlgG and amyloid |3 antibodies was observed, illustrating extensive amyloid |3 target engagement for BA101 in Tg-ArcSwe x hTfRl-KI brain (Figure 38A) while a low amyloid |3 plaque co-localization was seen for the BA102 comparator antibody lacking the hTfRl-binding module (Figure 38B) at 48 h post-dose. EXAMPLE 23

[0480] Evaluation of competition with transferrin

[0481] This example describes the competition of the bispecific binding molecule generated and produced in Example 16 with transferrin binding to hTfRl.

[0482] Materials and methods

[0483] In order to evaluate the binding competition between transferrin and the disclosed bispecific binding molecule BA101, K562 lymphoblast cells (Sigma / ECACC) were first incubated with human Fc block (BD Pharmingen, 564220) for 15 min at 4°C, to block non-specific Fc receptor-mediated antibody binding. The cells were subsequently incubated with serially diluted test constructs along with a fixed concentration of Alexa Fluor 647 conjugated, human holo-transferrin (Invitrogen / Thermo Fischer, T23366) for 30 min at 4°C. After the incubation, the cells were washed 3 times in staining buffer (1% BSA, 0.1% Sodium Azide in IX DPBS). Transferrin bound to hTfRl on cell surfaces was captured using a BD Lyric flow cytometer (BD Biosciences). The samples were analyzed using FlowJo software (BD Bioscience) and the mean fluorescence intensity of bound transferrin was plotted using GraphPad Prism software (GraphPad Software Inc).

[0484] Results

[0485] Figure 39 shows that there is no binding competition to TfRl between the bispecific binding molecule BA101 and transferrin. When non-labeled holo- transferrin was used as positive control for competition, the signal from binding of labeled (A647) transferrin was reduced in a concentration dependent way. The experiment illustrates that bispecific binding molecule BA101, partly directed against hTfRl, do not compete directly for the same epitope as transferrin and does not negatively affect the ability of the endogenous ligand transferrin to bind to the receptor. EXAMPLE 24

[0486] Characterization of binding to human Fc gamma receptors

[0487] This example describes the binding of the bispecific binding molecule BA101 generated and produced in Example 16 to human Fc gamma receptors (FcyR) by SPR.

[0488] Materials and methods

[0489] Human FcyRI (#HUGR1-U), human FcyRlla [167H] (WHUGR2AH-U), human FcyRlla [167R] (WHUGR2AR-U), human FcyRI I b (WHUGR2B-U), human FcyRI I la [176F] (WHUGR3AF-U), human FcyRllla [176V] (WHUGR3AV-U), human FcyRII lb [NA1] (#HUGR3B1-U), and human FcyRI II b [NA2] (#HUGR3B2-U) (all from Gamma Proteins) were reconstituted with water to a final concentration of 1 mg / ml. Aliquots were made and stored at -80 °C until analysis.

[0490] Binding interactions between FcyR and binding molecules BA101 and BA102 were evaluated by SPR using a Biacore 8K or 8K+ instrument (Cytiva) according to standard procedures. 5 pg / ml of BA101 or BA102 diluted in 10 mM sodium acetate, pH 4.5 (WBR100350, Cytiva) were immobilized on a CM5 sensor chip (WBR100399, Cytiva). FcyR was then injected over the chip using a 2-fold dilution series in five steps starting at 25 nM for FcyRI and 1000 nM for the other seven FcyR variants. The interaction was measured using the single cycle kinetics method with a contact time of 120 s of each concentration followed by a dissociation time of 600 s. Regeneration of the surface between cycles was done by injecting 10 mM glycine- HCI pH 3.0 (WBR-1003-57, Cytiva). The binding data was fitted to a 1:1 interaction model. In all SPR experiments, lxHBS-EP+ (# BR100669, Cytiva) was used as running buffer and to dilute analytes. Experiments were performed at 25 °C.

[0491] Results

[0492] The data confirm that BA101 binds to FcyRI with a similar affinity as the comparator molecule BA102 (Figure 40). Both BA101 and the comparator molecule BA102 bound weakly to the other seven FcyR variants. EXAMPLE 25

[0493] Characterization of binding to human neonatal Fc receptor

[0494] This example describes the binding of the bispecific binding molecule generated and produced in Example 16 to human neonatal Fc receptor (hFcRn).

[0495] Materials and methods

[0496] Binding to hFcRn was assessed at pH 6.0 using the Lumit® FcRn Binding Immunoassay kit (#W1151, Promega) according to manufacturer's instructions. The assay is based on a competition format, utilizing NanoBiT® technology to generate a bioluminescent signal that reflects FcRn-IgG binding interactions. Briefly, the binding molecules BA101 and BA102 were adjusted to pH 6.0 with pH adjustment buffer (Promega). The binding molecules were diluted down to 26.8 pM and titrated down a 12-point 4-fold dilution series. The pH adjusted sample dilution series was mixed with tracer human IgGl labeled with a large BiT subunit (Tracer-LgBiT; Promega) in a 1:1 volume ratio. Then, biotinylated hFcRn complexed with streptavidin-small BiT peptide (hFcRn-SmBiT; Promega) was added in a volume equal to the total volume of the sample and Tracer-LgBiT mixture. The plate was incubated for 30-60 min shaking at 300-400 rpm in the dark at room temperature. After the incubation, Lumit® FcRn Detection Reagent (Promega) was added to each well followed by another incubation at room temperature for 3-5 min. The luminescence signal was read with a Spark Multimode Microplate Reader (Tecan) and the data was analyzed with a four-parameter variable slope non-linear fit.

[0497] Results

[0498] The data confirmed that BA101 binds to hFcRn with a similar affinity as the comparator molecule BA102 (Figure 41).

[0499] EXAMPLE 26

[0500] Serum stability of bispecific binding molecule

[0501] This example describes the stability of the binding of bispecific binding molecule BA101 to human TfRl and A|3 protofibrils by SPR after incubation in human serum. Materials and methods

[0502] Serum stability was assessed by diluting the bispecific binding molecule BA101, generated and produced in Example 16, to 8 pM in human serum (#H45422, Sigma) or PBS (#14190, Thermo Scientific). Samples were incubated in the conditions described in Table 24. At the end of each timepoint, the sample was moved to -80 °C and stored until analysis.

[0503] Table 24: Serum stability sample conditions

[0504] Binding of the bispecific binding molecule BA101 to A|3 protofibrils and hTfRl was evaluated by SPR using a Biacore 8K or 8K+ instrument (Cytiva) according to standard procedures. In all SPR experiments, lxHBS-EP+ (#BR100669, Cytiva) was used as running buffer and to dilute analytes and capture ligands. Experiments were performed at 25 °C. Binding to A|3 protofibrils was measured according to the methods described in Example 17 with the only deviation being that 16 nM A 1-42 protofibrils were immobilized on the CM5 chip.

[0505] BA101 binding to hTfRl was determined using single cycle with capture. For measurement, 30 pg / ml of an anti-idiotypic antibody was immobilized on a CM5 chip (#BR100399, Cytiva) and 0.04 pg / ml BA101 was captured for each cycle. Following capture, hTfRl (produced as described in Example 17) was injected using a 3-fold dilution series in five steps starting at 1500 nM. Each concentration had an association time of 120 s, and the dissociation time was 600 s. Regeneration of the surface between each cycle was done by injecting 10 mM glycine-HCI, pH 1.5 (#BR100354, Cytiva). The binding data was fitted using a 1:1 interaction model. The normalized affinity highlights the change in affinity in the different samples as compared to the PBS sample stored directly at -80 °C (sample 1). Results

[0506] Storage under the tested conditions had no effect on the apparent affinity of BA101 binding to immobilized A|3 protofibrils or on the affinity of BA101 to hTfRl, as shown in Figure 42. Overall, the results demonstrate a high stability of BA101 in human serum for up to 3 weeks at 37 °C.

[0507] EXAMPLE 27

[0508] Plasma exposure in scid hFcRn mice

[0509] This example describes plasma exposure of the bispecific binding molecule BA101, generated and produced in Example 16, following intravenous dosing in scid hFcRn mice.

[0510] Materials and methods

[0511] To investigate any impact on the interaction of BA101 with the human neonatal Fc receptor (hFcRn) and to evaluate plasma pharmacokinetic (PK) doselinearity of BA101, the plasma concentrations of BA101 at three different dose levels, and of control compound BA102 at one dose level, were investigated up to 42 days (1008 h) post intravenous (i.v.) dosing in scid hFcRn mice.

[0512] The scid hFcRn mice (scid FcRn- / - hFcRn (32) Tg, HuPK™, #018441, The Jackson Laboratory) are homozygous for the knockout mutation of the FcRn a-chain (FcgrttmlDcr) and instead express a human FcRn a-chain (FCGRT) transgene. The expression of human FcRn (hFcRn) in these transgenic mice creates a better model for further prediction of antibody plasma PK in humans, compared to wild type mice. Furthermore, the scid allele makes the mice immunodeficient, preventing the formation of anti-drug antibodies upon treatment with humanized antibodies. However, the scid hFcRn mice only express endogenous mouse transferrin receptor 1 (TfRl) and lack human TfRl.

[0513] Ten weeks old scid hFcRn mice received an i.v. injection of BA101 at doses of 5.7, 17 or 57 nmol / kg, or of BA102 at a dose of 57 nmol / kg. Blood was sampled from the saphenous vein into Microvette CB300 K2-EDTA tubes at the following timepoints after dose administration: 5 min, 4 h, 24 h, 72 h, 168 h, 336 h, 672 h, 840 h, and 1008 h. The samples were inverted and centrifuged at 2400 x g for 10 min at 4 °C. Plasma was extracted and transferred to polypropylene tubes and frozen at -80 °C until further analysis of the concentrations of injected test constructs, using a custom Meso Scale Discovery (MSD) based assay for detecting the human Fc domain, as described in Example 21.

[0514] Results

[0515] Mean dose-normalized plasma concentration-time profiles of BA101 are shown in Figure 43A. The dose-normalized profiles of BA101 were overlapping, which supports the finding of a dose-linear PK in plasma of BA101, in the dose-range studied (5.7-57 nmol / kg) in scid hFcRn mice. Furthermore, the dose-normalized plasma concentration-time profiles of BA101 resembled the profile of control compound BA102 (Figure 43B), indicating no adverse impact on the interaction of BA101 with hFcRn, and hence antibody recycling, compared to control BA102.

[0516] EXAMPLE 28

[0517] Plasma and brain exposure in hTfRl-KI and 5xFAD x hTfRl-KI mice, and target engagement in 5xFAD x hTfRl-KI mice

[0518] This example describes the plasma and brain exposure as well as brain target engagement of the bispecific binding molecule BA101 generated and produced in Example 16, following intravenous dosing in hTfRl-KI and 5xFAD x hTfRl-KI mice.

[0519] Materials and methods

[0520] To further assess the plasma and brain exposure of BA101 and to evaluate brain target engagement of BA101 in comparison to control compound BA102 over time, brain and plasma samples were collected at multiple timepoints up to 168 h or 504 h, from hTfRl-KI (BA101 only) and 5xFAD x hTfRl-KI mice after i.v. dosing of the test constructs.

[0521] The 5xFAD x hTfRl-KI mice were generated by crossing 5xFAD male mice on a C57BL / 6J background (Northwestern University) with human transferrin receptor 1 knock-in (hTfRl-KI) females (see Example 6 for the generation of hTfRl-KI mice). The 5xFAD mouse model is an Alzheimer's disease (AD) model with mice expressing human APP and PSEN1 transgenes with a total of five AD-linked mutations, including the Swedish (K670N / M671L), Florida (1716V), and London (V717I) mutations in APP, and the M146L and L286V mutations in PSEN1. Seven months old, female hTfRl-KI mice and 5xFAD x hTfRl-KI female littermates received a single i.v. injection of BA101 or BA102 at a dose of 57 nmol / kg. Serial blood samples were collected from each animal up until its individual termination timepoint and the samples were processed to plasma as described in Example 27. Following termination by deep sedation and intracardiac perfusion with PBS, brains were extracted and separated into the left and right hemisphere. The left hemispheres were processed as described in Example 21 and the right hemispheres were placed in 4% formaldehyde and stored at 4 °C for 24 h, after which they were rinsed in cold PBS, transferred to cold 15% sucrose solution prepared in PBS and stored at 4 °C for further immunohistochemistry (IHC) processing.

[0522] Plasma and brain exposure: Plasma concentrations of BA101 and BA102 were determined using a custom MSD assay detecting the human Fc domain as described in Example 21. For brain concentration measurements, frozen left hemispheres were thawed on ice and homogenized at a weight to volume ratio (w / v) of 1:5 in Trisbuffered saline (TBS) containing complete protease inhibitor and PhosSTOP phosphatase inhibitor (#11836145001 and #04906837001, Roche) by automated bead homogenization using MP Biomedical's FastPrep-24 5G system with Lysing Matrix D for 5 s at 6 m / s. To determine the brain concentrations of BA101 and BA102, the homogenate samples and test construct calibrators were treated with 5% acetic acid and neutralized with IM Trizma buffer. Neutralized samples were further diluted with 1% Blocker A prior to loading samples to the MSD plate. The MSD assay setup and incubation conditions were the same as described for plasma samples (see Example 21).

[0523] Histology: In vivo target engagement of amyloid beta (AP) by the bispecific binding molecule BA101 and control molecule BA102 was studied using a quantitative IHC analysis. From 15% sucrose / PBS storage at 4 °C, right brain hemispheres were trimmed, transferred to cryomolds, embedded in OCT medium, frozen in isopentane, and stored at -80 °C until sectioned sagitta I ly at 10 pm thickness. Using a Leica CM1950 or Thermo Scientific NX70 cryotome, five consecutive sections were collected per level, discarding the next 25. This was repeated for 12 levels, resulting in 60 sections. Levels were chosen based on Paxinos and Franklin's atlas ("The Mouse Brain in Stereotaxic Coordinates", 2nd edition, 2001). Sections began ~0.2 mm lateral from the brain midline and were stored at -20 °C.

[0524] Five sections per mouse at the same level of sectioning were selected for systematic random sampling. Sections were air-dried for 45 min and washed in PBS for 10 min. Unspecific binding sites were blocked by incubating the sections with 10% normal donkey serum in PBS containing 0.1% Tergitol for 60 min. The sections were then washed three times in PBS for 5 min each. The sections were incubated with a secondary antibody, donkey anti-human Fey Fragment Specific Alexa 645- conjugated (#JAC709605098, Szabo-Scandic; 1:500 dilution), for 60 min in a light- protected, humidified chamber. After incubation, the sections were washed three times in PBS for 5 min each. Primary antibody incubation was conducted overnight at 4 °C with rabbit polyclonal anti-LOC (WAB2287, Sigma-Aldrich; 1:1000 dilution). The sections were washed three times in PBS for 5 min each before a subsequent incubation with a secondary antibody, donkey anti-rabbit IgG H+L Alexa 555- conjugated (#abl50066, Abeam; 1:500 dilution), for 60 min. This was followed by three washes in PBS, each lasting 5 min. The sections were incubated with DAPI working solution for 15 min, washed twice in PBS for 5 min each, and then washed in ddFhO for 5 min. Finally, sections were mounted with Mowiol and coverslips using a Leica CV5030 coverslipper. On a Zeiss AxioScan Z1 microscope, whole-brain images were captured at lOx magnification and high magnification images at 20x. Regions included the cortex, hippocampus, subiculum, and lateral ventricle.

[0525] Images from 5xFAD x hTfRl-KI mice, stained with LOC and secondary antihuman IgG, were quantitatively analyzed using Image Pro 10 software (Media Cybernetics). Regions of interest (ROIs) were selected to exclude artifacts. Target engagement and co-localization of test substances (IgG) on A|3 plaques (LOC) was assessed by quantifying the signal intensity of the test constructs (BA101 or BA102) on plaques, the average size, the fluorescence intensity, and the average number of signals per square millimeter. Background correction was applied, and immunoreactive objects were detected using appropriate thresholding and morphological filtering based on size and shape. Quantified object features reported in the current example cover i) the percentage of cumulative object area relative to the ROI size (immunoreactive area), giving the percentage of area covered by colocalized IgG and LOG signal points; and ii) the number of objects normalized to ROI size (object density), giving the density of colocalized IgG and LOG signal points.

[0526] Results

[0527] The plasma exposure of BA101 was similar in diseased (5xFAD x hTf Rl-KI) and non-diseased ( hTf Rl-KI ) mice, as seen in the plasma concentration versus time profiles shown in Figure 44. In the presence of A|3 pathology, and hence target, in 5xFAD x hTfRl-KI mice, BA101 brain concentrations accumulated over time, even when plasma concentrations decline, achieving a higher brain exposure compared to non-diseased hTfRl-KI mice (Figure 44). Brain concentrations of BA101 in nondiseased mice declined more in parallel with plasma (Figure 44).

[0528] As a result of TfR-mediated transport, plasma concentrations were lower, and brain concentrations substantially higher, for BA101 compared to BA102 in 5xFAD x hTfRl-KI mice after a single i.v. injection, as shown in Figures 45A and 45B. At 168 h (7 days) post-dose, the mean brain-to-plasma ratio was 43% for BA101 while it was around 0.9% for BA102 in 5xFAD x hTfRl-KI mice (Figure 450), demonstrating the substantial effect of hTfRl-mediated uptake with BA101, resulting in immense target engagement in mice with amyloid pathology.

[0529] When comparing A|3 target engagement (human IgG on LOG) between the bispecific binding molecule BA101 and control molecule BA102 at 24 h and 168 h in the cortex, a clear increase in immunoreactive area (Figure 46A) and object density (Figure 46B) were observed for BA101 at both timepoints. Similarly, when comparing the human IgG signal on LOG in the hippocampus, there was a noticeable increase in immunoreactive area as well as in object density in BA101 treated 5xFAD x hTfRl-KI mice compared to BA102 (Figure 46C-D), demonstrating a superior target engagement for the bispecific binding molecule BA101.

[0530] EXAMPLE 29

[0531] In vivo efficacy evaluation in 5xFAD x hTfRl-KI and littermate 5xFAD mice

[0532] This example describes the in vivo efficacy of the bispecific binding molecule BA101 generated and produced in Example 16, following single intravenous dosing in 5xFAD x hTfRl-KI mice. The control molecule BA102 was evaluated in parallel in 5xFAD littermates that lack the hTfRl expression. Materials and methods

[0533] To assess the in vivo efficacy of the bispecific binding molecule BA101 in comparison to the control antibody BA102, levels of AP protofibrils were investigated 168 h post intravenous dosing of BA101 or BA102, both at a dose of 57 nmol / kg, in 5xFAD x hTfRl-KI mice or 5xFAD littermates, respectively. The study was performed in 4.5 months old female mice and the 5xFAD x hTfRl-KI mouse line was generated as described in Example 28. A vehicle (PBS) treated group was included for both genotypes.

[0534] Serial blood samples were collected from each animal up until 168 h and the samples were processed to plasma as described in Example 27. Following termination with intracardiac perfusion, brains were extracted and separated into left and right hemispheres. The left hemispheres were processed as described in Example 21 and further homogenized as described in Example 28. The resulting homogenate was diluted 1:1 with TBS extraction buffer to generate a homogenate with a weight to volume ratio of 1:10. Homogenates were further processed by centrifugation at 16 000 x g for 1 h at 4 °C, after which supernatants were collected. The resulting TBS 16k extracts contain soluble AP and were used for AP protofibril concentration assessment.

[0535] Biochemical assessments: Plasma and brain homogenate concentrations of BA101 and BA102 were determined using a custom MSD assay detecting the human Fc domain as described in Example 28.

[0536] AP protofibril concentrations were determined using a custom MSD bridging assay utilizing the AP protofibril-binding murine antibody mAbl58 and Api-42 protofibrils prepared as described in Example 17. 0.5 pg / mL mAbl58 (BioArctic) in PBS was coated on a standard 96-well MSD plate and incubated overnight at 4 °C. The coat was removed, and wells were blocked with 1% Blocker A (#R93BA, MSD) in PBS-0.05% Tween20 (PBS-T) (#09-9410-100, Medicago AB). After incubation overnight at 4 °C and blocking in 1% Blocker A buffer, the plate was washed and the Api-42 protofibril calibrator and study samples diluted in 1% Blocker A were added and incubated for 2 h RT with shaking at 900 rpm. Detection of bound A protofibrils was performed by sequential incubations 1 h, RT at 900 rpm, first with the biotinylated mAbl58 (BioArctic), followed by the SULFO-TAG conjugated streptavidin (#R32AD, MSD), both at a concentration of 0.5 pg / mL in 1% Blocker A buffer. 4x wash with PBS-T was performed between all incubation steps. Following the last wash, 2X Read Buffer T (#R92TC, MSD) was added prior to reading the plate in an MSD SECTOR Imager. The A|3 protofibril concentration in the samples was evaluated with the MSD Discovery Workbench software, using a 4PL curve fitting algorithm and curve weighting 1 / Y2 for the A 1-42 protofibril calibrator curve.

[0537] Statistical analysis was performed in GraphPad Prism (v. 10.4.2) using one way ANOVA with Sidak post hoc test to compare treatment versus the PBS control in 5xFAD x hTfRl-KI and 5xFAD, respectively.

[0538] Results

[0539] Average brain and plasma exposure of the bispecific binding molecule BA101 and comparator antibody BA102 in 5xFAD x hTfRl-KI and 5xFAD, respectively, were confirmed to be comparable to the results reported in Example 28 and Figure 45.

[0540] Levels of A|3 protofibrils in soluble brain extracts were significantly reduced 168 h post BA101 treatment compared to vehicle (PBS) treated 5xFAD x hTfRl-KI mice (adjusted p-value <0.01) while no reduction was detected in 5xFAD mice treated with BA102 compared to PBS-treated group (Figure 47). The mean reduction of AP protofibril levels following BA101 treatment was 30% compared to PBS- treated animals, demonstrating a potent in vivo efficacy with significant lowering of target levels 7 days after a single i.v. dose.

[0541] EXAMPLE 30

[0542] Evaluation of reticulocyte counts post treatment in hTfRl-KI mice

[0543] This example describes the in vivo evaluation of circulating reticulocyte levels following single intravenous dosing of the bispecific binding molecule BA101, generated and produced in Example 16, in hTfRl-KI mice.

[0544] Materials and methods

[0545] To evaluate the potential effects of the bispecific binding molecule BA101 on circulating reticulocyte levels and other blood cells in vivo, blood samples were collected from hTfRl-KI mice 24 h after i.v. dosing of 114 or 28.5 nmol / kg BA101, corresponding to 20 and 5 mg / kg, respectively. Vehicle (PBS) treated and untreated animals were included as controls. In addition, the comparator antibody BA102 was dosed at 114 nmol / kg as a reference. hTfRl-KI mice were generated as described in Example 6. Blood samples were collected from the saphenous vein 14 days before i.v. injection (pre-dose samples) and at termination 24 h after i.v. injection (post-dose samples). Blood samples were divided into two separate Microvette CB300 K2 EDTA tubes, one tube for whole blood and one tube for further plasma processing. The blood sample used for plasma analysis was kept on ice before further processed to plasma by centrifugation at 2400 g at 4 °C for 10 min, whereas the whole blood sample for hematological analyses was kept at room temperature and analyzed within 4 h post collection.

[0546] Plasma samples were transferred to polypropylene tubes and frozen at -80 °C until further analysis of the concentrations of injected test constructs, using a custom Meso Scale Discovery (MSD) based assay detecting the human Fc domain as described in Example 21.

[0547] Blood samples were diluted 1:7 with cell-pack DCL (Sysmex) and analyzed with the Sysmex XN-1000 automated hematology analyzer according to the manufacturers' recommendations.

[0548] Results

[0549] Exposure of tested molecules in plasma 24 h post i.v. injection was confirmed. hTfRl-KI mice treated with BA101 had lower plasma exposure compared to an equimolar dose of BA102 due to hTfRl-mediated transport (Table 25).

[0550] Table 25: Plasma concentrations in hTfRl-KI mice, 24 h post dose

[0551] LLOQ: Lower limit of quantification, N / A: Not applicable, N.D.: Not determined Hematological analysis revealed no change in group mean either the white or red blood cell compartment, reticulocyte count (Table 26), or reticulocyte fractions including subpopulations 24 h after treatment with BA101. Hemoglobin content of reticulocytes and mature red blood cells were also unaltered following treatment. In addition, no change was seen in test construct treated groups compared to the PBS control group, when these measurements were related to the individual pre-dose sample exemplified by reticulocyte subpopulations in Figure 48.

[0552] Hematological analysis after treatment with the non TfRl-targeting comparator antibody BA102 also did not show any change compared to PBS-treated animals, as expected.

[0553] Table 26: Reticulocyte counts and reticulocyte fraction (%) of red blood cell compartment 24 h post dose

[0554] RET: Reticulocytes, RETfrac: Reticulocyte fraction, RBC: Red blood cells, N / A: Not applicable

[0555] EXAMPLE 31

[0556] In vivo evaluation in non-human primates

[0557] This example describes the plasma and brain exposure and primary in vivo evaluation of the bispecific binding molecule BA101, generated and produced in Example 16, and control antibody BA102, in non-human primates after single intravenous dosing.

[0558] Materials and methods

[0559] To further evaluate in vivo tolerability and exposure of BA101 in plasma and brain over time in relation to control antibody BA102, in vivo tolerability was assessed, and plasma and brain samples were collected at multiple timepoints up to 240 h after single i.v. dosing of non-human primates.

[0560] Female cynomolgus monkeys received a single i.v. bolus injection of BA101 at

[0561] 3 mg / kg (n=3) or 10 mg / kg (n=15), or control antibody BA102 at 20 mg / kg (n=12) . General in-life observations included mortality, cage-side observations, post-dose observations, detailed clinical observations, and individual body weights. Blood was collected at different time points for hematology, cytokine / chemokine and complement factor analysis and for concentration measurements of test constructs.

[0562] Hematology parameters were analyzed in animals up until each animal's individual termination timepoint during a pre-test period (at least 7 days before dosing day), pre-dose (Day 1), and at 24 h (Day 2), 72 h (Day 4) and 240 h (Day 11) after dose.

[0563] Blood samples for cytokine and chemokine concentration measurements were collected from all animals, pre-dose and 2 h after dose. Blood samples were collected in tubes with clot activator and centrifuged within 1 h after collection at

[0564] 4 °C, 3000 g for 10 min. The resulting serum was collected and stored at -80 °C until analyzed using a Luminex bead-based multiplex sandwich immunoassay (ProcartaPlex custom panel, ThermoFisher) according to the manufacturer's instructions.

[0565] Blood samples for the determination of complement factor (C3a and Bb) levels were collected from all animals, pre-dose and 2 h after dose. Blood samples were collected in tubes containing K3EDTA and centrifuged at 4 °C, 3000 g for 10 min. The resulting plasma was collected and stored at -80 °C until analyzed using validated ELISA methods (C3a Plus MicroVue™ EIA, #A032; and Bb Plus MicroVue™ EIA, #A027, both from QuidelOrtho). The minimal required dilution (MRD) was 1:10 for the Bb ELISA and 1:200 for the C3a ELISA.

[0566] Plasma pharmacokinetic (PK) dose linearity was assessed for the bispecific binding molecule BA101 at two dose levels, 3 and 10 mg / kg, and plasma PK was also compared to control antibody BA102 (20 mg / kg), in animals terminated 240 h postdose, with serial blood samples collected (in-life) at the following timepoints: approximately 0.083, 0.5, 2, 4, 8, 24, 48, 72, 120, 168, and 240 h post-dose. Blood samples were collected in K2EDTA containing tubes and gently inverted 10 times and placed on wet ice immediately after collection until centrifugation. The samples were then centrifuged at 4 °C, 2400 g for 10 min. Plasma was extracted and stored at -80 °C until analysis.

[0567] Animals were sacrificed at different timepoints in groups of three up to the final termination timepoint of 240 h to acquire composite brain exposure data over time. Terminal brain tissue (parietal cortex, hippocampus, and striatum) samples for measurements of test construct concentration were collected from animals administered BA101 (10 mg / kg) or BA102 (20 mg / kg) at 4 h (BA101 only), 24 h, 72 h, 168 h, and 240 h post-dose. At time of sacrifice, animals were euthanized by a pentobarbital sodium injection and subjected to a full body perfusion with PBS through the intracardiac route. Brains were collected after perfusion, and the left hemisphere was further dissected to collect parietal cortex, hippocampus, and striatum. Brain samples were weighed and fixed by snap freezing on dry ice and further stored at -80 °C until homogenization and analysis.

[0568] Homogenization and extraction of brain tissues were performed using the following process: samples from individual brain regions were added to TBS extraction buffer (TBS with complete protease inhibitor and PhosSTOP phosphatase inhibitor (#11836145001 and #04906837001, Roche)) at w / v 1:2.5 in lysing matrix D tubes (MP Biomedicals) and allowed to thaw for about 20 min. Homogenization was performed by using a Bead Mill 24 homogenizer at a speed of 6 m / s for 20 s and homogenates were transferred to 1.5 ml protein polypropylene LoBind tubes. An equal volume of 1% TBS-T extraction buffer (TBS extraction buffer w. 1% Triton X- 100) was added to the homogenate and mixed thoroughly, resulting in a final triton concentration of 0.5%. Samples were then centrifuged for 60 min, 4 °C at 16 000 x g. Supernatants (w / v 1:5) were aliquoted in protein polypropylene LoBind tubes and stored at -80 °C prior to analysis.

[0569] Analysis of the test construct concentrations in plasma and brain tissue samples was performed according to qualified methods. For quantification of levels in plasma, an electrochemiluminescence (ECL) ligand binding assay (LBA) was used in which an anti-human IgG antibody (#A80-319A, Bethyl Laboratories) diluted in PBS was coated at 1.0 pg / ml in a multi-array 96 well standard MSD plate overnight at 5 °C. The coat was removed, and wells were washed 3x with PBS 0.1% Tween20 (PBS-T 0.1%) wash buffer, followed by blocking in Blocker Casein in PBS (#37528, Thermo Scientific). Following 3x wash with PBS-T 0.1%, the samples and test construct calibrators diluted in surrogate matrix (Blocker Casein) were added to plate and incubated in RT for 2 h at 600 rpm. Detection of bound molecules was done by sequential incubations for 1 h at 600 rpm at RT with the biotinylated secondary antibody (goat anti-human IgG antibody #2049-08, Southern Biotech), followed by 1 h at 600 rpm at RT incubation with SULFO-TAG conjugated streptavidin (#R32AD, MSD). Secondary and detection reagents were diluted to 0.3 pg / ml and 0.1 pg / ml, respectively, in Blocker Casein and 3x wash with PBS-T 0.1% was performed between all incubation steps. Following the last wash, Read Buffer A (#R92TG, MSD) was added prior to reading the plates in an MSD MESOSCALE QuickPlex SQ120. For quantification of levels in brain tissue samples, a similar LBA approach was used; BA101 / BA102 anti-idiotypic antibody was diluted to 0.5 pg / ml in PBS and used to coat a multi-array 96 well standard MSD plate overnight at 5 °C. The coat was removed, and wells were washed 3x with PBS-T 0.1% wash buffer, followed by blocking in 1% Blocker A (#R93BA, MSD) in lxPBS-0.05% Tween20 (PBS- T, #28352 Thermo Scientific). Following 3x wash with PBS-T 0.1%, the samples and test construct calibrators diluted in surrogate matrix (1% Blocker A in lxPBS-T) were added to plate and incubated in RT for 2 h at 900 rpm. Detection of bound molecules was done by sequential incubations for 1 h at 900 rpm at RT with the biotinylated secondary antibody (goat anti-human IgG antibody #2049-08, Southern Biotech), followed by 1 h at 900 rpm at RT incubation with SULFO-TAG conjugated streptavidin (#R32AD, MSD). Secondary and detection reagents were diluted to 0.1 pg / ml and 0.03 pg / ml, respectively, in 1% Blocker A in lxPBS-T and 3x wash with PBS-T 0.1% was performed between all incubation steps. Following the last wash, 2xRead Buffer T (#R92TC, MSD) was added prior reading the plates in an MSD MESOSCALE QuickPlex SQ120.

[0570] The MRD of the methods were 1:100 for plasma and 1:20 for brain tissue samples. Concentrations in both methods were determined by interpolation from a calibration curve in which relative light units (RLU) values were plotted against test construct concentrations of calibration standards using a 5-parameter logistic (PL) non-linear regression model (weighting factor 1 / Y2, no blank subtracted). Results

[0571] There were no effects attributed to any one of test constructs BA101 or BA102 on mortality, clinical signs, body weight, or brain weights after i.v. dosing and up to the 240 h termination time point, independent of dose level. Similarly, no test construct related effects were observed on cytokine / chemokine (IFN-y, I L-ip, IL-2, IL-4, IL-5, IL-6, IL-8, MCP-1, and TNF-a) concentrations or complement factor (C3a and Bb) levels 2 h post-dose, indicating that no acute phase inflammatory response was elicited upon dosing with the test constructs.

[0572] No test construct related effects were observed on hematology over the course of the study, independent of test construct BA101 or BA102, or their dose level. Mean values of the reticulocyte counts are shown in Figure 49A. Individual post-dose reticulocyte counts were also normalized against individual pre-dose reticulocyte counts and the median values are shown in Figure 49B. A slight increase in reticulocyte counts was observed up to the last timepoint for all test constructs, independent of dose level. This non-adverse observation, together with a slight decrease in hematology parameters such as hematocrit, hemoglobin, and red blood cell counts, is consistent with frequent blood sampling and was not considered to be related to the test constructs.

[0573] The plasma exposure of BA101 increased with increasing dose in an approximately dose-proportional manner in the dose range studied (3 and 10 mg / kg). Mean dose-normalized plasma concentration versus time profiles of BA101 at 3 and 10 mg / kg, and of BA102 at 20 mg / kg, are shown in Figure 50. The lower dose-normalized exposure of the bispecific binding molecule BA101 in comparison to control compound BA102 also indicates peripheral engagement with the transferrin receptor (TfR) and hence TfR mediated disposition of BA101.

[0574] The concentrations of BA101 (10 mg / kg) and BA102 (20 mg / kg) in different brain regions (parietal cortex, hippocampus, and striatum) were measured at 4 h (BA101 only), 24 h, 72 h, 168 h, and 240 h after dose. Mean dose-normalized concentrations are shown in Figure 51. A higher exposure was observed in all investigated brain regions for BA101 compared to BA102. The data provides proof of increased brain uptake of the bispecific binding molecule BA101 with superior brain exposure compared to control antibody BA102 in cynomolgus monkey. EXAMPLE 32

[0575] Design, production and SEC analysis of additional stabilized hTfRl binding molecules In the single-chain variable fragment (scFv) format, hydrophobic patches within the former V / C interface of the full-length antibody become exposed (Nieba et al (1997), Prot Eng 10(4):435-444). These newly accessible hydrophobic areas, particularly within the VH domain, can serve as binding sites for preexisting antidrug antibodies (PE-ADA) when the scFv, or construct comprising it, is administered to a subject (Holland et al (2013), J Clin Immunol 33:1192-1203). Previous work has shown that mutation of certain hydrophobic residues within the former V / C interface of the VH domain to more hydrophilic residues leads to a reduction of such potential PE-ADA reactivity (Johansson et al (2023), mAbs 15( 1) :2215887) .

[0576] Eleven variants of the disulfide-stabilized VH domain of h26D3-HC6 were designed, containing either single or double substitutions of hydrophobic amino acid residues to more hydrophilic and / or less bulky amino acids residues. The variants are listed in Table 27. All VH domain variants were expressed in the form of scFv domains having the VL first orientation. A flexible (G4S)4 linker connects the VL domain to the VH domain within each scFv. For purification purposes, a Hise tag and (G4S)4 linker was added to the N-terminus of the VL domain (combined tag sequence given by SEQ ID NO:135).

[0577] Table 27: VH variants of the hTfRl binding scFv molecule h26D3-HC6_DS, VL-first These eleven scFv variants were produced in CHO cells and purified with IMAC plus preparative SEC as described in Example 10. The SEC fractions containing monomeric scFv were collected, sterile filtered and brought to 1 mg / ml final concentration in PBS, pH 7.4. The recovered molecules were analysed by CE-SDS, reducing and non-reducing, by loading 2.5 pg of respective molecule on a LabChip (GXII Touch™ HT Chip, PerkinElmer). Samples were prepared with the ProteinEXact Assay Reagent Kit (PerkinElmer). Instrument Assay used: LabChip GXII Touch HT Protein Characterization System (Perkin Elmer). Sample treatment: Before loading, samples were treated with DTT for reduced conditions, and both non-reduced and reduced samples were heated at 70 °C for 10 min. The results showed bands of expected size and high purity for all molecules.

[0578] Each scFv variant produced was then injected onto an SEC column and analyzed as described in Example 11. The results of the analytical SEC experiment are shown in Table 28 and demonstrate that all variants of the molecule were isolated with a high monomer content, similar to the DS-stabilized scFv molecules produced in Example 11.

[0579] Table 28: Results from aSEC of hTfRl binding scFv variants

[0580] EXAMPLE 33

[0581] Thermal stability of additional variants of hTfRl binding molecules

[0582] From the production and analysis of hTfR binding molecules in Example 32, six variant scFv:s (SEQ ID NO:153-158) were selected and subjected to a thermal stability study. Monomer stability of scFv samples was evaluated by HPLC SEC analysis. The scFv molecules, purified and stored in PBS, were subjected to temperature hold for one, two or four weeks at temperatures 4°C, 25°C, 40°C and frozen at -75°C. At each timepoint, samples of each variant from each temperature were analyzed by HPLC-SEC as described in Example 32. At the initiation of the study, frozen samples were thawed and analyzed, and denoted "TO". In addition, for each of the six molecules a sample was subjected to freeze-thaw stress. Molecules were subjected to 3x freeze-thaw cycles. Freezing was done at -75 °C for at least 16 h and thawing was done at ambient temperature until complete thawing of the sample (up to 60 min). Samples are denoted FT_3x and were analyzed immediately after the third freeze-thaw cycle completion.

[0583] All six scFv molecules were stable monomers at the tested conditions. The results for the six selected scFv molecules from samples collected as TO or after temperature hold at 40 °C for 1-4 weeks respectively are shown as stacked chromatograms in Figure 52A-F. As seen in Figure 52, the main peak for all samples has a retention time at ~11 min, as expected for a monomeric scFv. All samples have a high monomer content at all tested conditions, demonstrating that the stabilized DS design is maintained in all six variants.

[0584] EXAMPLE 34

[0585] Immunogenicity in silica of additional variants of hTfRl binding molecules The six scFv sequences analyzed in Example 33 (SEQ ID NO:153-158) and the variant "h26D3 HC6 DS, VL-first" (SEQ ID NO:131; Example 10) were subjected to an in silica analysis of immunogenicity risk, based on identification of potential T cell epitopes in the respective amino acid sequence. The assessment was done using iTope-AI (Abzena Ltd., Cambridge, UK). In addition, the sequences were analysed for homology to known T cell epitopes, previously identified by ex vivo EpiScreen™ analysis of other protein sequences. Homology scores against known T cell epitopes (Bryson et al (2010), BioDrugs 24(1) :l-8) are shown as TCED in Table 29.

[0586] The immunogenicity assessment of the respective protein sequence was performed using overlapping 9mer peptides, tested against 46 MHC class II allotypes used in the iTope-AI platform. Individual peptides, together spanning the whole sequence, were given a binding score from 0 to 3 for each allotype, and those scores were added together to provide a "Position Risk Score". The "Total Score" for the respective test protein in Table 29 was calculated by adding the "Position Risk Scores" obtained for all individual peptides. The highest "Position Risk Score" for each sequence was denoted "Hotspot Max" as also shown in Table 29.

[0587] 5 Taken together, all seven tested protein sequences show very similar results, indicating that no novel risk sites of concern were identified in any of the assessed variants of the TfR binding molecule.

[0588] Table 29: In silico immunogenicity assessment of hTfRl-binding scFv molecules

[0589] 10

[0590] EXAMPLE 35

[0591] SPR analysis of binding of additional scFv variants to hTfRl and cTfRl

[0592] The 11 scFv molecules produced and purified as described in Example 32 and the variant "h26D3 HC6 DS, VL-first" (SEQ ID NO:131; Example 10) were evaluated

[0593] 15 for binding to human and cynomolgus TfRl using SPR in a Biacore 8K instrument (Cytiva). For this, 30 pg / ml of DYKDDDDK Tag Monoclonal Antibody (FG4R) (ThermoFisher SCIENTIFIC. #14-6681-80) was immobilized on a CM5 sensor chip (Cytiva. #BR100399) using the amine coupling kit type 2 (Cytiva. #BR100633) according to the manufacturer's instruction. hTfRl (SEQ ID NO:99) or cTfRl (SEQ ID

[0594] 20 NQ:100), each containing a FLAG tag, was captured on the chip via injection of a 5 pg / ml solution for 60 s at 10 pl / min flow rate. Each scFv variant was injected over the chip using a 3-fold dilution series in five steps starting at 700 nM (hTfRl) or 1500 nM (cTfRl). The interaction was measured using the single cycle kinetics method with a contact time of 120 s at a flow rate of 30 pl / min followed by a dissociation

[0595] 25 time of 600 s. Regeneration of the surface between cycles was done by injecting 10 mM glycine-HCI, pH 1.7. The binding data was fitted to a 1:1 interaction model. The scFv variants were diluted in HBS-EP+ (Cytiva. #BR100669). Experiments were performed at 25 °C. As expected, the binding to TfR was similar for all analyzed molecules. The results for binding to hTfR are shown in Figure 53A-L, and to cTfR in Figure 54A-L. The calculated KD values for binding to hTfR and cTfR are given below in Tables 30 and 31, respectively.

[0596] 5

[0597] Table 30: Affinity constants for binding to hTfRl

[0598] Table 31: Affinity constants for binding to cTfRl

[0599] 10 Next, in order to compare binding parameters with more certainty, a followup SPR analysis with additional replicates (N=4), was conducted for six selected molecules (SEQ ID NO:153-158) and the variant "h26D3 HC6 DS, VL-first". The selected molecules were produced as described above. As shown in Table 32 below, the measured affinity KD values are very similar between all selected variants.

[0600] Table 32: Affinity constants for binding to hTfRl measured in SPR with 4 replicates

[0601] 5

[0602] EXAMPLE 36

[0603] PE-ADA assay in serum

[0604] To investigate the degree of reactivity from pre-existing anti-drug antibodies among non-exposed individuals to a set of scFv molecules, a representative subset

[0605] 10 of 21 human serum samples was used to screen for binding response to 11 new scFv variants (SEQ ID NO:153-163) produced as described in Example 32. The response was measured using a direct ELISA as described below. Results from the direct ELISA screen show that the reactivity frequences differ between the screened variants, and that the response frequency is reduced for some of the investigated variants in

[0606] 15 line with previous findings (Johansson et al (2023), mAbs 15(1):2215887). The binding reactivity is shown in Figure 55A.

[0607] From the results of this initial ELISA screen, six variants (SEQ ID NO:153-158) were selected and subjected to additional ELISA measurements of PE-ADA reactivity in a larger set of 107 human serum samples, as described below. As seen in Figure

[0608] 20 55B, all six variants have a reduced reactivity compared to "h26D3-HC6_DS, VL-first" (SEQ ID NO:131).

[0609] To further evaluate the observed PE-ADA reactivity, a competition ELISA assay was conducted, as described below. The result is shown in Figure 55C-D and verify that the reactivity is directed to "h26D3-HC6_DS, VL-first" and could be largely

[0610] 25 blocked by competition with "h26D3-HC6_DS, VL-first" in solution. Serum samples: Serum was obtained from healthy donors giving blood during 2023-2025 (ethical permission D-nr 2018 / 804-31 from the regional ethics board in Stockholm, Sweden).

[0611] Screening of PE-ADA response by direct ELISA: 96-well half-area ELISA plates (Corning. #3690) were coated with 50 pl per well of 3.4 nM scFv variant in PBS for 2 h at RT with shaking at 600-900 rpm. Two wells per plate were instead coated with anti-human IgG F(ab')2 (Jacksonlmmuno; #109-005-097) to serve as positive control wells. For blocking, plates were emptied and tapped dry on paper towels prior to addition of 150 pl per well of blocking solution (1% BSA, 0.01% Tween20 in PBS). Plates were incubated with blocking solution for at least 1 h at RT. shaking at 600- 900 rpm. Plates were then washed 4 times with 150 pl per well of ELISA wash buffer with an automated plate washer (Tecan). Plates were tapped dry on paper towels and 25 pl of LowCross buffer (Candor Bioscience; #100-050) were added to each well. Then, 25 pl of 1:20 diluted serum samples in LowCross Buffer were added in duplicates to reach a final serum dilution of 1:40. Rituximab diluted in LowCross buffer was added to positive control wells. Samples were incubated for 25 min at RT without shaking. Plates were washed 4 times as above prior to addition of 50 pl per well of HRP-F(ab')2 goat anti-human IgG Fey specific polyclonal antibody (Jacksonlmmuno #109-036-008) diluted 1:5000 in LowCross buffer for 1 h at RT without shaking. Plates were washed 4 times as above prior to addition of 50 pl per well of TMB (Neogen; #331177). The colorimetric signal was allowed to develop for 5 min prior to stopping with 50 pl per well of sulfuric acid (Honeywell; #35354-lL). Plates were read immediately after addition of sulfuric acid on a plate reader at 450nM (Tecan).

[0612] Confirmatory competition ELISA assay: The competition ELISA was performed analogous to the direct ELISA screening assay described above, with the following adaptation: during blocking of plates, serum diluted 1:20 in LowCross buffer was either mixed with an equal volume of LowCross buffer or with an equal volume of 3400 nM "h26D3-HC6_DS, VL-first" scFv, resulting in a final serum dilution of 1:40 and 500x excess scFv compared to coating. The samples were pre-incubated in 96- well PP plate for 1 h, with shaking at 900 rpm. After blocking and washing, duplicates of either serum only or serum with soluble scFv were added to the plate and incubated for 25 min at RT without shaking. From here, the protocol for screening was followed.

[0613] Calculation of assay cut-off points: In order to determine the assay cut-off point, technical outliers with a %CV larger than 20 were first removed. For this, the mean of the sample duplicates was calculated, followed by calculation of the standard deviation. %CV was calculated as %CV = STDEV / MEAN*100.

[0614] The sample population minus technical outliers was then analyzed for statistical outliers by ROUT analysis in GraphPad Prism (Q=l%; allowing for 1% of false positive identified outliers). The sample population minus technical and statistical outliers made up the pseudo-negative population and was used to calculate the assay cut-off point. In case of high PE-ADA prevalence, the cut-off point was based on the competition assay: cut-off point = mean (of pseudo negative population) + 2.33 x STDEV (of pseudo negative population), allowing for a 1% false positive rate.

[0615] In case of low PE-ADA prevalence, the cut-off point can be directly calculated from the screening assay: cut-off point = mean (screening population with outliers removed) + 1.645 x STDEV (of screening population with outliers removed), allowing for a 5% false positive rate.

[0616] EXAMPLE 37

[0617] Generation of additional bispecific binding molecules

[0618] This example describes the design and production of a bispecific binding molecule denoted BA103 and BA104, which each incorporate an A|3 protofibrilspecific antibody described in W02016 / 005466 and a humanized, stabilized hTfRl- binding scFv variant with reduced PE-ADA reactivity as described in Examples 32-36.

[0619] Materials and methods

[0620] Design of constructs: The bispecific binding molecules BA103 and BA104 were designed as knob-into-hole variants of the A|3 protofibril-specific antibody A17D / R79T_DI8 described in W02016 / 005466, each with a different hTfRl-binding scFv linked to the C-terminal amino acid residue of the knob heavy chain of the antibody. The amino acid sequences of the molecules produced are provided in the sequence listing as noted in Table 33. Table 33: Amino acid sequences of bispecific binders BA103 and BA 104

[0621] Expression from transient transfection: The BA103 and BA104 molecules were expressed in CHO cells (ExpiCHO; ThermoFisher) and purified by affinity chromatography, cation exchange chromatography (CIEX), followed by preparative size-exclusion chromatography (SEC) and buffer exchange into phosphate buffered saline (PBS) solution. The purified molecules were characterized using SDS-PAGE, SEC, and UV protein determination.

[0622] EXAMPLE 38

[0623] In vivo efficacy evaluation in 5xFAD x hTfRl-KI after repeated treatment

[0624] This example describes the in vivo efficacy of the bispecific binding molecule BA101, generated and produced in Example 16, following repeated intravenous dosing in 5xFAD x hTfRl-KI mice. The control molecule BA102 was evaluated in parallel.

[0625] Materials and methods

[0626] To further assess the in vivo efficacy of the bispecific binding molecule BA101 in comparison to the control antibody BA102, levels of A|3 protofibrils and diffuse A|3 plaques were investigated in 5xFAD x hTfRl-KI mice. The mice received 12 weekly i.v. injections with BA101 at doses of 5.7 nmol / kg, 17 nmol / kg, or 57 nmol / kg, BA102 at a dose of 57 nmol / kg, or vehicle (PBS) and were terminated 7 days after the last injection. The study was initiated at the age of 4.5 months in female mice, and the 5xFAD x hTfRl-KI mouse line was generated as described in Example 28. On Day -6 and Day 0 all mice received 1 mg of the anti-mouse CD4 antibody GK1.5 (#BEOOO3-1, BioXcell) in order to deplete CD4+ T cells and consequently to prevent any generation of anti-drug antibodies. On Day 1 the first treatment with BA101, BA102 or PBS was performed, and one group of mice were terminated to serve as a baseline reference for pathology measurements.

[0627] Blood samples were collected from each animal at termination, and samples were processed to plasma as described in Example 27. Following termination by deep sedation and intracardiac perfusion with PBS, brains were extracted and separated into the left and right hemisphere which were further processed as described in Example 28.

[0628] Biochemical assessments: Plasma and brain homogenate concentrations of BA101 and BA102 were determined using a custom MSD assay detecting the human Fc domain as described in Example 28. A|3 protofibril concentrations were assessed as described in Example 29.

[0629] Histology: Amyloid plaque load was determined using quantitative IHC analysis. Right brain hemispheres were prepared and sectioned as described in Example 28.

[0630] Five sections per mouse at the same level of sectioning were selected for systematic random sampling. Sections were air-dried for 45 min and washed in PBS for 10 min. Sections were incubated for 7 min in 0.5% Thioflavin S (#T1892, Sigma- Aldrich) in ddH2O and then differentiated for 2 x 3 minutes in 80% ethanol, followed by 2 x 5 min wash in PBS. Unspecific binding sites were blocked by incubating the sections with 10% normal donkey serum in PBS containing 0.1% Tergitol for 60 min, followed by 3 x 5 min wash in PBS. Primary antibody incubation was conducted overnight at 4 °C with rabbit polyclonal anti- amyloid |3 fibrils LOC (WAB2287, Sigma- Aldrich; 1:1000 dilution). The sections were washed three times in PBS for 5 min each before a subsequent incubation with a secondary antibody, donkey anti-rabbit IgG H+L Alexa 555-conjugated (#abl50066, Abeam; 1:500 dilution), for 60 min. This was followed by three washes in PBS, each lasting 5 min. The sections were incubated with DAPI working solution for 15 min, washed twice in PBS for 5 min each, and then washed in ddH2O for 5 min. Finally, sections were mounted with Mowiol and coverslips using a Leica CV5030 coverslipper. Whole-brain images were captured at lOx magnification using a Zeiss AxioScan Z1 microscope.

[0631] Images from 5xFAD x hTfRl-KI mice, stained with Thioflavin S and LOC, were quantitatively analyzed using Image Pro 10 software (Media Cybernetics). Regions of interest (ROIs) were selected to exclude artifacts. Background correction was applied, and immunoreactive objects were detected using appropriate thresholding and morphological filtering based on size and shape. Signals of LOC-stained fibrillary amyloid (diffuse plaques and dense-core plaques) were quantified within the identified area and mask images were prepared, then ThioS-stained beta-sheets (core plaques) were quantitatively evaluated on the LOG mask. Quantified object features reported in the current example cover i) the percentage of cumulative object area relative to the ROI size (immunoreactive area); ii) the number of objects normalized to ROI size (object density); and iii) the size of above-threshold objects. Diffuse plaques were defined as LOCpos- (LOCpos, Thioflavin Spos) plaques.

[0632] Statistical analysis was performed in GraphPad Prism (v. 10.4.2) using one way ANOVA with Tukey's multiple comparisons on log-transformed data for all groups, including baseline.

[0633] Results

[0634] Following repeated doses, the brain concentrations of BA101 and BA102 accumulated compared to single i.v. dosing (Example 28). The bispecific binding molecule BA101 accumulated substantially compared to BA102, resulting in 10-fold higher brain exposure at equimolar doses of 57 nmol / kg (Figure 56). Consequently, the median brain-to-plasma ratio at the 57 nmol / kg dose was 207% for BA101 and 2.1% for BA102, representing a 100-fold difference.

[0635] Levels of AP protofibrils in soluble brain extracts were significantly reduced for all dose levels of BA101 and BA102 compared to the vehicle (PBS) treated group. Notably, the 5.7 nmol / kg dose of BA101 generated a similar mean decrease of A|3 protofibril levels as the 57 nmol / kg dose of BA102, compared to the PBS treated group (Figure 57).

[0636] Levels of diffuse A|3 plaques in cortex were also significantly decreased after BA101 treatment. All three doses of BA101 resulted in lower cortical plaque load compared to vehicle control when quantified as immunoreactive area and object density. The mean object size was also decreased in the groups receiving 17 or 57 nmol / kg BA101. In contrast, the group treated with 57 nmol / kg BA102 was significantly altered from vehicle control based on object density quantification, but no difference was detected on immunoreactive area nor object size. Results indicate that the increased brain uptake and brain exposure of BA101 results in superior in vivo efficacy (Figure 58).

[0637] ITEMIZED LISTING OF EMBODIMENTS

[0638] 1. A bispecific binding molecule, comprising

[0639] - a first moiety Ml, which is an A|3 protofibril binding moiety comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), said VH and VL domains forming a VH / VL pair comprising an antigen-binding surface, said VH domain in Ml comprising the amino acid sequence SEQ ID NO:1:

[0640] EVQLVESGGG LVQPGGSLRL SCSASGFTFS SFGMHWVRQXaPGKGLEWVAY I SSGSSTIYY GDTVKGRFTI SRDNAKNSLF LQMSSLRAED TAVYYCAREG GYYYGRSYYT MDYWGQGTTV TVS , wherein

[0641] Xais selected from the group consisting of A, N and T; said VL domain in Ml comprising the amino acid sequence SEQ ID NO:5:

[0642] DWMTQSPLS LPXbTPGDPAS XcSCRSSQSIV HSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLXdI XeXfVXgAEDVGI YYCFQGSHVP PTFGPGTKLE IK, wherein

[0643] Xb is selected from the group consisting

[0644] Xcis selected from the group consisting

[0645] Xd is selected from the group consisting

[0646] Xeis selected from the group consisting

[0647] Xf is selected from the group consisting

[0648] Xgis selected from the group consisting

[0649] - a second moiety M2, which is a human transferrin receptor 1 (hTfRl) binding moiety comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), said VH and VL domains forming a VH / VL pair comprising an antigen-binding surface, in which said antigenbinding surface provides the binding protein with the capacity to bind selectively to an epitope located in the protease-like domain of hTfRl defined by amino acid residues 121-183 and 384-605 in SEQ ID NO:98. 2. Bispecific binding molecule according to item 1, wherein said VH domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 3.

[0650] 3. Bispecific binding molecule according to item 2, wherein said VH domain in Ml comprises the amino acid sequence SEQ ID NO:2.

[0651] 4. Bispecific binding molecule according to item 2, wherein said VH domain in Ml comprises the amino acid sequence SEQ ID NO:3.

[0652] 5. Bispecific binding molecule according to item 1, wherein said VH domain in Ml comprises the amino acid sequence SEQ ID NO:4.

[0653] 6. Bispecific binding molecule according to any preceding item, wherein, in SEQ ID NO:5 of said VL domain in Ml, one of the following alternatives applies:

[0654] Xd is R and Xf is R;

[0655] Xd is T and Xf is R;

[0656] Xd is R and Xf is S; and

[0657] Xd is T and Xf is S.

[0658] 7. Bispecific binding molecule according to item 6, wherein said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6-13.

[0659] 8. Bispecific binding molecule according to item 6, wherein, in SEQ ID NO:5 of said VL domain in Ml, Xd is T and Xf is R.

[0660] 9. Bispecific binding molecule according to item 8, wherein said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 and 10-13.

[0661] 10. Bispecific binding molecule according to any preceding item, wherein, in SEQ ID NO:5 of said VL domain in Ml, Xb is A. 11. Bispecific binding molecule according to item 10, wherein said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NQ:10-ll.

[0662] 12. Bispecific binding molecule according to any preceding item, wherein, in SEQ ID NO:5 of said VL domain in Ml, Xgis D.

[0663] 13. Bispecific binding molecule according to item 12, wherein said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NQ:10 and 12, such as wherein said VL domain in Ml comprises the amino acid sequence SEQ ID NQ:10

[0664] 14. Bispecific binding molecule according to any preceding item, wherein, in SEQ ID NO:5 of said VL domain in Ml, Xeis Q.

[0665] 15. Bispecific binding molecule according to item 14, wherein said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11 and 13.

[0666] 16. Bispecific binding molecule according to any preceding item, wherein, in SEQ ID NO:5 of said VL domain in Ml, Xcis V.

[0667] 17. Bispecific binding molecule according to item 16, wherein said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12-13.

[0668] 18. Bispecific binding molecule according to any preceding item, wherein said Ml VH domain is as defined in any one of items 2-5 and said Ml VL domain is as defined in any one of items 6-17.

[0669] 19. Bispecific binding molecule according to item 18, wherein the Ml VH domain and the Ml VL domain are represented by one of the following VH / VL combinations: a) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10; b) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:11; c) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:12; d) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:13; e) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NQ:10; f) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:11; g) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:12; h) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:13; and i) a VH domain comprising SEQ ID NO:4 and a VL domain comprising SEQ ID NO:7.

[0670] 20. Bispecific binding molecule according to item 19, wherein the Ml VH domain and the Ml VL domain are represented by one of the following VH / VL combinations: a) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10; b) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:11; c) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:12; d) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:13; e) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NQ:10; f) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:11; g) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:12; and h) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:13.

[0671] 21. Bispecific binding molecule according to item 20, wherein the Ml VH domain and the Ml VL domain are represented by one of the following VH / VL combinations: a) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10; b) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NQ:10; and c) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:11.

[0672] 22. Bispecific binding molecule according to item 21, wherein the Ml VH domain and the Ml VL domain are represented by the VH / VL combination of a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10. 23. Bispecific binding molecule according to any preceding item, wherein said epitope of M2 located in the protease-like domain of hTfRl comprises or consists of the amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO:98.

[0673] 24. Bispecific binding molecule according to item 23, in which said antigen-binding surface of M2 is composed of three complementarity-determining regions (CDRs) from said VH domain and three CDRs from said VL domain, and wherein said CDRs comprise the following:

[0674] VHCDR1: X1X2NMX3 (SEQ ID NO:14), wherein

[0675] XI is selected from D and A;

[0676] X2 is selected from Y and A; and X3 is selected from D and A;

[0677] VHCDR2: X4INPX5X6X7TTSX8X9X10KFKG (SEQ ID NO:15), wherein 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;

[0678] X9 is selected from N and S; and X10 is selected from E and Q;

[0679] VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID NO:17), wherein

[0680] XII 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;

[0681] VLCDR2: X15ASTRES (SEQ ID NO:18) wherein X15 is selected from W and A; and

[0682] VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:19) wherein X16 is selected from X17 is selected from X18 is selected from X19 is selected from X20 is selected from

[0683] 25. Bispecific binding molecule according to item 24, said antigen-binding surface of M2 further comprising VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:16) wherein X21 is selected from Y and A; X22 is selected from Y and A; X23 is selected from Y and A;

[0684] X24 is selected from D and A; and X25 is selected from Y and A.

[0685] 26. Bispecific binding molecule according to any one of items 24-25, in which said

[0686] VHCDR2 in moiety M2 is:

[0687] VHCDR2: X4INPX5X6X7TTSX8NEKFKG (SEQ ID NQ:20), wherein X4 is selected from D and A;

[0688] X5 is selected from D and A;

[0689] X6 is selected from Y and A;

[0690] X7 is selected from D and A; and X8 is selected from Y and A.

[0691] 27. Bispecific binding molecule according to any one of items 24-26, in which said

[0692] VLCDR1 in moiety M2 is:

[0693] VLCDR1: KSSQSLLX11STNQKNX14LA (SEQ ID NO:21), wherein

[0694] Xll is selected from Y and A; and X14 is selected from Y and A.

[0695] 28. Bispecific binding molecule according to any one of items 24-27, in which said

[0696] VLCDR3 in moiety M2 is:

[0697] VLCDR3: QQX16FIX19PRT (SEQ ID NO:22) wherein

[0698] X16 is selected from Y and A; X19 is selected from Y and A.

[0699] 29. Bispecific binding molecule according to any one of items 24-28, in which the amino acid sequence of said VHCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:23 and 29-31.

[0700] 30. Bispecific binding molecule according to any one of items 24-29, in which the amino acid sequence of said VHCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:24, 32-36 and 47, for example selected from the group consisting of SEQ ID NO:24 and 32-36.

[0701] 31. Bispecific binding molecule according to any one of items 24, 26-30, in which the amino acid sequence of said VHCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:25, 37-41 and 48, for example selected from the group consisting of SEQ ID NO:25 and 37-41.

[0702] 32. Bispecific binding molecule according to any one of items 24-31, in which the amino acid sequence of said VLCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:26, 42, 43 and 49, for example selected from the group consisting of SEQ ID NO:26, 42 and 43.

[0703] 33. Bispecific binding molecule according to any one of items 24-32, in which the amino acid sequence of said VLCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:27 and 44.

[0704] 34. Bispecific binding molecule according to any one of items 24-33, in which the amino acid sequence of said VLCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:28, 45, 46 and 50, for example selected from the group consisting of SEQ ID NO:28, 45 and 46.

[0705] 35. Bispecific binding molecule according to any one of items 24-34, in which the amino acid sequences of the six CDRs in moiety M2 are the following:

[0706] VHCDR1: DYNMD (SEQ ID NO:23),

[0707] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:24),

[0708] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:25)

[0709] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:26),

[0710] VLCDR2: WASTRES (SEQ ID NO:27), and

[0711] VLCDR3: QQYFIYPRT (SEQ ID NO:28).

[0712] 36. Bispecific binding molecule according to any one of items 24-34, in which the amino acid sequences of the six CDRs in moiety M2 are the following:

[0713] VHCDR1: DYNMD (SEQ ID NO:23),

[0714] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:34), VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:25),

[0715] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:26),

[0716] VLCDR2: WASTRES (SEQ ID NO:27), and

[0717] VLCDR3: QQYFIYPRT (SEQ ID NO:28).

[0718] 37. Bispecific binding molecule according to any one of items 24, 26-34, in which the amino acid sequences of the six CDRs in moiety M2 are the following:

[0719] VHCDR1: DYNMD (SEQ ID NO:23),

[0720] VHCDR2: DINPNYDTTSYSQKFKG (SEQ ID NO:47),

[0721] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:48),

[0722] VLCDR1: KSSQSLLYSSNRKNYLA (SEQ ID NO:49),

[0723] VLCDR2: WASTRES (SEQ ID NO:27), and

[0724] VLCDR3: QQYYNYPYT (SEQ ID NQ:50).

[0725] 38. Bispecific binding molecule according to any preceding item, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0726] (i) the group consisting of SEQ ID NQ:57-70, 78 and 80, for example the group consisting of SEQ ID NQ:57-70, for example the group consisting of SEQ ID NO:57 and 63; and

[0727] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i).

[0728] 39. Bispecific binding molecule according to any preceding item, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0729] (i) the group consisting of SEQ ID NO:71-77, 79 and 81, for example the group consisting of SEQ ID NO:71-77; and

[0730] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions a sequence defined in (i). 40. Bispecific binding molecule according to any one of items 38-39, wherein said VH domain in moiety M2 is as defined in item 38 and said VL domain in moiety M2 is as defined in item 39.

[0731] 41. Bispecific binding molecule according to item 40, in which said VH domain in moiety M2 comprises SEQ ID NO:57 and said VL domain in moiety M2 comprises a sequence selected from SEQ ID NO:71-77.

[0732] 42. Bispecific binding molecule according to item 40, in which said VH domain in moiety M2 comprises a sequence selected from SEQ ID NQ:57-70 and said VL domain in moiety M2 comprises SEQ ID NO:71.

[0733] 43. Bispecific binding molecule according to any one of items 41-42, in which said VH domain in moiety M2 comprises SEQ ID NO:57 and said VL domain in moiety M2 comprises SEQ ID NO:71.

[0734] 44. Bispecific binding molecule according to item 42, in which said VH domain in moiety M2 comprises SEQ ID NO:63 and said VL domain in moiety M2 comprises SEQ ID NO:71.

[0735] 45. Bispecific binding molecule according to any one of items 1-37, which comprises one first cysteine residue in said VH domain in moiety M2 and one second cysteine residue in said VL domain in moiety M2, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL domains.

[0736] 46. Bispecific binding molecule according to item 45, wherein said first cysteine residue is located at an amino acid position selected from M2 VH position 39-49, such as selected from M2 VH position 41-47, such as selected from M2 VH position 43-45, such as at M2 VH position 44, all as determined by reference to the Kabat numbering scheme. 47. Bispecific binding molecule according to item 45 or 46, wherein said second cysteine residue is located at an amino acid position selected from M2 VL position 95-105, such as selected from M2 VL position 97-103, such as selected from M2 VL position 99-101, such as at M2 VL position 100, all as determined by reference to the Kabat numbering scheme.

[0737] 48. Bispecific binding molecule according to any one of items 45- 47, wherein said first cysteine residue is located at M2 VH position 44 and said second cysteine residue is located at M2 VL position 100, as determined by reference to the Kabat numbering scheme.

[0738] 49. Bispecific binding molecule according to any one of items 45-48, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0739] (i) the group consisting of SEQ ID NO:101-116, for example the group consisting of SEQ ID NO:101-114, for example the group consisting of SEQ ID NQ:101 and 107; and

[0740] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 44.

[0741] 50. Bispecific binding molecule according to any one of items 45-49, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0742] (i) the group consisting of SEQ ID NO:118-126, for example the group consisting of SEQ ID NO:118-124; and

[0743] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 106. 51. Bispecific binding molecule according to any one of items 49-50, wherein said VH domain in moiety M2 is as defined in item 49 and said VL domain in moiety M2 is as defined in item 50.

[0744] 52. Bispecific binding molecule according to item 51, in which said VH domain in moiety M2 comprises SEQ ID NO:101 and said VL domain in moiety M2 comprises a sequence selected from SEQ ID NO:118-124.

[0745] 53. Bispecific binding molecule according to item 51, in which said VH domain in moiety M2 comprises a sequence selected from SEQ ID NQ:101-114 and said VL domain in moiety M2 comprises SEQ ID NO:118.

[0746] 54. Bispecific binding molecule according to any one of items 52-53, in which said VH domain in moiety M2 comprises SEQ ID NQ:101 and said VL domain in moiety M2 comprises SEQ ID NO:118.

[0747] 55. Bispecific binding molecule according to item 53, in which said VH domain in moiety M2 comprises SEQ ID NQ:107 and said VL domain in moiety M2 comprises SEQ ID NO:118.

[0748] 56. Bispecific binding molecule according to any one of items 45-48, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0749] (i) the group consisting of SEQ ID NO:142-152, for example the group consisting of SEQ ID NO:142-147, for example the group consisting of SEQ ID NO:142-143; and

[0750] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 44. 57. Bispecific binding molecule according to item 56, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0751] (i) the group consisting of SEQ ID NO:118-126, for example the group consisting of SEQ ID NO:118-124; and

[0752] (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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 106.

[0753] 58. Bispecific binding molecule according to item 57, in which said VH domain in moiety M2 comprises SEQ ID NO:142 and said VL domain in moiety M2 comprises a sequence selected from SEQ ID NO:118-124.

[0754] 59. Bispecific binding molecule according to item 57, in which said VH domain in moiety M2 comprises a sequence selected from SEQ ID NO:142-143 and said VL domain in moiety M2 comprises SEQ ID NO:118.

[0755] 60. Bispecific binding molecule according to any one of items 58-59, in which said VH domain in moiety M2 comprises SEQ ID NO:142 and said VL domain in moiety M2 comprises SEQ ID NO:118.

[0756] 61. Bispecific binding molecule according to any one of items 58-59, in which said VH domain in moiety M2 comprises SEQ ID NO:143 and said VL domain in moiety M2 comprises SEQ ID NO:118.

[0757] 62. Bispecific binding molecule according to any one of items 1-24, in which said antigen-binding surface in moiety M2 is composed of three complementaritydetermining regions (CDRs) from said VH domain and three CDRs from said VL domain, wherein said CDRs comprise the following:

[0758] VHCDR1: NYWLG (SEQ ID NO:51),

[0759] VHCDR2: DIFPGSDNTYYNEKFKG (SEQ ID NO:52),

[0760] VHCDR3: SGNFYAMDY (SEQ ID NO:53),

[0761] VLCDR1: SASSSVNYMN (SEQ ID NO:54), VLCDR2: DTSKLAS (SEQ ID NO:55), and

[0762] VLCDR3: FQGSGYPFT (SEQ ID NO:56).

[0763] 63. Bispecific binding molecule according to item 62, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:82 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 to SEQ ID NO:82, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NO:82.

[0764] 64. Bispecific binding molecule according to any one of items 62-63, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:83 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 to SEQ ID NO:83, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NO:83.

[0765] 65. Bispecific binding molecule according to any one of items 63-64, wherein said VH domain in moiety M2 is as defined in item 63 and said VL domain in moiety M2 is as defined in item 64.

[0766] 66. Bispecific binding molecule according to item 62, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:117 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 to SEQ ID NO:117, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NO:117, and provided that the sequence comprises a cysteine residue at position 44.

[0767] 67. Bispecific binding molecule according to any one of items 62 and 66, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:127 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 to SEQ ID NO:127, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NO:127, and provided that the sequence comprises a cysteine residue at position 99.

[0768] 68. Bispecific binding molecule according to any one of items 66-67, wherein said VH domain in moiety M2 is as defined in item 66 and said VL domain in moiety M2 is as defined in item 67.

[0769] 69. Bispecific binding molecule according to any preceding item, in which the VH / VL pair in moiety M2 forms part of an antibody construct.

[0770] 70. Bispecific binding molecule according to item 69, wherein the VH / VL pair in moiety M2 is present in an antibody fragment selected from the group consisting of a Fab fragment, a single chain Fab (scFab) fragment, an Fv fragment and a single chain (scFv) fragment.

[0771] 71. Bispecific binding molecule according to item 70, wherein the VH / VL pair of the moiety M2 forms part of an scFv, in which the VH and VL domains are coupled together by a peptide scFv linker.

[0772] 72. Bispecific binding molecule according to item 71, wherein said scFv linker is attached to the N-terminal amino acid residue of the VH domain and to the C- terminal amino acid residue of the VL domain.

[0773] 73. Bispecific binding molecule according to item 71, wherein said scFv linker is attached to the C-terminal amino acid residue of the VH domain and to the N- terminal amino acid residue of the VL domain.

[0774] 74. Bispecific binding molecule according to any one of items 71-73, in which said scFv linker is a flexible peptide linker consisting of from 5 to 40 amino acid residues, for example from 10 to 30 amino acid residues, for example from 15 to 25 amino acid residues, for example about 15 amino acid residues, for example 15 amino acid residues, for example comprising or consisting of the sequence (648)3 (SEQ ID NO:137).

[0775] 75. Bispecific binding molecule according to any preceding item, in which Ml and M2 are connected to each other by at least one peptide linker between Ml and M2.

[0776] 76. Bispecific binding molecule according to item 75, in which said at least one peptide linker between Ml and M2 is attached, on the M2 side, to the C-terminal amino acid residue of the VH domain of M2 or to the N-terminal amino acid residue of the VL domain of M2.

[0777] 77. Bispecific binding molecule according to any one of items 75-76, wherein said at least one peptide linker between Ml and M2 is a flexible linker.

[0778] 78. Bispecific binding molecule according to item 77, wherein said flexible linker(s) comprise(s) glycine, serine, alanine and / or threonine residues.

[0779] 79. Bispecific binding molecule according to item 78, wherein said linker(s) has a general formula selected from (GnSm)p and (SnGm)p, wherein, independently, n = 1-7, m = 0-7, n + m < 8 and p = 1-10.

[0780] 80. Bispecific binding molecule according to any one of items 75-78, wherein said at least one linker is between 10 and 50 amino acid residues long, such as between 10 and 30 amino acid residues long, such as between 15 and 25 amino acid residues long or between 10 and 20 amino acids long.

[0781] 81. Bispecific binding molecule according to any preceding item, in which Ml is provided as a knob-into-hole antibody comprising two identical antibody light chains; one antibody hole heavy chain; and one antibody knob heavy chain; and M2 is provided as an scFv linked to the C-terminal amino acid residue of the knob heavy chain of Ml. 82. Bispecific binding molecule according to item 81, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:138, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:139, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NQ:140.

[0782] 83. Bispecific binding molecule according to item 81, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:138, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:139, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:164.

[0783] 84. Bispecific binding molecule according to item 81, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:138, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:139, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:165.

[0784] 85. Bispecific binding molecule according to any preceding item, which has a higher affinity for A|3 protofibrils than for A|3 monomers.

[0785] 86. Bispecific binding molecule according to item 85, which has at least 2x higher affinity for A|3 protofibrils than for A|3 monomers, such as at least lOx higher, such as at least 40x higher, such as at least lOOx higher, such as at least 200x higher, such as at least lOOOx higher, such as at least 2000x higher, such as at least 3000x higher affinity.

[0786] 87. Bispecific binding molecule according to any preceding item, which has a binding affinity for A|3 protofibrils that corresponds to a KD value of no more than 5 nM, such as no more than 2 nM, such as no more than 1 nM, such as between 10 and 500 pM, such as between 200 and 400 pM, such as between 250 and 350 pM, as determined by surface plasmon resonance. 88. Pharmaceutical composition, comprising a bispecific binding molecule according to any preceding item and a pharmaceutically acceptable carrier or excipient.

[0787] 89. Bispecific binding molecule according to any one of items 1-87 or a pharmaceutical composition according to item 88 for use in treatment, such as for use in therapeutic treatment and / or for use in prophylactic treatment.

[0788] 90. Bispecific binding molecule according to any one of items 1-87 or pharmaceutical composition according to item 88 for use in diagnosis in vivo and / or prognosis in vivo.

[0789] 91. Bispecific binding molecule or pharmaceutical composition for use according to any one of items 89-90, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a neurodegenerative disorder associated with amyloid beta peptide aggregation, for example a disorder selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), Lewy body dementia, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataract due to A|3 deposition, traumatic brain injury with an accumulation of A|3, adult onset diabetes, senile cardiac amyloidosis and macular degeneration.

[0790] 92. Bispecific binding molecule or pharmaceutical composition for use according to item 91, wherein said neurodegenerative disorder is Alzheimer's disease.

[0791] 93. A method of therapeutic and / or prophylactic treatment of a mammal having, or being at risk of developing, a neurodegenerative disorder, said method comprising administering to said mammal a therapeutically effective amount of a bispecific binding molecule according to any one of items 1-87 or a pharmaceutical composition according to item 88. 94. Method according to item 93, wherein said neurodegenerative disorder is a disorder associated with amyloid beta peptide aggregation, for example a disorder selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), Lewy body dementia, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataract due to A|3 deposition, traumatic brain injury with an accumulation of A|3, adult onset diabetes, senile cardiac amyloidosis and macular degeneration.

[0792] 95. Method according to item 94, wherein said neurodegenerative disorder is Alzheimer's disease.

[0793] 96. A method of detecting A|3 protofibrils in vitro, comprising providing a sample suspected to contain A|3 protofibrils, contacting said sample with a bispecific binding molecule according to any one of items 1-87, and detecting the binding of said binding molecule to indicate the presence of A|3 protofibrils in the sample.

[0794] 97. A method of determining the amount of A|3 protofibrils present in a subject, comprising the steps of: a) contacting the subject, or a sample isolated from the subject, with a bispecific binding molecule according to any one of items 1-87 or a pharmaceutical composition according to item 88, and b) obtaining a value corresponding to the amount of the antibody or antigenbinding fragment thereof or pharmaceutical composition that has bound in said subject or to said sample.

[0795] 98. Method according to item 97, further comprising a step of comparing said value to a reference.

Claims

CLAIMS1. A bispecific binding molecule, comprising- a first moiety Ml, which is an A|3 protofibril binding moiety comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), said VH and VL domains forming a VH / VL pair comprising an antigen-binding surface, said VH domain in Ml comprising the amino acid sequence SEQ ID NO:1:EVQLVESGGG LVQPGGSLRL SCSASGFTFS SFGMHWVRQXaPGKGLEWVAY I SSGSSTIYY GDTVKGRFTI SRDNAKNSLF LQMSSLRAED TAVYYCAREG GYYYGRSYYT MDYWGQGTTV TVS , whereinXais selected from the group consisting of A, N and T; said VL domain in Ml comprising the amino acid sequence SEQ ID NO:5:DWMTQSPLS LPXbTPGDPAS XcSCRSSQSIV HSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLXdI XeXfVXgAEDVGI YYCFQGSHVP PTFGPGTKLE IK, whereinXb is selected from the group consisting of V and A;Xcis selected from the group consisting of I and V;Xd is selected from the group consisting of R and T;Xeis selected from the group consisting of S and Q;Xf is selected from the group consisting of R and S; andXgis selected from the group consisting of E and D; and- a second moiety M2, which is a human transferrin receptor 1 (hTfRl) binding moiety comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), said VH and VL domains forming a VH / VL pair comprising an antigen-binding surface, in which said antigenbinding surface provides the binding protein with the capacity to bind selectively to an epitope located in the protease-like domain of hTfRl defined by amino acid residues 121-183 and 384-605 in SEQ ID NO:98.

2. Bispecific binding molecule according to claim 1, wherein said VH domain in Ml comprises the amino acid sequence SEQ ID NO:2.

3. Bispecific binding molecule according to any preceding claim, wherein, in SEQ ID NO:5 of said VL domain in Ml, one of the following alternatives applies:Xd is R and Xf is R;Xd is T and Xf is R;Xd is R and Xf is S; andXd is T and Xf is S.

4. Bispecific binding molecule according to claim 3, wherein, in SEQ ID NO:5 of said VL domain in Ml, Xd is T and Xf is R.

5. Bispecific binding molecule according to claim 4, wherein said VL domain in Ml comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7 and 10-13.

6. Bispecific binding molecule according to claim 5, wherein said VL domain in Ml comprises the amino acid sequence SEQ ID NQ:10.

7. Bispecific binding molecule according to any preceding claim, wherein said Ml VH domain is as defined in any one of claims 1-2 and said Ml VL domain is as defined in any one of claims 3-6.

8. Bispecific binding molecule according to claim 7, wherein the Ml VH domain and the Ml VL domain are represented by one of the following VH / VL combinations: a) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10; b) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:11; c) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:12; d) a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NO:13; e) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NQ:10; f) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:11;g) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:12; h) a VH domain comprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:13; and i) a VH domain comprising SEQ ID NO:4 and a VL domain comprising SEQ ID NO:7, for example by the VH / VL combination of a VH domain comprising SEQ ID NO:2 and a VL domain comprising SEQ ID NQ:10.

9. Bispecific binding molecule according to any preceding claim, in which said antigen-binding surface of M2 is composed of three complementarity-determining regions (CDRs) from said VH domain and three CDRs from said VL domain, wherein said CDRs comprise the following: VHCDR1: X1X2NMX3 (SEQ ID NO:14), whereinXI 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:15), wherein 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:17), whereinXII 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:18) wherein X15 is selected from W and A; andVLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:19) wherein X16 is selected from X17 is selected from X18 is selected from X19 is selected from X20 is selected fromoptionally further comprising VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:16) whereinX21 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.

10. Bispecific binding molecule according to claim 9, in which the amino acid sequences of the six CDRs in moiety M2 are the following:VHCDR1: DYNMD (SEQ ID NO:23),VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:34),VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:25),VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:26),VLCDR2: WASTRES (SEQ ID NO:27), andVLCDR3: QQYFIYPRT (SEQ ID NO:28).

11. Bispecific binding molecule according to any preceding claim, which comprises one first cysteine residue in said VH domain in moiety M2 and one second cysteine residue in said VL domain in moiety M2, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL domains, for example wherein said first cysteine residue is located at M2 VH position 44 and said second cysteine residue is located at M2 VL position 100, as determined by reference to the Kabat numbering scheme.

12. Bispecific binding molecule according to claim 11, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NQ:101-116, for example the group consisting of SEQ ID NQ:101-114, for example the group consisting of SEQ ID NQ:101 and 107; 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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 44.

13. Bispecific binding molecule according to any one of claims 11-12, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NO:118-126, for example the group consisting of SEQ ID NO:118-124; 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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 106.

14. Bispecific binding molecule according to any one of claims 12-13, in which said VH domain in moiety M2 comprises SEQ ID NQ:107 and said VL domain in moiety M2 comprises SEQ ID NO:118.

15. Bispecific binding molecule according to claim 11, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NO:142-152, for example the group consisting of SEQ ID NO:142-147, for example the group consisting of SEQ ID NO:142-143; 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 to a sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 44.

16. Bispecific binding molecule according to claim 15, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NO:118-126, for example the group consisting of SEQ ID NO:118-124; 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 to a142 sequence defined in (i), provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in a sequence defined in (i), and provided that the sequence comprises a cysteine residue at position 106.

17. Bispecific binding molecule according to claim 16, in which said VH domain in moiety M2 comprises SEQ ID NO:142 and said VL domain in moiety M2 comprises SEQ ID NO:118.

18. Bispecific binding molecule according to claim 16, in which said VH domain in moiety M2 comprises SEQ ID NO:143 and said VL domain in moiety M2 comprises SEQ ID NO:118.

19. Bispecific binding molecule according to any preceding claim, in which Ml is provided as a knob-into-hole antibody comprising two identical antibody light chains; one antibody hole heavy chain; and one antibody knob heavy chain; and M2 is provided as an scFv linked to the C-terminal amino acid residue of the knob heavy chain of Ml.

20. Bispecific binding molecule according to claim 19, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:138, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:139, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NQ:140.

21. Bispecific binding molecule according to claim 19, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:138, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:139, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:164.

22. Bispecific binding molecule according to claim 19, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:138, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists143 of SEQ ID NO:139, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:165.

23. Pharmaceutical composition, comprising a bispecific binding molecule according to any preceding claim and a pharmaceutically acceptable carrier or excipient.

24. Bispecific binding molecule according to any one of claims 1-22 or a pharmaceutical composition according to claim 23 for use in treatment, such as for use in therapeutic treatment and / or for use in prophylactic treatment.

25. Bispecific binding molecule according to any one of claims 1-22 or a pharmaceutical composition according to claim 23 for use in diagnosis in vivo and / or prognosis in vivo.

26. Bispecific binding molecule or pharmaceutical composition for use according to any one of claims 24-25, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a neurodegenerative disorder associated with amyloid beta peptide aggregation, for example a disorder selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), Lewy body dementia, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataract due to A|3 deposition, traumatic brain injury with an accumulation of A|3, adult onset diabetes, senile cardiac amyloidosis and macular degeneration.

27. Bispecific binding molecule or pharmaceutical composition for use according to claim 26, wherein said neurodegenerative disorder is Alzheimer's disease.

28. A method of therapeutic and / or prophylactic treatment of a mammal having, or being at risk of developing, a neurodegenerative disorder, said method comprising administering to said mammal a therapeutically effective amount of a bispecific144 binding molecule according to any one of claims 1-22 or a pharmaceutical composition according to claim 23.

29. Method according to claim 28, wherein said neurodegenerative disorder is a disorder associated with amyloid beta peptide aggregation, for example a disorder selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), Lewy body dementia, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataract due to A|3 deposition, traumatic brain injury with an accumulation of A|3, adult onset diabetes, senile cardiac amyloidosis and macular degeneration.

30. Method according to claim 29, wherein said neurodegenerative disorder is Alzheimer's disease.

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