Bispecific binding molecule

Abstract

The present disclosure relates to a bispecific binding molecule, which binds to α-synuclein protofibrils (α-synuclein aggregates) 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 a-synuclein protofibrils (a-synuclein aggregates) and to the protease-like domain of human transferrin receptor 1 (hTfRl), as well as therapeutic and diagnostic uses thereof.

[0004] Background

[0005] Neuronal aggregates of misfolded, pathological species of a-synuclein in Lewy bodies and Lewy neurites are a common pathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Therapies specifically intended to target a-synuclein and thereby slow disease progression are under development. The prevalence of PD and DLB are increasing and may affect more than 10 million people and their families worldwide. This number is expected to increase due to a higher diagnosis rate as well as increasing life expectancy. Available pharmacological treatments for PD focus on symptom management mainly by targeting the dopaminergic pathway, whereas symptomatic treatments for DLB mainly address cognitive issues and have adopted treatment options shown to be efficacious in Alzheimer's disease, such as acetylcholine esterase inhibitors.

[0006] There is a major unmet need for a disease-modifying treatment for patients with PD and DLB to address the underlying pathophysiology of these and other conditions characterized by aggregated a-synuclein, also referred to as a-synuclein protofibrils. Therapeutic approaches that act on the underlying pathophysiology have the potential to slow disease progression. Pathological, genetic, and mechanistic data suggest that accumulation of aggregated a-synuclein plays a key role in the pathogenesis of PD. Consequently, investigating potential disease modification through removal of aggregated a-synuclein by immunotherapy has become one of the highest priority therapeutic targets in PD.

[0007] Exidavnemab (BAN0805, ABBV-0805) is a humanized lgG4 mAb that binds to a broad spectrum of aggregated a-synuclein, including small and large aggregates with different conformations. Exidavnemab has a very high affinity (KD = 18 pM) and a more than 100,000-fold selectivity for pathological aggregated forms of a- synuclein versus monomers, thus enabling a high target engagement of pathological a-synuclein aggregates in the CNS while sparing the physiological monomers. Binding of exidavnemab to pathological a-synuclein has further been demonstrated in post-mortem brains of patients with PD, and the murine version of exidavnemab (mAb47) decreased a-synuclein aggregates in a dose-dependent manner, delayed the development of motor symptoms, and prolonged survival in mouse models.

[0008] Murine antibodies exhibiting the same CDR sequences as exidavnemab, e.g. mAb47, are disclosed in W02011 / 104696. The humanized sequences of the heavy and light chains of exidavnemab are disclosed in WO2021 / 260434.

[0009] Treatment modalities for brain and neurological diseases are limited, due to 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.

[0010] 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.

[0011] There are two forms of the human transferrin receptor (hTf R), namely hTfRl and hTfR2. hTfRl is the target for the binding molecule of the present disclosure. hTfRl 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. hTfRl is expressed in numerous cells and organs, but expression levels vary and, importantly, hTfRl is expressed to a higher degree on BBB endothelial cells than on other endothelial cells, making the receptor a target for neuropharmaceutical delivery. Structurally, hTfRl is a dimeric transmembrane glycoprotein comprising the amino acid sequence SEQ ID NO:85, 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).

[0012] In the context of BBB transport via the hTfRl, antibodies and fragments thereof which have affinity for hTfRl have been described. By way of example, a number of hTfRl 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 hTfRl binding antibodies are disclosed in EP3088518, EP3315606 and EP3560958, however without any information about the epitope specificity of these disclosed antibodies.

[0013] Thus, most work on using hTfRl 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 hTfRl 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 hTfRl 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).

[0014] Furthermore, the detailed structure of the hTfRl and ferritin complex was recently determined (Montemiglio et al (2019), Nat Commun 10:1121), showing that the interface between hTfRl and ferritin is located within the apical domain. This suggests that hTfRl apical binders could potentially interfere with the binding of ferritin to hTfRl 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 hTfRl (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 hTfRl, 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 hTfRl.

[0015] It has been reported that hTfRl apical binders can induce both acute clinical signs and a decrease in circulating reticulocytes (Couch et al (2013), Sci Transl Med 5:183ra57). The hTfRl 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 hTfRl has also been described (Hyman et al (1984), N Engl J Med 311:214-218). Taken together, the data suggest that hTfRl 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.

[0016] To date, the focus within the field has been to avoid interfering with one of the described hTfRl ligands, namely transferrin. This has guided the field to utilize binding sites in the apical domain of hTfRl, distant from the binding site of transferrin. However, such apical binders may still interfere with the other important hTfRl ligand, ferritin, leading to interference in iron transport and function.

[0017] 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 PD, LBD, MSA and other neurodegenerative diseases characterized by aggregated or protofibril la r a-synuclein. 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.

[0018] Disclosure of the invention

[0019] One object of the disclosure is to provide binding molecules having one or more novel and useful binding specificity / specificities.

[0020] Another object of the disclosure is to provide novel candidate molecules for the treatment of neurodegenerative diseases via targeting of aggregates of a- synuclein, for example a-synuclein, with a beneficial and unique binding profile.

[0021] Another object of the disclosure is to enable the diagnosis of PD, LBD, MSA and other neurodegenerative disorders via detection of aggregates of a-synuclein, for example a-synuclein protofibrils, implicated in disease formation and / or progression.

[0022] Another object of the disclosure is to provide molecules that bind to aggregates of a-synuclein, for example a-synuclein protofibrils, with high affinity.

[0023] Another object of the disclosure is to provide molecules that bind to aggregates of a-synuclein, for example a-synuclein protofibrils, with high specificity.

[0024] Another object of the disclosure is to provide molecules that bind to aggregates of a-synuclein, for example a-synuclein protofibrils, with high selectivity with respect to other a-synuclein variants, for example a-synuclein monomers or fibrils.

[0025] Another object of the disclosure is to provide a-synuclein-binding molecules that combine desirable properties for development into a biopharmaceutical product.

[0026] Another object of the disclosure is to provide a-synuclein-binding molecules that exhibit little or no immunogenicity upon administration in human subjects.

[0027] 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.

[0028] 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. One such object is to provide a TfRl-binding molecule which utilizes a different binding site on TfRl than transferrin.

[0029] Another such object is to provide a TfRl-binding molecule which utilizes a different binding site on TfRl than ferritin.

[0030] Yet another such object is to provide a TfRl-binding molecule which utilizes a different binding site on TfRl than HFE (homeostatic iron regulator).

[0031] 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.

[0032] 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.

[0033] Another 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.

[0034] Another object of the disclosure is to provide a hTfRl binding molecule which exhibits a higher affinity at a physiological pH value than at the lower pH value found in endosomes.

[0035] A related object of the disclosure is to provide a hTfRl binding molecule for which the ratio of the affinity at physiological pH to the affinity at the lower endosome pH may be adjusted in order to adjust the properties of the binding molecule with regard to endosomal escape into the cytoplasmic environment of a cell.

[0036] 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.

[0037] 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.

[0038] One or more of these objects, and other objects that are evident to the skilled person from the teachings herein, are met by all or some of the embodiments of the various aspects of the disclosure. Thus, in a first aspect, the present disclosure provides a bispecific binding molecule, comprising

[0039] - a first moiety Ml, which is an a-synuclein protofibril binding moiety comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, said VH region in Ml comprising the amino acid sequence SEQ ID NO:447:

[0040] QVQLQESGPG LVKPSETLSL TCTVSGFSLT SYGVHWIRQP PGKGLEWSGV IWRGGSTDYS AAFMSRLTI S KDTSKNQVSL KLSSVTAADT AVYYCAKLLR SVGGFADWGQ GTMVTVSS said VL region in Ml comprising the amino acid sequence SEQ ID NO:448:

[0041] DIVMTQSPLS LPVTPGEPAS I SCRSSQTIV HNNGNTYLEW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCFQGSHVP FTFGQGTKLE IK and

[0042] - a second moiety M2, which is a human transferrin receptor 1 (hTfRl) binding moiety comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, in which said antigen-binding 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:85.

[0043] In a second aspect, the present disclosure provides a binding molecule which is an a-synuclein protofibril binding antibody or a fragment thereof, comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, said VH region comprising the amino acid sequence SEQ ID NO:447:

[0044] QVQLQESGPG LVKPSETLSL TCTVSGFSLT SYGVHWIRQP PGKGLEWSGV IWRGGSTDYS AAFMSRLTI S KDTSKNQVSL KLSSVTAADT AVYYCAKLLR SVGGFADWGQ GTMVTVSS said VL region comprising the amino acid sequence SEQ ID NO:448:

[0045] DIVMTQSPLS LPVTPGEPAS I SCRSSQTIV HNNGNTYLEW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCFQGSHVP FTFGQGTKLE IK . In one embodiment of said second aspect, the binding molecule comprises an antibody heavy chain represented by SEQ ID NO:449 and an antibody light chain represented by SEQ ID NO:450.

[0046] In a third aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific binding molecule in accordance with the first aspect, or a binding molecule in accordance with the second aspect, and a pharmaceutically acceptable excipient or carrier.

[0047] In further aspects, the present disclosure provides bispecific binding molecules, 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. a-synuclein 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-synuclein protofibrils, and in which the sequences of the VH and VL regions are as defined above with reference to SEQ ID NO:447 and SEQ ID NO:448, respectively. a-synuclein protofibril binding moiety Ml was developed starting from the exidavnemab (BAN0805) antibody disclosed in WQ2021 / 260434, hereby incorporated in its entirety into the present disclosure. As disclosed in Example 1 herein, the exidavnemab sequence was further improved herein by the present inventors, creating a novel antibody with a significantly reduced immunogenic potential.

[0049] 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 through specific and selective binding to a-synuclein aggregates, such as a-synuclein protofibrils, by way of the a-synuclein protofibrilbinding moiety Ml.

[0050] 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.

[0051] As a person of skill in the art is aware, a-synuclein peptides may exist in various forms along the progressive aggregation thereof from monomers to fibrils. Of particular relevance to the present disclosure, soluble forms of a-synuclein may be present in monomer form, or in various oligomeric or further aggregated forms. Soluble forms of polymerized or aggregated a-synuclein 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. The bispecific binding molecule of the first aspect has an affinity for a-synuclein protofibrils through the presence of moiety Ml.

[0052] The bispecific binding molecule may exhibit a preference or selectivity for one form of a-synuclein over another. In one such embodiment, the bispecific binding molecule has a higher affinity for a-synuclein protofibrils than for a- synuclein monomers. In certain embodiments, the bispecific binding molecule of the first aspect binds selectively to a-synuclein protofibrils. As used herein, the term "bind selectively" refers to the preferential binding to the a-synuclein protofibril target. In certain embodiments, the bispecific binding molecule of the first aspect does not bind to any appreciable extent to a-synuclein monomers. In one embodiment, the bispecific binding molecule has at least 2x higher affinity for a- synuclein protofibrils than for a-synuclein 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, such as at least lOOOOx higher, such as at least 25000x higher, such as at least 50000x higher, such as at least 75000x higher, such as at least lOOOOOx higher affinity.

[0053] In one embodiment of the bispecific binding molecule, it has a binding affinity for a-synuclein 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 50 and 150 pM, such as between 75 and 125 pM, as determined by surface plasmon resonance. What is said above about the target binding properties of the Ml moiety of the bispecific binding molecule according to the first aspect applies in equal measure to the binding molecule according to the second aspect. hTfRl-binding moiety M2

[0054] 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:85. 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.

[0055] 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:85. 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:85. 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:85. 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.

[0056] 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.

[0057] In one embodiment, the binding to hTfRl by the M2 binding moiety is monovalent.

[0058] 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 regions in the polypeptide chain, but is only used to convey that both the VH and VL regions 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 region precedes the VH region in a single chain Fv, constructs in which the VH region precedes the VL region in a single chain Fv, and constructs in which the VH and VL regions are non- covalently associated with each other. In a specific embodiment of the binding protein, the VH / VL pair in M2 is arranged such that the VL region precedes the VH region in a single chain Fv construct.

[0059] 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 regions. In one embodiment, said CDRs comprise the following amino acid sequences:

[0060] VHCDR1: X1X2NMX3 (SEQ ID NO:1), wherein XI is selected from D and A; X2 is selected from Y and A; and X3 is selected from D and A;

[0061] VHCDR2: X4INPX5X6X7TTSX8X9X10KFKG (SEQ ID NO:2), 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;

[0062] X9 is selected from N and S; and X10 is selected from E and Q;

[0063] VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID NO:4), wherein

[0064] Xll is selected from Y and A;

[0065] X12 is selected from T and S;

[0066] X13 is selected from Q and R; and

[0067] X14 is selected from Y and A;

[0068] VLCDR2: X15ASTRES (SEQ ID NO:5) wherein X15 is selected from W and A; and

[0069] VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:6) wherein X16 is selected from

[0070] X17 is selected from

[0071] X18 is selected from X19 is selected from X20 is selected from

[0072] In one embodiment, the antigen-binding surface of moiety M2 further comprises:

[0073] VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:3) wherein X21 is selected from Y and A;

[0074] X22 is selected from Y and A;

[0075] X23 is selected from Y and A; X24 is selected from D and A; and X25 is selected from Y and A.

[0076] In an alternative embodiment, the antigen-binding surface of moiety M2 further comprises:

[0077] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:35)

[0078] 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 2-21. Further development of these antibodies into bispecific binding molecules of the present disclosure is detailed in Examples 22-29.

[0079] 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.

[0080] In one embodiment, said VHCDR2 of moiety M2 is:

[0081] VHCDR2: X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO:7), wherein X4 is selected from D and A;

[0082] X5 is selected from D and A;

[0083] X6 is selected from Y and A; X7 is selected from D and A; and X8 is selected from Y and A.

[0084] In one embodiment, said VLCDR1 of moiety M2 is:

[0085] VLCDR1: KSSQSLLX11STNQKNX14LA (SEQ ID NO:8), wherein Xll is selected from Y and A; and X14 is selected from Y and A.

[0086] In one embodiment, said VLCDR3 of moiety M2 is:

[0087] VLCDR3: QQX16FIX19PRT (SEQ ID NO:9) wherein X16 is selected from Y and A; X19 is selected from Y and A.

[0088] In one embodiment, the amino acid sequence of said VHCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:10 and 16-18.

[0089] In one embodiment, the amino acid sequence of said VHCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:11, 19-23 and 34, for example selected from the group consisting of SEQ ID NO:11 and 19-23.

[0090] In one embodiment, the amino acid sequence of said VHCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:12, 24-28 and 35, for example selected from the group consisting of SEQ ID NO:12 and 24-28.

[0091] In one embodiment, the amino acid sequence of said VLCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:13, 29, 30 and 36, for example selected from the group consisting of SEQ ID NO:13, 29 and 30.

[0092] In one embodiment, the amino acid sequence of said VLCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:14 and 31. In one embodiment, the amino acid sequence of said VLCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:15, 32, 33 and 37, for example selected from the group consisting of SEQ ID NO:15, 32 and 33.

[0093] 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 10 for alanine substituted variants of the representative M2 moiety h26D3.

[0094] 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:

[0095] VHCDR1: DYNMD (SEQ ID NO:10),

[0096] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0097] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12),

[0098] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0099] VLCDR2: WASTRES (SEQ ID NO:14), and

[0100] VLCDR3: QQYFIYPRT (SEQ ID NO:15).

[0101] 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:

[0102] VHCDR1: DYNMD (SEQ ID NQ:10),

[0103] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0104] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12),

[0105] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0106] VLCDR2: WASTRES (SEQ ID NO:14), and

[0107] VLCDR3: QQYFIYPRT (SEQ ID NO:15).

[0108] 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:

[0109] VHCDR1: DYNMD (SEQ ID NQ:10),

[0110] VHCDR2: DINPNYDTTSYSQKFKG (SEQ ID NO:34),

[0111] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:35),

[0112] VLCDR1: KSSQSLLYSSNRKNYLA (SEQ ID NO:36),

[0113] VLCDR2: WASTRES (SEQ ID NO:14), and

[0114] VLCDR3: QQYYNYPYT (SEQ ID NO:37).

[0115] 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:

[0116] VHCDR1: NYWLG (SEQ ID NO:38),

[0117] VHCDR2: DIFPGSDNTYYNEKFKG (SEQ ID NO:39), VHCDR3: SGNFYAMDY (SEQ ID NQ:40),

[0118] VLCDR1: SASSSVNYMN (SEQ ID NO:41),

[0119] VLCDR2: DTSKLAS (SEQ ID NO:42), and

[0120] VLCDR3: FQGSGYPFT (SEQ ID NO:43).

[0121] In one embodiment of the bispecific binding molecule of the disclosure, the VH region in moiety M2 comprises or consists of an amino acid sequence selected from

[0122] (i) the group consisting of SEQ ID NO:44-57, 65 and 67, for example the group consisting of SEQ ID NO:44-57, for example the group consisting of SEQ ID NO:44 and 50; and

[0123] (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).

[0124] In one embodiment of the bispecific binding molecule of the disclosure, the VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0125] (i) the group consisting of SEQ ID NO:58-64, 66 and 68, for example the group consisting of SEQ ID NO:58-64; and

[0126] (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).

[0127] In a particular such embodiment, the VH region and VL region in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NO:44-57 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NO:58-64 and sequences having at least 80 % sequence identity thereto.

[0128] In another embodiment of the bispecific binding molecule of the disclosure, said VH region in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:69 and a sequence having at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:69, provided that the sequences of the CDR regions are 100% identical to those of SEQ ID NO:69.

[0129] In another embodiment of the bispecific binding molecule of the disclosure, said VL region of moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NQ:70 and a sequence having at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NQ:70, provided that the sequences of the CDR regions are 100% identical to those of SEQ ID NQ:70.

[0130] In a particular such embodiment, the M2 VH region and VL region are both as defined immediately above, i.e. a VH comprising or consisting of the sequence SEQ ID NO:69 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of the sequence SEQ ID NQ:70 and sequences having at least 80 % sequence identity thereto.

[0131] In one embodiment of the bispecific binding molecule of the disclosure, said VH region in M2 comprises SEQ ID NO:44 and said VL region in M2 comprises a sequence selected from SEQ ID NO:58-64.

[0132] In one embodiment of the bispecific binding molecule of the disclosure, said VH region in M2 comprises a sequence selected from SEQ ID NO:44-57 and said VL region in M2 comprises SEQ ID NO:58.

[0133] In one embodiment of the bispecific binding molecule of the disclosure, said VH region in M2 comprises SEQ ID NO:44 and said VL region in M2 comprises SEQ ID NO:58.

[0134] In one embodiment of the bispecific binding molecule of the disclosure, said VH region in M2 comprises SEQ ID NQ:50 and said VL region in M2 comprises SEQ ID NO:58.

[0135] In some embodiments of the bispecific binding molecule of the disclosure, the VH / VL pair in moiety M2 is stabilized by a disulfide bridge. In such embodiments, the hTfRl-binding moiety M2 comprises one first cysteine residue in the VH region thereof, and one second cysteine residue in the VL region thereof, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL regions.

[0136] Without wishing to be bound by theory, the provision of the first and second cysteine residues in the VH and VL regions 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.

[0137] Importantly, the increased size and avidity of dimers, or further multimers, of binding molecules comprising pairs of VH and VL regions 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. 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 regions, 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.

[0138] In one embodiment, said first cysteine (in the M2 VH region) 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.

[0139] In one embodiment, said second cysteine (in the M2 VL region) 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.

[0140] 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.

[0141] In one embodiment of the bispecific binding molecule of the disclosure, the

[0142] VH region in moiety M2 comprises or consists of an amino acid sequence selected from (i) the group consisting of SEQ ID NO:88-103, for example the group consisting of SEQ ID NO:88-101, for example the group consisting of SEQ ID NO:88 and 94; and

[0143] (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).

[0144] In one embodiment of the bispecific binding molecule of the disclosure, the VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0145] (i) the group consisting of SEQ ID NQ:105-113, for example the group consisting of SEQ ID NQ:105-lll; and

[0146] (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).

[0147] In a particular such embodiment, the VH region and VL region in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NQ:88-103 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NQ:105-lll and sequences having at least 80 % sequence identity thereto, subject to the defined provisos.

[0148] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH region comprises SEQ ID NO:88 and the M2 VL region comprises a sequence selected from SEQ ID NQ:105-lll.

[0149] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH region comprises a sequence selected from SEQ ID NQ:88-103 and the M2 VL region comprises SEQ ID NQ:105.

[0150] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH region comprises SEQ ID NO:88 and the M2 VL region comprises SEQ ID NQ:105. In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH region comprises SEQ ID NO:94 and the M2 VL region comprises SEQ ID NO:105.

[0151] 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

[0152] (i) the group consisting of SEQ ID NO:457-467, for example the group consisting of SEQ ID NO:457-462, for example the group consisting of SEQ ID NO:457-458; and

[0153] (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).

[0154] 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

[0155] (i) the group consisting of SEQ ID NQ:105-113, for example the group consisting of SEQ ID NQ:105-lll; 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), and provided that the sequence comprises a cysteine residue at position 106 (Kabat position 100).

[0157] 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:457-467 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NQ:105-113 and sequences having at least 80 % sequence identity thereto, subject to the defined provisos.

[0158] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises a sequence selected from SEQ ID NO:457-458 and the M2 VL domain comprises SEQ ID NQ:105. In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises SEQ ID NO:457 and the M2 VL domain comprises SEQ ID NQ:105.

[0159] In one embodiment of a bispecific binding molecule of the disclosure, the M2 VH domain comprises SEQ ID NO:458 and the M2 VL domain comprises SEQ ID NQ:105.

[0160] In another embodiment of the bispecific binding molecule of the disclosure, the VH region in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NQ:104 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 NQ:104, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NQ:104, and provided that the sequence comprises a cysteine residue at position 44 (Kabat position 44).

[0161] In another of the bispecific binding molecule of the disclosure, the VL region in moiety M2 comprises or consists of an amino acid sequence selected from SEQ ID NO:114 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:114, provided that the sequences of the CDR regions are 100% identical to those of the CDR regions in SEQ ID NO:114, and provided that the sequence comprises a cysteine residue at position 99 (Kabat position 100).

[0162] In a particular such embodiment, the M2 VH region and M2 VL region are both as defined immediately above, i.e. a VH comprising or consisting of the sequence SEQ ID NQ:104 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of the sequence SEQ ID NO:114 and sequences having at least 80 % sequence identity thereto, subject to the defined provisos.

[0163] In other embodiments of the bispecific binding molecule of the disclosure, which have a pH sensitive affinity for hTfRl, said VH / VL pair in moiety M2 comprises from two to four histidine residues, and wherein the affinity of the binding molecule for hTfRl is higher at pH 7.4 than at pH 5.5.

[0164] In one embodiment, the VH region of said VH / VL pair in moiety M2 comprises from one to four histidine residues, such as from one to three histidine residues or from two to four histidine residues, such as from one to two histidine residues, from two to three histidine residues or from three to four histidine residues, such as comprising one, two, three or four histidine residues, subject to the proviso that the total number of histidine residues in the VH / VL pair in moiety M2 is from two to four.

[0165] In one embodiment, the VL region of said VH / VL pair in moiety M2 comprises from zero to three histidine residues, such as from zero to two histidine residues or from one to three histidine residues, such as from zero to one histidine residues, from one to two histidine residues or from two to three histidine residues, such as comprising zero, one, two or three histidine residues, subject to the proviso that the total number of histidine residues in the VH / VL pair in moiety M2 is from two to four.

[0166] In one embodiment, said VH region in moiety M2 comprises two histidine residues and said VL region in moiety M2 comprises zero histidine residues.

[0167] In another embodiment, said VH region in moiety M2 comprises three histidine residues and said VL region in moiety M2 comprises zero histidine residues.

[0168] In yet another embodiment, said VH region in moiety M2 comprises two histidine residues and said VL region in moiety M2 comprises one histidine residue.

[0169] In some embodiments of the bispecific binding molecule of the first aspect of the disclosure in which the hTfRl affinity provided by moiety M2 is higher at pH 7.4 than at pH 5.5, said affinity of the binding molecule for hTfRl at pH 7.4 is characterized by a first KD value and said affinity of the binding molecule for hTfRl at pH 5.5 is characterized by a second KD value. Because the affinity of the binding molecule for hTfRl is higher at pH 7.4, the first KD value (i.e. the KD at pH 7.4) is lower than the second KD value (i.e. the KD at pH 5.5). As readily understood by a person of skill in the art, the first and second KD values may suitably be determined under conditions that are identical except for the pH value. According to one embodiment, the KD values are determined using bio-layer interferometry. As explained below, however, the skilled person is aware of other methods for measuring affinity, either directly or indirectly.

[0170] In one embodiment, said first KD value is lower than said second KD value by a factor of at least 1.5, for example by a factor of at least 2, for example by a factor of at least 3, for example by a factor of at least 4, for example by a factor of at least 5. In one embodiment, said first KD value is no more than 1 x IO-6M, such as no more than 1 x IO-7M, such as no more than 1 x IO-8M.

[0171] In one such pH sensitive embodiment, said CDRs comprise the following:

[0172] VHCDR1: X1X2NMX3 (SEQ ID NO:1), wherein

[0173] XI is selected from D and A;

[0174] X2 is selected from Y and A; and

[0175] X3 is selected from D and A;

[0176] VHCDR2: X4IX26PX5X6X7X27TSX8X9X10KFKG (SEQ ID NO:468), wherein X4 is selected from D and A;

[0177] X26 is selected from N and H;

[0178] X5 is selected from D, N and A;

[0179] X6 is selected from Y, H and A;

[0180] X7 is selected from D and A;

[0181] X27 is selected from T and H;

[0182] X8 is selected from Y and A;

[0183] X9 is selected from N and S; and X10 is selected from E and Q;

[0184] VHCDR3: GGX21X28GSX29X22X23HPMX24X25 (SEQ ID NO:469) wherein

[0185] X21 is selected from Y and A;

[0186] X28 is selected from S and H;

[0187] X29 is selected from S and H;

[0188] X22 is selected from Y, H and A;

[0189] X23 is selected from Y, H and A;

[0190] X24 is selected from D and A; and

[0191] X25 is selected from Y and A.

[0192] VLCDR1: KSSQSLLX11X30X12NX13KNX14LA (SEQ ID NO:470), wherein

[0193] Xll is selected from Y and A;

[0194] X30 is selected from S and H;

[0195] X12 is selected from T, H and S;

[0196] X13 is selected from Q and R; and

[0197] X14 is selected from Y, H and A;

[0198] VLCDR2: X15ASTRES (SEQ ID N0:5) wherein X15 is selected from W and A; and

[0199] VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:471) wherein X16 is selected from

[0200] X17 is selected from

[0201] X18 is selected from X19 is selected from X20 is selected from

[0202] In one embodiment, the amino acid sequence of said VHCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:10 and 16-18.

[0203] In one embodiment, the amino acid sequence of said VHCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:11, 19-23, 34 and 472-474, for example selected from the group consisting of SEQ ID NO:472-474.

[0204] In one embodiment, the amino acid sequence of said VHCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:12, 24-28, 35 and 475-478, for example selected from the group consisting of SEQ ID NO:475-478.

[0205] In one embodiment, the amino acid sequence of said VLCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:13, 29, 30, 36 and 479-481, for example selected from the group consisting of SEQ ID NO: 479-481.

[0206] In one embodiment, the amino acid sequence of said VLCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:14 and 31.

[0207] In one embodiment, the amino acid sequence of said VLCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:15, 32, 33, 37 and 482, for example being SEQ ID NO:482.

[0208] In some embodiments, the CDR sequences can be freely combined among the options listed above.

[0209] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0210] VHCDR1: DYNMD (SEQ ID NQ:10),

[0211] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0212] VHCDR3: GGYHGSSYYHPMDY (SEQ ID NO:475) VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID N0:13),

[0213] VLCDR2: WASTRES (SEQ ID N0:14)

[0214] VLCDR3: QQYFIYPRT (SEQ ID N0:15)

[0215] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0216] VHCDR1: DYNMD (SEQ ID NQ:10),

[0217] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0218] VHCDR3: GGYHGSSYYHPMDY (SEQ ID NO:475)

[0219] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0220] VLCDR2: WASTRES (SEQ ID NO:14)

[0221] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0222] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0223] VHCDR1: DYNMD (SEQ ID NQ:10),

[0224] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0225] VHCDR3: GGYSGSSHYHPMDY (SEQ ID NO:477)

[0226] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0227] VLCDR2: WASTRES (SEQ ID NO:14)

[0228] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0229] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0230] VHCDR1: DYNMD (SEQ ID NQ:10),

[0231] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0232] VHCDR3: GGYSGSSHYHPMDY (SEQ ID NO:477)

[0233] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0234] VLCDR2: WASTRES (SEQ ID NO:14)

[0235] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0236] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0237] VHCDR1: DYNMD (SEQ ID NQ:10),

[0238] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11), VHCDR3: GGYSGSSYYHPMDY (SEQ ID N0:12)

[0239] VLCDR1: KSSQSLLYHTNQKNYLA (SEQ ID NO:479),

[0240] VLCDR2: WASTRES (SEQ ID N0:14)

[0241] VLCDR3: QQYFIYPRT (SEQ ID N0:15)

[0242] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0243] VHCDR1: DYNMD (SEQ ID NQ:10),

[0244] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0245] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0246] VLCDR1: KSSQSLLYHTNQKNYLA (SEQ ID NO:479),

[0247] VLCDR2: WASTRES (SEQ ID NO:14)

[0248] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0249] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0250] VHCDR1: DYNMD (SEQ ID NQ:10),

[0251] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0252] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0253] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0254] VLCDR2: WASTRES (SEQ ID NO:14)

[0255] VLCDR3: QQYFIHPRT (SEQ ID NO:482)

[0256] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs in moiety M2 are the following:

[0257] VHCDR1: DYNMD (SEQ ID NQ:10),

[0258] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0259] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0260] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0261] VLCDR2: WASTRES (SEQ ID NO:14)

[0262] VLCDR3: QQYFIHPRT (SEQ ID NO:482)

[0263] In one such pH sensitive embodiment, said VH region in moiety M2 comprises or consists of an amino acid sequence selected from

[0264] (i) the group consisting of SEQ ID NO:44, 50, 129-145, 163-178, 195-211, 229- 245, 263-279, 297-312, 329-344 and 442, for example the group consisting of SEQ ID NO: 129-145, 163-178, 195-211, 229-245, 263-279, 297-312, 329-344 and 442, for example the group consisting of SEQ ID NO:129-142, 163-175, 195-208, 229-242, 263-276, 297-309, 329-341 and 442, for example the group consisting of SEQ ID NO:229-242, 263-276 and 297-309 or the group consisting of SEQ ID NO:129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329 and 335, for example the group consisting of SEQ ID NO:229, 235, 263, 269, 297 and 303; and

[0265] (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 any histidine residue in the sequence defined in (i) is present.

[0266] In one pH sensitive embodiment, said VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0267] (i) the group consisting of SEQ ID NO:58, 361-370, 381-390, 401-409 and 419- 427, for example the group consisting of SEQ ID NQ:361-370, 381-390, 401-409 and 419-427, for example the group consisting of SEQ ID NO:361-367, 381-387, 401-406 and 419-424, for example the group consisting of SEQ ID NO:419-424 or the group consisting of SEQ ID NO:361, 381, 401 and 419, for example SEQ ID NO:419; and

[0268] (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 any histidine residue in the sequence defined in (i) is present.

[0269] In a particular such embodiment, said VH and VL regions in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NO:44, 50, 129-145, 163-178, 195-211, 229-245, 263-279, 297- 312, 329-344 and 442 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NO:58, 361- 370, 381-390, 401-409 and 419-427 and sequences having at least 80 % sequence identity thereto, subject to the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0270] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329, 335 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58, 361-367, 381-387, 401-406 and 419-424, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0271] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129-142, 163-175, 195-208, 229-242, 263-276, 297- 309, 329-341 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58 and 419-424, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0272] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129-142, 163-175, 195-208, 229-242, 263-276, 297- 309, 329-341 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58, 361, 381, 401 and 419, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0273] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329, 335 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58 and 419-424, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0274] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329, 335 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58, 361, 381, 401 and 419, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0275] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 229, 263, 297, 329 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO: 58, 361, 381, 401 and 419, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0276] In one embodiment, the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:58; b) a VH region comprising SEQ ID NO:263 and a VL region comprising SEQ ID NO:58; c) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:361; d) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:381; e) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NQ:401; f) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:419; g) a VH region comprising SEQ ID NO:297 and a VL region comprising SEQ ID NO:419; and h) a VH region comprising SEQ ID NO:442 and a VL region comprising SEQ ID NO:58.

[0277] In a more specific embodiment, the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:58; b) a VH region comprising SEQ ID NO:263 and a VL region comprising SEQ ID NO:58; and c) a VH region comprising SEQ ID NO:297 and a VL region comprising SEQ ID NO:419.

[0278] In an individual embodiment, said VH region in moiety M2 comprises SEQ ID NO:229 and said VL region in moiety M2 comprises SEQ ID NO:58.

[0279] In another individual embodiment, said VH region in moiety M2 comprises SEQ ID NO:263 and said VL region in moiety M2 comprises SEQ ID NO:58.

[0280] In still another individual embodiment, said VH region in moiety M2 comprises SEQ ID NO:297 and said VL region in moiety M2 comprises SEQ ID NO:419.

[0281] In a further individual embodiment, said VH region in moiety M2 comprises SEQ ID NO:442 and said VL region in moiety M2 comprises SEQ ID NO:58.

[0282] In still other embodiments of the bispecific binding molecule, the hTfRl binding moiety M2 is both stabilized by a disulfide bridge and provided with a pH sensitive affinity for its target.

[0283] In one such embodiment, said VH region in moiety M2 comprises or consists of an amino acid sequence selected from

[0284] (i) the group consisting of SEQ ID NO:88, 94, 146-162, 179-194, 212-228, 246- 262, 280-296, 313-328, 345-360 and 443, for example the group consisting of SEQ ID NO:146-162, 179-194, 212-228, 246-262, 280-296, 313-328, 345-360 and 443, for example the group consisting of SEQ ID NO:146-159, 179-191, 212-225, 246-259, 280-293, 313-325, 345-357 and 443, for example the group consisting of SEQ ID NO:246-259, 280-293 and 313-325 or the group consisting of SEQ ID NO:146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345 and 351, for example the group consisting of SEQ ID NO:246, 252, 280, 286, 313 and 319; and

[0285] (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), provided that any histidine residue in the sequence defined in (i) is present, and provided that the sequence comprises a cysteine residue at position 44.

[0286] In one embodiment, said VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0287] (i) the group consisting of SEQ ID NQ:105, 371-380, 391-400, 410-418 and 428-436, for example the group consisting of SEQ ID NQ:371-380, 391-400, 410-418 and 428-436, for example the group consisting of SEQ ID NO:371-377, 391-397, 410- 415 and 428-433, for example the group consisting of SEQ ID NO:428-433 or the group consisting of SEQ ID NO:371, 391, 410 and 428, for example SEQ ID NO:428; and

[0288] (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), provided that any histidine residue in the sequence defined in (i) is present, and provided that the sequence comprises a cysteine residue at position 106.

[0289] In a particular such embodiment, the VH region and VL region in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NO:88, 94, 146-162, 179-194, 212-228, 246-262, 280-296, 313-328, 345-360 and 443 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NQ:105, 371-380, 391-400, 410-418 and 428-436 and sequences having at least 80 % sequence identity thereto, subject to the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105, and provided that the sequences comprise the defined cysteine residues at VH position 44 and VL position 106.

[0290] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345, 351 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:105, 371-377, 391-397, 410-415 and 428-433, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0291] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146-159, 179-191, 212-225, 246-259, 280-293, 313- 328, 345-360 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0292] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146-159, 179-191, 212-225, 246-259, 280-293, 313- 325, 345-357 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371, 391, 410 and 428, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0293] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345, 351 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0294] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345, 351 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371, 391, 410 and 428, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0295] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 179, 212, 246, 280, 313, 345 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO: 105, 371, 391, 410 and 428, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NO:105.

[0296] In one embodiment, the VH region and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NQ:280 and a VL region comprising SEQ ID NQ:105; c) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:371; d) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:391; e) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:410; f) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:428; g) a VH region comprising SEQ ID NO:313 and a VL region comprising SEQ ID NO:428; and h) a VH region comprising SEQ ID NO:443 and a VL region comprising SEQ ID NQ:105.

[0297] In one embodiment, the VH region and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NQ:280 and a VL region comprising SEQ ID NQ:105; and c) a VH region comprising SEQ ID NO:313 and a VL region comprising SEQ ID NO:428.

[0298] In a specific embodiment, said VH region in moiety M2 comprises SEQ ID NO:246 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0299] In another specific embodiment, said VH region in moiety M2 comprises SEQ ID NQ:280 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0300] In yet another specific embodiment, said VH region in moiety M2 comprises SEQ ID NO:313 and said VL region in moiety M2 comprises SEQ ID NO:428.

[0301] In still another specific embodiment, said VH region in moiety M2 comprises SEQ ID NO:443 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0302] In one embodiment, said VH region in moiety M2 comprises or consists of an amino acid sequence selected from

[0303] (i) the group consisting of SEQ ID NQ:483-530, for example the group consisting of SEQ ID NO:483-506, for example the group consisting of SEQ ID NO:483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503 and 505, for example the group consisting of SEQ ID NO:483, 487, 491, 495, 499 and 503, or the group consisting of SEQ ID NO:485, 489, 493, 497, 501 and 505; or the group consisting of SEQ ID NQ:507-530, for example the group consisting of SEQ ID NQ:507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527 and 529, for example the group consisting of SEQ ID NQ:507, 511, 515, 519, 523 and 527 or the group consisting of SEQ ID NQ:509, 513, 517, 521, 525 and 529; and

[0304] (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), provided that any histidine residue in the sequence defined in (i) is present, and provided that the sequence comprises a cysteine residue at position 44.

[0305] In one such embodiment, said VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0306] (i) the group consisting of SEQ ID NQ:105, 371-380, 391-400, 410-418 and 428-436, for example the group consisting of SEQ ID NQ:371-380, 391-400, 410-418 and 428-436, for example the group consisting of SEQ ID NO:371-377, 391-397, 410- 415 and 428-433, for example the group consisting of SEQ ID NO:428-433 or the group consisting of SEQ ID NO:371, 391, 410 and 428, for example SEQ ID NO:428; and

[0307] (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), provided that any histidine residue in the sequence defined in (i) is present, and provided that the sequence comprises a cysteine residue at position 106.

[0308] In a particular such embodiment, the VH region and VL region in moiety M2 are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NQ:483-530 and sequences having at least 80 % sequence identity thereto, and a VL comprising or consisting of a sequence selected from SEQ ID NQ:105, 371-380, 391-400, 410-418 and 428-436 and sequences having at least 80 % sequence identity thereto, provided that the sequences comprise the defined cysteine residues at VH position 44 and VL position 106.

[0309] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483-530, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:105, 371-377, 391-397, 410-415 and 428-433.

[0310] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:483-506, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0311] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503 and 505, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0312] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483, 487, 491, 495, 499 and 503, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0313] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:485, 489, 493, 497, 501 and 505, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0314] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507-530, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0315] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527 and 529, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0316] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507, 511, 515, 519, 523 and 527, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0317] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:509, 513, 517, 521, 525 and 529, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:105, 371-377, 391-397, 410-415 and 428-433.

[0318] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483-530, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0319] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:483-506, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0320] In one embodiment, said VH region in moiety M2 comprises a sequence selected SEQ ID NO:483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503 and 505, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0321] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483, 487, 491, 495, 499 and 503, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0322] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:485, 489, 493, 497, 501 and 505, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0323] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507-530, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0324] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527 and 529, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0325] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507, 511, 515, 519, 523 and 527, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0326] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:509, 513, 517, 521, 525 and 529, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428-433. In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483-530, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:105, 371 and 428.

[0327] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:483-506, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0328] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503 and 505, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0329] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:483, 487, 491, 495, 499 and 503, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0330] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:485, 489, 493, 497, 501 and 505, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0331] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507-530, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0332] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527 and 529, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0333] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:507, 511, 515, 519, 523 and 527, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0334] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:509, 513, 517, 521, 525 and 529, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0335] In one embodiment, said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:441 and 531, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:371 and 428. In one embodiment, the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:483 and a VL region comprising SEQ ID NO:105; b) a VH region comprising SEQ ID NO:491 and a VL region comprising SEQ ID NQ:105; c) a VH region comprising SEQ ID NQ:507 and a VL region comprising SEQ ID NQ:105; d) a VH region comprising SEQ ID NO:515 and a VL region comprising SEQ ID NQ:105; e) a VH region comprising SEQ ID NO:483 and a VL region comprising SEQ ID NQ:106; f) a VH region comprising SEQ ID NO:491 and a VL region comprising SEQ ID NQ:106; g) a VH region comprising SEQ ID NQ:507 and a VL region comprising SEQ ID NQ:106; h) a VH region comprising SEQ ID NO:515 and a VL region comprising SEQ ID NQ:106; i) a VH region comprising SEQ ID NO:441 and a VL region comprising SEQ ID NO:371; j) a VH region comprising SEQ ID NO:441 and a VL region comprising SEQ ID NO:428; k) a VH region comprising SEQ ID NO:531 and a VL region comprising SEQ ID NO:371; and l) a VH region comprising SEQ ID NO:531 and a VL region comprising SEQ ID NO:428.

[0336] In one embodiment, the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:483 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NO:491 and a VL region comprising SEQ ID NQ:105; c) a VH region comprising SEQ ID NQ:507 and a VL region comprising SEQ ID NQ:105; and d) a VH region comprising SEQ ID NO:515 and a VL region comprising SEQ ID NQ:105.

[0337] In a specific embodiment, said VH region in moiety M2 comprises SEQ ID NO:483 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0338] In another specific embodiment, said VH region in moiety M2 comprises SEQ ID NO:491 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0339] In yet another specific embodiment, said VH region in moiety M2 comprises SEQ ID NQ:507 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0340] In still another specific embodiment, said VH region in moiety M2 comprises SEQ ID NO:515 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0341] In certain embodiments, the VH and VL sequences, when present in moiety M2 of the bispecific binding molecule, are 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.

[0342] Linkage of Ml and M2

[0343] 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.

[0344] 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 regions 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 region and to the N-terminal amino acid residue of the VL region, or to the N-terminal amino acid residue of the VH region and to the C-terminal amino acid residue of the VL region. In the first configuration, the VH region precedes the VL region in the polypeptide chain constituting the scFv, while in the second configuration, the VL region precedes the VH region. The two different configurations are sometimes denoted "VH first" and "VL first" in the present disclosure.

[0345] 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:455).

[0346] The same or similar design considerations apply to linkers used to attach the a-synuclein-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 region of M2 or to the N-terminal amino acid residue of the VL region of M2.

[0347] 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.

[0348] 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.

[0349] 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:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of an amino acid sequence selected from SEQ ID NO:124-128 and 452-454, or selected from SEQ ID NO:124-127 and 452-454, or selected from SEQ ID NO:124-128, or selected from SEQ ID NO:124-127, or selected from SEQ ID NO:452-454. 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:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:124.

[0350] 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 NQ:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:125.

[0351] 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 NQ:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:126.

[0352] 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 NQ:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:127.

[0353] 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 NQ:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:452.

[0354] 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 NQ:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:453.

[0355] 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 NQ:450; the amino acid sequence of the antibody hole heavy chain of Ml comprises or consists of SEQ ID NO:451, and the combined amino acid sequence of the Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:454.

[0356] Amino acid sequences

[0357] 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 regions 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.

[0358] 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).

[0359] Affinity for a target

[0360] 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- synuclein 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.

[0361] According to some embodiments of the bispecific binding molecule of the disclosure, moiety M2 has an affinity for hTfRl which is different at different pH values. Specifically, in such embodiments, the bispecific binding molecule has a higher affinity for hTfRl at pH 7.4 than at pH 5.5. As readily realized by the skilled person, this property is readily tested by measuring the affinity at said different pH values, while keeping all other experimental conditions identical, or as close to identical as possible in the chosen experimental setup. Measuring affinity at different pH values may be done using any of the following general methods for measuring affinity.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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.

[0370] Stability of a bispecific binding molecule of the disclosure

[0371] The introduction of cysteine residues and the resulting formation of a disulfide bridge in moiety M2 in some embodiments of the bispecific binding molecule of the disclosure is contemplated to increase stability. 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 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.

[0372] Polynucleotides, vectors and cells

[0373] 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:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:124. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NQ:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:125. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NQ:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:126. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:127. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NQ:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:128. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NQ:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:452. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NQ:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:453. In another specific embodiment, such a set comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NQ:450; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:451; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:454.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] Pharmaceutical compositions

[0379] In a third aspect, there is provided a pharmaceutical composition comprising a bispecific binding molecule or binding molecule as described herein and at least one pharmaceutically acceptable excipient or carrier.

[0380] 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.

[0381] 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.

[0382] 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.

[0383] Methods of prevention, treatment, diagnosis, prognosis and detection

[0384] The bispecific binding molecules and binding molecules according to the present disclosure may be useful as therapeutic and / or diagnostic agents.

[0385] Hence, in a further aspect of the disclosure, there is provided a bispecific binding molecule according to the first aspect, a binding molecule according to the second aspect, or a pharmaceutical composition according to the third aspect, for use as a medicament.

[0386] In yet another aspect of the disclosure, there is provided a bispecific binding molecule according to the first aspect, a binding molecule according to the second aspect, or a pharmaceutical composition according to the third aspect, for use as a diagnostic agent.

[0387] In yet another aspect of the disclosure, there is provided a bispecific binding molecule according to the first aspect, a binding molecule according to the second aspect, or a pharmaceutical composition according to the third aspect, for use as a prognostic agent.

[0388] Also provided are methods of preventing disease, treating disease or diagnosing disease or assessing disease prognosis, wherein a molecule as disclosed herein is administered to a subject, typically a human subject.

[0389] Also provided is the use of the disclosed molecules 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.

[0390] 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 molecule as described herein. These methods are typically in vitro methods.

[0391] Thus, said bispecific binding molecule, binding molecule or pharmaceutical composition comprising either of them, 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 a-synuclein, such as formation of a-synuclein fibrils. Such diseases or conditions include but are not limited to Parkinson's disease (RD), multiple system atrophy (MSA) and Lewy body dementia (LBD), including dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).

[0392] Thus, in one embodiment, there is provided a molecule of the disclosure, or pharmaceutical composition comprising it, for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of an a-synuclein-associated condition. In one embodiment, there is provided a molecule of the disclosure, or pharmaceutical composition comprising it, for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of an a-synuclein-associated condition, selected from the group consisting of Parkinson's disease (PD), multiple system atrophy (MSA) and Lewy body dementia (LBD), including dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).

[0393] In one specific embodiment, said molecule, or pharmaceutical composition comprising it, is provided for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of Parkinson's disease (PD).

[0394] In one specific embodiment, said molecule, or pharmaceutical composition comprising it, is provided for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of multiple system atrophy (MSA).

[0395] In another aspect, there is provided a method of therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of an a-synuclein-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 according to the first aspect, a binding molecule according to the second aspect, or a pharmaceutical composition comprising either molecule. In one embodiment, said a-synuclein-associated condition is, for example, selected from the group consisting of Parkinson's disease (PD), multiple system atrophy (MSA) and Lewy body dementia (LBD), including dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD). In one more specific embodiment, said a-synuclein-associated condition is Parkinson's disease (PD). In another more specific embodiment, said a-synuclein-associated condition is multiple system atrophy (MSA).

[0396] With regard to diagnostic and / or prognostic use of the disclosed bispecific binding molecule, binding molecule or composition in neurodegenerative diseases, the harmful a-synuclein 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-synuclein protofibrils in blood, plasma, CSF and other fluids, using such methods as ELISA, Mesoscale Discovery (MSD), SMCxPro or Simoa.

[0397] Kit

[0398] 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.

[0399] Incorporation by reference

[0400] Various publications are cited in the present application, each of which is incorporated by reference herein in its entirety.

[0401] Brief description of the figures

[0402] Figure 1 shows the results of a binding screen of the indicated IgG antibodies from the immunization described in Example 2 towards human (hTfRl), cyno (cTfRl) and mouse (mTfRl) TfRl in crude hybridoma supernatants by biolayer interferometry (BLI).

[0403] Figure 2 shows the result of the BLI binding analysis described in Example 3 for the indicated Fab fragments of mouse antibodies 24B4, 26D3 and 37D10 as well as for a Fab fragment of control antibody 8D3.

[0404] Figure 3 shows mapping of antibody binding epitopes to the protease-like domain of hTfRl as described in Example 3, 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).

[0405] 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.

[0406] Figure 5 shows the result of carrying out the epitope binding assay as described in Example 3, 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).

[0407] Figure 6 shows binding by the indicated binders to hTfRl on the surface of cells, studied as described in Example 3. 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).

[0408] Figure 7 shows the result of competition analysis of indicated binders with ferritin and transferrin as described in Example 4. The diagrams show (A) MFI of the indicated binders binding to hTfRl expressed on THP-1 cell surfaces, (B) MFI of ferritin on cell surface when exposed to the indicated binders, with the positive control antibody MA-712 competing with ferritin, and (C) MFI of transferrin on cell surfaces when exposed to the indicated binders.

[0409] Figure 8 is a collection of sensorgrams showing the result of SPR analysis of original 26D3 and 26D3 humanized as described in Example 5 (h26D3) in Fab formats when binding to hTfRl and cTfRl as indicated.

[0410] 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 5. (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.

[0411] Figure 10 are depictions of the x-ray structure of the complex of h26D3-Fab and hTfRl, determined as described in Example 6. 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.

[0412] Figure 11 is a ribbon representation of the h26D3-Fab human TfRl complex determined with x-ray crystallography as described in Example 6. h26D3-Fab is depicted in dark gray and hTfRl in white. The binding interface (epitope / paratope) is encircled.

[0413] 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.

[0414] Figure 13 illustrates the work on generating and characterizing an hTfRl-KI mouse model as described in Example 7. (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 p-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).

[0415] 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 8. (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".

[0416] 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 9. 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.

[0417] Figure 16 shows BLI sensorgrams for the indicated alanine variants of h26D3 as described in Example 10. 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.

[0418] 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 10.

[0419] 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 10.

[0420] 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 10.

[0421] Figure 20 shows chromatograms from preparative SEC of (A) h26D3-HC6_DS and (B) h26D3-HC6, carried out as described in Example 11.

[0422] 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

[0423] 12. 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 13.

[0424] 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 13.

[0425] 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 13. 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.

[0426] Figure 25 demonstrates the results of the ELISA experiment described in Example 14, 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.

[0427] Figure 26 shows representative SPR sensorgrams of the interaction between the indicated variants of h26D3 with hTfRl, measured as described in Example 16.

[0428] Figure 27 shows representative SPR sensorgrams of the interaction between the indicated pH sensitive "single mutant" variants of h26D3 with human TfRl, measured as described in Example 18.

[0429] Figure 28 shows representative SPR sensorgrams of the interaction between the indicated pH sensitive "single mutant" variants of h26D3 with cynomolgus TfRl, measured as described in Example 18.

[0430] Figure 29 shows representative SPR sensorgrams of the interaction between the indicated pH sensitive "double mutant" variants of h26D3 with hTfRl, measured as described in Example 18.

[0431] Figure 30 shows representative BLI sensorgrams of the interaction at pH 5.5 and pH 7.4 between the indicated pH sensitive variants of h26D3 with hTfRl, measured as described in Example 18. Figure 31 shows the results of competitive ELISA at pH 6 and pH 7.4, carried out as described in Example 18.

[0432] Figure 32 shows the result of capillary electrophoresis analysis (CE-SDS) of S102H-DS after expression and purification as described in Example 19, at nonreduced (NR) and reduced (R) conditions. A size marker (M) was loaded for reference.

[0433] Figure 33 shows chromatograms from analytical SEC of S102H-DS scFv, on samples analyzed before (A) and after (B) 3 freeze / thawing cycles as described in Example 19.

[0434] Figure 34 shows the results of analytical SEC of S102-DS scFv samples subjected to temperature hold as described in Example 20, for 1, 2 or 4 weeks at - 75 °C (A), 4 °C (B) or 40 °C (C). Graphs show the monomer content as grey bars and high molecular weight content as black bars, as calculated from analytical SEC peak areas.

[0435] Figure 35 shows representative SPR sensorgrams of the interaction between the indicated test molecules with hTfRl, measured as described in Example 21.

[0436] Figure 36 shows representative SPR sensorgrams of the interaction between the indicated test molecules with monomers of human a-synuclein, measured as described in Example 21.

[0437] Figure 37 shows representative SPR sensorgrams of the interaction between the indicated test molecules with protofibrils of human a-synuclein, measured as described in Example 21.

[0438] Figure 38 shows the results of immunodepletion of a-synuclein protofibrils in brain extracts from patients with Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), using the indicated test molecules as described in Example 22.

[0439] Figure 39 shows immunohistochemical (IHC) analysis of tissues carried out as described in Example 22. A) Binding of BA201 to Lewy bodies and neurites in PDD cortex. B) Binding of BA202 to Lewy bodies and neurites in PDD. C) Binding of BAN0805 to Lewy bodies and neurites in PDD cortex. D) Negative control staining using an IgGl isotype control. Figure 40 shows images from target engagement by the indicated test molecules on living SH-SY5Y a-synuclein tRFP cells 1 h after exposure, obtained as described in Example 23. Scale bar 20 pm.

[0440] Figure 41 shows the results of quantification of a-synuclein seeding in living SH-SY5Y cells expressing a-synuclein-tRFP after exposure to the respective indicated binding molecule as described in Example 23.

[0441] Figure 42 shows uptake of a-synuclein protofibrils by THP-1 cells after exposure to increasing concentrations of the indicated test molecules, measured as described in Example 24.

[0442] Figure 43 shows the brain to plasma ratios over time of the respective indicated test molecule after administration of a single i.v. dose of 40 nmol / kg to hTfRl transgenic mice as described in Example 25.

[0443] Figure 44 shows the total brain exposure up to 168 h (area under the curve) of the respective indicated test molecule after administration of a single i.v. dose of 40 nmol / kg to hTfRl transgenic mice as described in Example 25.

[0444] Figure 45 shows images from sections of cerebral cortex of brains from hTfRl transgenic mice after administration of the indicated test molecules, obtained as described in Example 25. Images show localization of the tested molecules in neurons (NeuN positive cells) in cerebral cortex. Scale bar 20 pm. Zoomed areas to the right show nuclei, NeuN and BA201 / 202 / 203 (top to bottom). Scale bar 5 pm.

[0445] Figure 46 shows the results of the ADCC assay described in Example 26, showing fold induction over unstimulated cells as a function of the concentration of the indicated test construct and controls, for A) BA202 and BA203, and B) BA201, in both cases contrasted to the positive control rituximab.

[0446] Figure 47 shows representative SPR sensorgrams of the interaction between the indicated test molecules with hTfRl, measured as described in Example 29.

[0447] Figure 48 shows representative SPR sensorgrams of the interaction between the indicated test molecules with monomers of human a-synuclein, measured as described in Example 29.

[0448] Figure 49 shows representative SPR sensorgrams of the interaction between the indicated test molecules with protofibrils of human a-synuclein (HNE-PF), measured as described in Example 29. Figure 50 shows the normalized reactivity by pre-existing anti-drug antibodies (PE ADA) in human serum against the indicated test molecules, measured as described in Example 30.

[0449] Figure 51 shows TH staining in substantia nigra in ipsi- and contralateral sides in AAV-A53T a-synuclein inoculated hTfRl transgenic mice, treated as described in Example 31.

[0450] Figure 52 shows quantification of TH object density in substantia nigra of AAV-A53T a-synuclein inoculated hTfRl transgenic mice, treated as described in Example 31. Data is presented as mean ± standard deviation with individual values plotted, * represents a p-value < 0.05.

[0451] Examples

[0452] 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.

[0453] 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 error 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.

[0454] EXAMPLE 1

[0455] Generation of humanized a-synuclein antibodies with reduced immunogenicity This example describes the generation of a deimmunized variant of the a- synuclein protofibril binding antibody BAN0805 (described in WO2021 / 260434, also known by the INN "exidavnemab").

[0456] Ex vivo T-cell proliferation assay

[0457] Peripheral blood mononuclear cells (PBMC) from 50 donors, representing the number and frequency of HLA-DR and DQ allotypes in the world population, were thawed, counted and viability was assessed. Cells were revived in room temperature in AIM V® culture medium (Invitrogen, Paisley, UK) before adjusting the cell density to 2.5-3.5 x 106PBMC / ml (proliferation cell stock). Seventy 15-mer peptides covering the VH and VL regions of BAN0805 (comprising SEQ ID NO:1 and SEQ ID NO:2 in WO2021 / 260434, respectively) were synthesized on a 1-4 mg scale with free N-terminal amine and C-terminal carboxylic acid. Peptides were dissolved in DMSO to a concentration of 10 mM and peptide culture stocks prepared by diluting into AIM V® culture medium to a final concentration of 5 pM in each well. For each peptide and each donor, sextuplicate cultures were established in a flat bottomed 96 well plate. Both positive and negative control cultures were also tested in sextuplicate. For each donor, three controls (KLH protein, final assay concentration 100 pg / ml) and peptides derived from IFV and EBV (final assay concentration 5 pM) were also included. For a positive control, PHA (Sigma, Poole, UK) was used at a final concentration of 2.5 pg / ml.

[0458] Cultures were incubated for a total of 6 days before adding 0.75 pCi3H- thymidine (Perkin Elmer, Beaconsfield, UK) to each well. Cultures were incubated for a further 18 h before harvesting onto filter mats using a TomTec Mach III cell harvester. Counts per minute (CPM) for each well were determined by Meltilex™ (Perkin Elmer, Beaconsfield, UK) scintillation counting on a Microplate Beta Counter (Perkin Elmer, Beaconsfield, UK) in paralux, low background counting mode. Proliferation assays were set up in sextuplicate cultures ("non-adjusted data"). To ensure that intra-assay variability was low, the data were also analyzed after removing the maximum and minimum cpm values ("adjusted data"). Peptide 15 (SEQ ID NO:444) induced T cell proliferation (SI >2.00, p <0.05) above the

[0459] 5 background threshold with positive responses in 8% of the study cohort in both the non-adjusted and adjusted data sets (Table 1). In silica MHC class II binding analysis using iTope™ revealed two non-germline MHC class II binding sequences, LEWIGVIWR (SEQ ID NO:445) and IGVIWRGGS (SEQ ID NO:446), predicted to bind 24 and 26 of 34 MHC class II alleles, respectively. The 9-mer sequence LEWIGVIWR was also present in peptide 14 (overlapping the N-terminal of peptide 15), which did not induce significant donor responses. The other potential core 9-mer sequence, IGVIWRGGS, was present in peptide 16 (overlapping the C-terminal of peptide 15). This peptide stimulated sub threshold number of donors to proliferate (4% and 6% of the cohort in the non-adjusted and adjusted data sets respectively) and is

[0460] 15 therefore the more likely T cell epitope.

[0461] The magnitude of T cell proliferation responses can provide an indication of the T cell precursor frequency. In general, peptides that induce high magnitude and high frequency T cell proliferation responses correspond to high T cell precursor frequencies and this is often a characteristic of memory-like or recall T cell

[0462] 20 responses. In contrast, naive T cell responses with low T cell precursor frequencies are generally characterized by low magnitude T cell proliferation responses. The mean magnitude of the Sis for peptide 15 were 2.46 and 2.12 (non-adjusted and adjusted data sets, respectively). Low magnitude responses are generally consistent with naive T cell epitopes (mean SI <3.00), as seen from Table 1, which shows a summary of the magnitude (mean SI and standard deviation) and frequency (% donor response) of positive T cell proliferation responses to peptide 15. The potential pl anchor residue as predicted by iTope™ is the isoleucine residue at position 6.

[0463] 30 Table 1: T cell proliferation responses to peptide 15 Design of deimmunized antibody variants

[0464] Selection of specific amino acid changes was influenced by the available biophysical and biochemical data, e.g. constraints on modification of the parental sequence taking into consideration secondary and tertiary protein structures as well as potential interactions of amino acid side chains with the core of the protein. Additionally, selection of amino acid changes was influenced by the frequency of occurrence at any particular amino acid at any given position. The aim was therefore to avoid introducing, where possible, amino acids that typically do not occur at a given position and that would be more likely to adversely affect the structure. Each epitope was analyzed individually to identify residues that would remove promiscuous MHC class II binding.

[0465] The iTope™ software predicts favorable interactions between amino acid side chains of a peptide and specific binding pockets (in particular pocket positions; pl, p4, p6, p7 and p9) within the open-ended binding grooves of 34 human MHC class II alleles. These alleles represent the most common HLA-DR alleles found worldwide with no weighting attributed to those found most prevalently in any particular ethnic population. Twenty of the alleles contain the "open" pl configuration and 14 contain the "closed" configuration where glycine at position 83 is replaced by a valine. The location of key binding residues is achieved by in silica generation of 9- mer peptides that overlap by one amino acid and span the test protein sequence. Inhouse comparisons with physical MHC class II binding experiments have shown that iTope™ can be used to successfully discriminate with high accuracy between peptides that either bind or do not bind MHC class II molecules. However, the results should be assessed in the light of the fact that all predictive methods for MHC class II binding inherently over-predict the number of T cell epitopes, because they do not allow for other important processes during antigen presentation such as protein / peptide processing, recognition by the T cell receptor or T cell tolerance to the peptide. The TCED™ contains the sequences of all the peptides previously screened in EpiScreen™ T cell epitope mapping assays. The TCED™ epitope database is used to search any test sequence against a large (>10,000 peptides) database of peptides derived from unrelated protein and antibody sequences which have been tested in EpiScreen™ T cell epitope mapping assays. Analysis of the VH and VL sequences of BAN0805 was performed using iTope™ with overlapping 9-mers spanning the HLA-DR-restricted peptide 15 and tested against each of the 34 MHC class II alleles. Each 9-mer was scored based on the potential fit and interactions with the MHC class II molecules. The peptide scores calculated by the software lie between 0 and 1. Peptides that produced a high mean binding score (>0.55 in the iTope™ scoring function) were highlighted, and if >50% of the MHC class II binding peptides (i.e. 17 out of 34 alleles) had a high binding affinity (score >0.6), such peptides were defined as "promiscuous high affinity" MHC class II binding peptides and considered at high risk for containing CD4+ T cell epitopes. "Promiscuous moderate affinity" MHC class II binding peptides bind a high number of alleles (>50%) with a binding score of >0.55 (but without a majority at >0.6).

[0466] The identified potential HLA-DR restricted epitope in BAN0805, i.e. peptide 15 (SEQ ID NO:444), was identified at the junction of framework region 2 and CDR2 in the heavy chain variable region (VH). As VHCDR2 may contribute to antigen binding, mutations were chosen outside of this area when possible, in order to minimize any loss of binding; however, residues within the CDR2 were also considered. In silica MHC class II binding analysis using iTope™ revealed one possible core 9-mer, HLA-DR restricted epitope, with one associated pl anchor residue at 148. The in-silico data suggested several changes to reduce predicted HLA-DR binding and hence the immunogenicity. Following one such suggestion, the isoleucine anchoring residue at position 48 was substituted with a serine, generating the novel VH sequence SEQ ID NO:447. Using iTope™, 9-mers from the original (IGVIWRGGS; SEQ ID NO:446) and novel (SGVIWRGGS; SEQ ID NO:456) sequences were compared, and the total number of MHC Class II alleles binding to the peptides were counted. Binding scores of >0.55 as determined by the iTope™ scoring function were classified as binding, whereas scores of >0.6 were classified as high binding. The original 9-mer sequence showed a total number of 26 binding MHC Class II ligands, with 17 of these classified as high binding. By contrast, the novel sequence yielded no binding ligands at all, showing that deimmunization had been successful.

[0467] A variant a-synuclein-binding antibody incorporating the mutation in the VH sequence was thus created, comprising the novel VH region sequence SEQ ID NO:447 and the BAN0805 VL region sequence SEQ ID NO:448. This VH / VL pair was combined with constant regions into a standard antibody format, with complete heavy and light chains represented by SEQ ID NO:449 and SEQ ID NO:450, respectively. The resulting antibody is denoted BA201 herein.

[0468] EXAMPLE 2

[0469] Identification of binders of human TfRl by immunization and screening Immunization and hybridoma screening

[0470] 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:71). 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 hTfRl 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.

[0471] 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.

[0472] 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.

[0473] 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.

[0474] Indirect ELISA screening

[0475] 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 hTfRl target protein. Briefly, 96-well half area plates (Corning) were coated with 1 pg / ml Hisio-mTfRl (SEQ. ID NO:72) or Hisw-hTfRl (SEQ ID NO:73). 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.

[0476] 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).

[0477] Examples of clones considered to be positive in binding mouse TfRl and human TfRl are shown in Table 2. 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.

[0478] Table 2: Examples of identified clones from hybridoma screening

[0479] Biolayer interferometry measurements

[0480] 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.

[0481] 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. Sequencing of selected clones

[0482] 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) regions of these antibodies are given in Table 3 below:

[0483] Table 3: Variable region amino acid sequences for selected primary antibodies The complementarity determining regions (CDRs) of these antibodies were identified using the Kabat definition, and are given in Table 4 below.

[0484] Table 4: CDR sequences of primary antibodies

[0485] In accordance with some embodiments of the present disclosure, the VH and

[0486] VL regions of the identified antibodies may be mutated to introduce cysteine residues for the provision of a disulfide bridge between the VH and VL regions. The resulting sequences, variously denoted "disulfide stabilized variants", "DS stabilized variants", "DS versions" or similar herein, are given in Table 5.

[0487] Table 5: Disulfide stabilized variants of primary antibodies EXAMPLE 3

[0488] In vitro binding to human and cynomolgus TfRl and epitope screen

[0489] 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.

[0490] 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 2. Briefly, ELISA plates were coated with the following His-tagged antigens at 1 pg / ml: ectodomain of human TfRl (HislO-hTfRl; SEQ ID NO:74), ectodomain of mouse TfRl (HislO-mTfRl; SEQ ID NO:75), chimeric TfRl consisting of human apical domain grafted on mouse TfRl ectodomain (h / m apical domain chimera, mhHD_TFRl; SEQ ID NO:76), chimeric TfRl consisting of human helical domain grafted on mouse TfRl ectodomain (h / m helical domain chimera, mhHD_TfRl; SEQ ID NO:77) 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:78). 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.

[0491] 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.

[0492] 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.

[0493] 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). These data illustrate that both 24B4 and 26D3 bind to hTfRl expressed on a cell surface.

[0494] EXAMPLE 4

[0495] 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 2, 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 5 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).

[0496] For transferrin competition, 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 hTfRl does not compete directly for the same epitope as transferrin. 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.

[0497] EXAMPLE 5

[0498] Humanization of hTfRl binder 26D3

[0499] The Fab sequence of mouse antibody 26D3, identified and characterized as described in Examples 2-4, 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 regions of 26D3 (see Table 3; SEQ ID NO:10-15) 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 region sequence defined in SEQ ID NO:44 and the VL region sequence defined in SEQ ID NO:58. According to some embodiments of this disclosure, a DS version of h26D3 has the VH and VL amino acid sequences SEQ ID NO:88 and 105, respectively.

[0500] 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).

[0501] 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:86) or cynomolgus TfRl (truncated cTfRl of SEQ ID NO:87) was immobilized on a Cm5 sensor chip (Cytiva, WBR100399) using the amine coupling kit type 2 (Cytiva, #BR100633) 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 6 below.

[0502] Table 6: SPR analysis of murine and humanized 26D3 Fabs vs. hTfRl and cTfRl

[0503] 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:79 and SEQ ID NO:80 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:81), while the hole half of Fc (SEQ ID NO:82) 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:83, whereas the h26D3 scFv fused to the knob half of the Fc has the complete amino acid sequence SEQ ID NO:84. 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 2.

[0504] EXAMPLE 6

[0505] Crystallization and structure determination of h26D3-Fab in complex with hTfRl

[0506] 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:74) 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 5.

[0507] 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 5.

[0508] 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.

[0509] 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 7 below. Table 7: X-ray diffraction data collection and refinement statistics

[0510] 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.

[0511] 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 8.

[0512] Table 8: Amino acid residues involved in interaction between h26D3 and hTfRl

[0513] Table 8 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 10 below, several positions outside the observed binding interaction show important participation in binding of h26D3 to hTfRl.

[0514] In Table 9 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 9: Amino acid residues in hTfRl which interact with the respective ligand

[0515] * Montemiglio et al (2019), Nat Commun 10:1121

[0516] # 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.

[0517] EXAMPLE 7

[0518] 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.

[0519] EXAMPLE 8

[0520] Brain uptake of hTfRl binding constructs in vivo

[0521] To evaluate hTfRl-mediated brain uptake in vivo, monovalent Fc-scFv constructs (see Example 5) 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.

[0522] 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 9 below).

[0523] 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.

[0524] 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.

[0525] 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.

[0526] EXAMPLE 9

[0527] Immunohistochemistry data on brain exposure

[0528] 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 8 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).

[0529] 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 8) 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 10

[0530] Generation of affinity variants and affinity determinations

[0531] 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 regions were denoted HC1-HC13 and their amino acid sequences are provided in the sequence listing as SEQ ID NO:45-57, respectively. Disulfide stabilized versions of these variant VH regions have the amino acid sequences SEQ ID NO:89-101, respectively. Variant CDR sequences comprised in these variant VH regions are listed as SEQ ID NO:16-28, respectively. The resulting variant VL regions were denoted LC1-LC6 and their amino acid sequences are provided in the sequence listing as SEQ ID NO:59-64, respectively. Disulfide stabilized versions of these variant VL regions have the amino acid sequences SEQ ID NQ:106-lll, respectively. Variant CDR sequences comprised in these variant VL regions are listed as SEQ ID NO:29-33, respectively. Table 10 below provides a summary of the specific mutations in each of the alanine variants.

[0532] 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 NO:74), 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 extent (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 11 below provides a summary of the specific mutations in each of the alanine variants that were selected.

[0533] 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.

[0534] 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:86) or cTfRl (SEQ ID NO:87) 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 12 below.

[0535] Table 12: 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:74) 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.

[0536] 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.

[0537] 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 13 below, the different tested variants exhibited a range of affinities for the hTfRl target. Table 13: SPR analysis of variant h26D3 scFv in bispecific format vs. hTfRl

[0538] EXAMPLE 11

[0539] Design, production and preparative SEC of disulfide-stabilized hTfRl binding molecules

[0540] 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 14.

[0541] Table 14: hTfRl binding scFv molecules and their amino acid sequences

[0542] The scFv variants whose respective designation includes the "_DS" suffix all comprise two mutations which introduce cysteine residues at position 44 of the VH region and at position 106 of the VL region 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 regions.

[0543] 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:122), 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:123) for site directed in vivo biotinylation, and was expressed in 1 1 culture volume.

[0544] 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.

[0545] EXAMPLE 12

[0546] Analytical SEC of disulfide-stabilized hTfRl binding molecules

[0547] Following three freeze / thaw cycles between room temperature and -80 °C, 1 pg of each scFv variant produced in Example 11 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.

[0548] 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 15. 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.

[0549] 5

[0550] Table 15: Distribution of peak areas from SEC chromatograms

[0551] EXAMPLE 13

[0552] Thermal stability of disulfide-stabilized hTfRl binding molecules 0 Monomer stability of scFv samples was evaluated by HPLC SEC analysis. The panel of scFv molecules produced and studied in Examples 11-12, 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 5 each timepoint, samples of each variant from each temperature were analyzed by HPLC-SEC as described in Example 12. At the initiation of the study, frozen samples were thawed and analyzed, and are denoted TO.

[0553] 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 0 monomers in the preceding preparative SEC purification described in Example 11. 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 5 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.

[0554] 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-B). 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.

[0555] 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.

[0556] As can be seen in Table 16 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 16: Percentage of monomer forms of scFv samples

[0557] EXAMPLE 14

[0558] Serum stability of disulfide-stabilized hTfRl binding molecules 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, Thermo Fisher Scientific) 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:

[0559] Serum stability = (ELISA OD450 at 37 °C) / (ELISA OD450 at 4 °C) x 100 % 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).

[0560] EXAMPLE 15

[0561] 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 11) 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.

[0562] The results are shown in Table 17. 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 15), 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).

[0563] Table 17: Dynamic light scattering analysis of scFv variants

[0564] EXAMPLE 16

[0565] Surface plasmon resonance analysis of disulfide-stabilized hTfRl binding molecules

[0566] Binding to hTfRl of six different purified scFv variants from Example 11 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 p.l / min . The binding modules were diluted in HBS-EP+ (Cytiva, WBR100669). Experiments were performed at 25 °C.

[0567] 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.

[0568] EXAMPLE 17

[0569] Generation of pH sensitive mutants

[0570] This example describes the design, production and purification of pH sensitive variants of the hTfRl binding molecules described in the previous examples. pH sensitive variants and controls were designed and produced in a Fab format, i.e. with one polypeptide chain comprising the VH and CHI domains of a traditional antibody molecule, and another polypeptide chain comprising the corresponding VL and CL domains. All of these Fab constructs had the same CHI and CL domains, represented by SEQ ID NO:437 and SEQ ID NO:438, respectively. The Fab constructs were expressed with His tag in 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) and buffer exchanged to lx dPBS (Thermo Fisher Scientific) using Zeba™ Spin Desalting Columns (Thermo Fisher Scientific) according to the manufacturer's instructions. 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.

[0571] A large number of variants were successfully produced and purified. Their designations and the amino acid sequences of their VH and VL regions are listed in Table 18. EXAMPLE 18

[0572] Affinity determination using SPR, BLI and competitive ELISA

[0573] This example describes the binding of molecules generated and produced in Example 17 to human TfRl by SPR, BLI and competitive ELISA.

[0574] Human and cynomolgus TfRl: The human transferrin receptor 1 (hTfRl; SEQ ID NO:86) and cynomolgus transferrin receptor 1 (cTfRl; SEQ ID NO:87) were successfully expressed in human embryonic kidney (HEK293) cells (Expi293; Thermo Fisher Scientific) 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 (Thermo Fisher Scientific). Purified hTfRl and cTfRl were concentrated to 2 mg / ml and stored at -80 °C until used in the affinity measurements below.

[0575] 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.

[0576] Single cycle kinetics were used to measure binding to immobilized hTfRl and cTfRl. For immobilization, 1.5 pg / ml of hTfRl or cTfRl was immobilized on a CM5 chip using NHS / EDC coupling. For interaction measurement, a 2-fold dilution in five steps starting at 50 nM for wt Fab and Fab constructs having a pH sensitive variant in either VH or VL ("single mutants"), and at 200 nM for Fab constructs having a pH sensitive variant in both VH and VL ("double mutants"), using a 120 s injection of every concentration and a 1000 s dissociation time. Regeneration of the surface between each cycle was done by injecting 3 pl 3 M MgCl2 (Cytiva, cat. No. BR100839) for 30 s. The binding data was fitted using a 1:1 interaction model and reported as KD in Table 19. Representative sensorgrams are shown in Figure 27 for binding of single mutants to hTfRl, in Figure 28 for binding of single mutants to cTfRl, and in Figure 29 for binding of double mutants to hTfRl. Table 19: Affinities by SPR of pH sensitive variants for immobilized hTfRl and cTfRl . KDVS. hTfRl KDVS. cTfRl

[0577] Designation(M) (M)

[0578] N52H 1.68e-7 3.30e-5

[0579] S105H 1.30e-6 9.70e-8

[0580] T33H 6.83e-9 1.38e-8 wt 4.81e-9 3.14e-9

[0581] S32H 5.55e-9 3.72e-9

[0582] S102H 7.77e-8 1.44e-7

[0583] T57H 1.32e-5 4.01e-6

[0584] Y100H 7.02e-9 2.00e-8

[0585] Y107H 6.20e-9 3.19e-8

[0586] Y38H 1. Ole-7 1.61e-7

[0587] Y55H 1.15e-6 5.81e-7

[0588] Y106H 3.82e-9 2.16e-8

[0589] S32H x N52H 2.57e-7

[0590] S32HxS102H 8.75e-8

[0591] S32HxY55H 3.58e-7

[0592] S32HxY106H 2.28e-8

[0593] S32HxY107H 2.91e-8

[0594] T33H x N52H Poor fit

[0595] T33HxS102H 2.14e-7

[0596] T33HxY55H 1.99e-7

[0597] T33HxY106H 1.09e-7

[0598] T33HxY107H 7.08e-8

[0599] Y38H x N52H 8.65e-9

[0600] Y38H x S102H 8.01e-9

[0601] Y38HxY55H 1.07e-8

[0602] Y38HxY106H 2.12e-7

[0603] Y38HxY107H 1.76e-7

[0604] Y100H x N52H Poor fit

[0605] Y100HxS102H Poor fit

[0606] Y100HxY55H Poor fit

[0607] Y100HxY106H 1.34e-7

[0608] Y100HxY107H 4.95e-8

[0609] Evaluation of affinity pH sensitivity by biolayer interferometry: Bi ndi ng interactions between antigens and binding molecules at different pH values were evaluated by BLI using an Octet RED384 (FORTEBIO) instrument.

[0610] Octet SAX (Sartorius, cat. No.18-5117) biosensors were equilibrated for 60 s, 1000 rpm in IX Kinetics buffer. Biotinylated hTfRl at a concentration of 7.5 pg / ml was loaded for 600 s, 1000 rpm. Baseline was recorded for 120 s, 1000 rpm in citrate buffer BSA pH 7.4 or citrate buffer BSA pH 5.5, respectively. Association of analyte was recorded for 300 s, 1000 rpm. Dissociation was recorded for 900 s, 1000 rpm. For each row in the sample plate, a new set of SAX biosensors was used (e.g. there was no regeneration of sensor surface). Analyte stock solutions (e.g. variant and control Fabs) were diluted to 500 nM in citrate buffer BSA pH 7.4 or in citrate buffer BSA pH 5.5. A 3-fold dilution series in the citrate buffer with the respective pH was performed for each analyte, resulting in three different concentrations per analyte to be investigated. Data was fitted using a 1:1 binding model and are reported in Table 20. Representative sensorgrams are shown in Figure 30.

[0611] Table 20: Determination of affinity pH sensitivity by BLI

[0612] KDVS. hTfRl KDVS. hTfRl „ .

[0613] Ratio

[0614] Designation at pH 7.4 at pH 5.5 .u_ „

[0615] , . .. , . .. KDat pH5.5 / KDat pH7.4

[0616] (nM) (nM) wt 23 26 1.1

[0617] HC6 280 450 1.6

[0618] S102H 68 690 10

[0619] Y106H 28 120 4.2

[0620] Y38H 150 230 1.5

[0621] Y100H 36 89 2.5

[0622] S32H x Y55H 1000 2200 2.2

[0623] S32H x S102H 68 420 6.2

[0624] S32H x Y106H 32 120 3.6

[0625] S32H x Y107H 33 140 4.2

[0626] T33H x S102H 760 ND1

[0627] T33H x Y106H 170 330 1.9

[0628] T33H x Y107H 110 340 3.3

[0629] Y100H x Y106H 270 ND1

[0630] Y100H x Y107H 74 240 3.3

[0631] 1ND: the affinity could not be determined pH sensitivity evaluation by enzyme-linked immunosorbent assay: A competitive ELISA was carried out to evaluate pH sensitivity of the binding interaction between antigens and a subset of the pH sensitive binding molecules produced herein. ELISA plates (Corning, cat. No 3690) were coated using 50 pl / well of a 1 pg / ml solution of hTfRl in PBS at 2-8 °C overnight. Plates were then emptied and blocked using 150 pl / well of Pierce™ Protein-Free Blocking Buffer (Thermo Fisher Scientific, cat. No 37572) for 1 h, 900 rpm, RT. 3-fold dilution series of the tested binding molecules were prepared in PBS containing 0.1% (w / v) BSA and 0.05% (v / v) Tween at pH 7.4 or pH 6.0. Titrated binding molecules or isotype controls (human non-specific IgG; Invitrogen cat. no. 001-7102) were then mixed with a biotinylated, high-affinity binding molecule, competing for the same epitope, which was diluted to 0.5 pM in 0.1% (w / v) BSA and 0.05% (v / v) Tween at pH 7.4 or pH 6.0, respectively. 50 pl / well of this solution was incubated on the hTfRl plate for 2 h, 900 rpm, RT. Detection was carried out using Streptavidin-HRP (Mabtech, cat. No 3310-9-1000) and TMB (Neogen Corporation, 331177-77).

[0632] The results are shown in Figure 31 and Table 21, and demonstrate that the introduction of additional histidine residues into the VH and / or VL regions of hTfRl binding molecules renders these molecules susceptible to pH change with regard to their affinity for the hTfRl target. The S102H and Y100H x Y106H variants bound immobilized hTfRl so weakly at pH 6 that no curve fits could be obtained.

[0633] Table 21: IC50 for hTfRl at different pH values

[0634] , , , IC50 at pH 7.4 IC50 at pH 6

[0635] Test molecule . ... . ...

[0636] (nM) (nM)

[0637] Wt 6.16 4.73

[0638] HC6 838 211

[0639] S102H 584 ND

[0640] Y100H x Y106H 950 ND

[0641] EXAMPLE 19

[0642] Stability assessment of pH sensitive module in an scFv format

[0643] Production, purification and thermal stability assessment: One of the pH sensitive hTfRl binding variants studied in Examples 17-18 was selected for stabilization using a disulfide bridge as tested for other binding molecules in Examples 11-16. The test item comprised the scFv amino acid sequence SEQ ID NO:440. It was designated S102H-DS and produced as a Hise tagged, VL-first scFv construct 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:122), by transient transfection of CHO cells in 1000 ml culture volume.

[0644] The His-tagged scFv was recovered by immobilized metal ion affinity chromatography (IMAC) purification from clarified cell supernatants. For IMAC, supernatants were loaded on a HiTrap 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) and protein containing fractions were pooled and, in a following step, passed over a preparative SEC column (HiLoad 16 / 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. Purity of recovered protein was analyzed by capillary electrophoresis (CE-SDS) in reducing (R) and nonreducing (NR) conditions with a LabChip GXII Touch HT Protein Characterization System (Perkin Elmer) using ProteinEXact Assay Reagent Kit (PerkinElmer, CLS150466) for sample preparation. Samples containing 2.5 pg protein per lane was loaded on a LabChip® GXII Touch™ HT Chip (PerkinElmer, CLS150337). The results show that a highly pure protein of the expected size was recovered as seen in Figure 32.

[0645] Analytical SEC for short term thermal stability assessment of scFv : M o n o m e r stability of the purified scFv was evaluated by analytical SEC measurement before and after three freeze / thaw cycles between room temperature and -75 °C. For this, 1 pg scFv samples were 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. The results are exhibited in Figure 33 and show that the recovered molecule is stable, exhibiting an intact monomeric state over freeze / thawing cycles.

[0646] To further assess the thermal stability, samples of the scFv were subjected to temperature hold for one, two or four weeks at temperatures 4 °C, 40 °C or frozen at -75 °C. At each timepoint, samples from each temperature were analyzed by analytical SEC as described above. At the initiation of the study, frozen samples were thawed and analyzed, and are denoted TO. Results are shown in Figure 34 and demonstrate that the monomer content (grey bars) of scFv is 97.4% or higher in all tested conditions, and that refrigerated or frozen samples maintained a monomer content of >99%, similar to that observed at the start of the study (TO). A slow and limited increase in high molecular weight (HMW) content (black bars) is seen in some samples, with a highest content of 2.66% measured after 4 w at 40 °C (Figure 34C).

[0647] EXAMPLE 20

[0648] Generation of bispecific binding molecules

[0649] This example describes the design and production of bispecific binding molecules which incorporate the a-synuclein-specific antibody described in Example 1 with stabilized hTfRl binding scFv variants described in Examples 2-19.

[0650] Design of constructs: Bispecific binding molecules BA202, BA203 and BA204 were designed as knob-into-hole variants of the a-synuclein-specific antibody BA201, having a hTfRl-binding scFv linked to the C-terminal amino acid residue of the knob heavy chain of the antibody. As control, the a-synuclein-specific antibody BA201 was used without any hTfRl-binding scFv fusion partner. Amino acid sequences of the tested binding molecules are listed in Table 22.

[0651] Table 22: Amino acid sequences of tested bispecific and control binding molecules

[0652] Expression from transient transfection: The designed binding molecules were expressed in CHO cells and purified by affinity chromatography, followed by preparative size-exclusion chromatography (SEC) and buffer exchange into phosphate buffered saline (PBS) solution. The purified binding molecules were characterized using SDS-PAGE, SEC, and UV protein determination.

[0653] Results: Bispecific and control binding molecules were successfully produced and purified to a final concentration of 3.5 mg / ml. Protein purity, as defined by SEC- HPLC, was >99 % monomer for all constructs. EXAMPLE 21 Characterization of target binding

[0654] This example describes the binding of the bispecific antibodies generated and produced in Example 20 to human TfRl and to a-synuclein monomers and protofibrils by surface plasmon resonance (SPR). Binding interactions between the tested binding molecules and their antigens were evaluated by SPR using a Biacore 8K or 8K+ instrument (Cytiva) according to standard procedures.

[0655] Binding to hTfRl: Single cycle kinetics using capture was used to measure binding to hTfRl by SPR. For measurement, 30 pg / ml FLAG-Tag mAb FG4R (Invitrogen) was immobilized on a CM5 chip. 5 pg / ml hTfRl (SEQ ID NO:86; prepared as described in Example 18) 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 BA202 and 500 nM for BA203 and BA204, 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. KD values were determined and demonstrated binding to hTfRl with different affinities. The calculated ka, kd and KD values are shown in Table 23. Representative sensorgrams are shown in Figure 35.

[0656] Table 23: SPR analysis of binding to human TfRl

[0657] Binding to a-synuclein monomers: Single cycle kinetics using capture was used to measure binding to a-synuclein monomers. For measurement, a human antibody capture chip, CM5, was prepared according to manufacturer's instructions (Cytiva, cat. No. 29234600). 3 pg / ml of the respective tested binding molecule BA201, BA202, BA203 and BA204 was captured for each cycle followed by injection of the analyte using a 2-fold dilution in five steps starting at 3000 nM, 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 3 M MgCl2 (Cytiva, cat. No. 29234600). The binding data was fitted using a 1:1 interaction model. All four tested binding molecules had a similar affinity for the a-synuclein monomer. The calculated ka, kd and KD values are shown in Table 24. Representative sensorgrams are shown in Figure 36.

[0658] Table 24: SPR analysis of binding to a-synuclein monomer

[0659] Binding to a-synuclein protofibrils: Single cycle kinetics was used to measure binding of the binding molecules BA201, BA202, BA203 and BA204 to HNE-stabilized a-synuclein protofibrils ("a-synuclein HNE-PF"). For measurement, 1.5 pg / ml a- synuclein HNE-PF was immobilized on a CM5 chip using the method "Immobilization low levels" according to manufacturer's instructions. Each cycle of injection of analytes was done using a 2-fold dilution in five steps starting at 10 nM, using a 2 min injection of every concentration and a 60 min dissociation time. Regeneration of the surface between each cycle was done by injecting 30 pl 3 M MgCl2 (Cytiva, cat. No. 29234600). The binding data was fitted using a 1:1 interaction model. All four tested binding molecules had similar affinity for the a-synuclein HNE-PF. The calculated ka, kd and KD values are shown in Table 25. Representative sensorgrams are shown in Figure 37. Table 25: SPR analysis of binding to a-synuclein HNE-PF

[0660] In addition to the SPR analysis, the cross-reactivity of the binding molecules BA201, BA202 and BA203 to homologous proteins, such as |3- or y-synuclein, was tested using direct ELISA in which dense coating mimics aggregated forms of the coated protein. The results indicate that there was no detectable binding of BA201, BA202 or BA203 to |3- or y-synuclein (not shown).

[0661] EXAMPLE 22

[0662] Target binding in brain from human synucleinopathy and non-diseased controls This example describes target binding of bispecific antibodies, generated as described in Example 20, by immunodepletion in human brain extracts and on tissue sections from synucleinopathy patients.

[0663] Immunodepletion: The binding to pathological a-synuclein by control binding molecule BA201 and the bispecific binding molecules BA202 and BA203 was analyzed by immunodepletion of target protein in solution using BA201, BA202 and BA203 covalently coupled to magnetic beads, followed by protofibril measurement using an a-synuclein protofibril assay. BAN0805 and isotype controls were used as positive and negative controls, respectively. In the assay procedure, the magnetic beads coupled to binding molecules were allowed to incubate with brain extracts for 60-90 min at room temperature in a rotating mixer. Complexes of binding molecule and a-synuclein protofibrils were depleted from brain extracts from PD, PDD, DLB and MSA patients by placing the test tubes on a magnetic stand, thereby removing the magnetic beads and bound complexes of binding molecule and protofibril.

[0664] The levels of remaining a-synuclein protofibrils in depleted supernatants were determined in the protofibril assay using mAb38F capture antibody and biotinylated mAb38F (mAb38F-bio) as detection antibody (Fagerqvist et al (2013), J Neurochem 126(1):131-144). The calibrator used is a-synuclein protofibrils made from recombinant human a-synuclein and 4-hydroxynonenal (HNE). The assay measures exclusively aggregated a-synuclein.

[0665] The results are shown in Figure 38, and indicate that binding molecules BA201, BA202 and BA203 depleted pathological a-synuclein protofibrils in PD, PDD, DLB and MSA brain extracts to a similar extent as BAN0805.

[0666] Immunohistochemistry: Formalin-fixed, proteinase-treated sections of temporal cortex from a PDD patient was used to perform immunohistochemical (IHC) analysis of aggregated a-synuclein in diseased human brain. As control, temporal cortex from an age-matched non-diseased control was used. BAN0805 and isotype controls were used as positive and negative controls, respectively. Binding molecules BA201 and BA202 were tested, and used at a concentration of 1 pg / ml. Bound test agents were detected using a rabbit anti-Human IgG Fc gamma (Invitrogen, #31142) as primary antibody and the OmniMap goat anti-rabbit HRP (Ventana, 760-4311) as secondary antibody.

[0667] The results are shown in Figure 39, and demonstrate that BA201 and BA202 exhibited a-synuclein positive IHC staining with strong intensity in Lewy bodies and Lewy neurites in the brain from one PDD case (Figure 39A and 39B). IHC staining was positive in nerve fiber-like structures (axons, dendrites) with a punctate appearance intraneuronal. BA201 and BA202 exhibited IHC binding profiles very similar to that of BAN0805 (Figure 39C). No positive staining was observed in non-diseased brain using isotype control (Figure 39D).

[0668] EXAMPLE 23

[0669] In vitro target binding in cellular model

[0670] The effect of BA201, BA202 and BA203 on a-synuclein aggregation and seeding was examined in vitro using human recombinant a-synuclein pre-formed fibrils (PFF) and SH-SY5Y cells stably expressing a-synuclein linked to Tag Red Fluorescent Protein (tRFP).

[0671] Fluorescence labeling of binding molecules: BA201, BA202 and BA203 were fluorescently labeled with Alexa Fluor 647 following manufacturer's instructions (Thermo Fisher Scientific, A20186). Antibody concentrations were determined using Nanodrop 2000 / 2000c (Thermo Fisher Scientific). Target binding in living cells: SH-SY5Y cells stably expressing a-synuclein-tRFP (Innoprot, P30707-02R) were seeded on an optically clear flat bottom 96 well plate (PerkinElmer, 6055302) and cultured for 24 h. ATTO-488-labelled a-synuclein PFF (StressMarq, SPR-322-A488) were sonicated (Hielscher Ultrasonic, 200 W, 75% amplitude 30 s ON / 30 s OFF for a total of 5 cycles) and incubated with the cells at a final concentration of 4 pg / ml at 37°C, 5% CO2for 72 h. a-synuclein monomers (2 pg / ml) were used as control. BA201, BA202 and BA203 (2 pg / ml), labelled with Alexa Fluor 647 or non-labelled, were then added to the cells for 1 h followed by live confocal microscopy imaging, or for 24 h followed by fixation with 4% PFA (Thermo Fisher Scientific, J91899AP), immunocytochemistry and confocal microscopy imaging.

[0672] Aggregation of a-synuclein-tRFP was observed in SH-SY5Y cells exposed for 72 h with a-synuclein-PFF-ATTO-488, while no aggregation was observed in cells exposed to a-synuclein monomers or vehicle. Target engagement was demonstrated with all three binding molecules tested, using both Alexa Fluor 647-labelled and unlabeled forms. One hour after exposure, binding of BA201, BA202 and BA203 to a- synuclein aggregates in both membranes and vesicles was observed in living cells as exemplified with BA203 in Figure 40. No binding was observed in cells exposed to a- synuclein monomers or vehicle. Target binding was confirmed by immunocytochemistry in fixed cells.

[0673] Target binding and seeding inhibition in living cells: SH-SY5Y cells stably expressing a-synuclein-tRFP were seeded and cultured as described above. ATTO- 488-labelled a-synuclein PFF were sonicated as described above and mixed with Alexa Fluor 647-labelled or non-labelled BA201, BA202 or BA203 (a-synuclein PFF 4 pg / ml, a-synuclein PFF:binder ratio of 5:1, 1:1 or 1:5). a-synuclein PFF and a- synuclein monomers (4 pg / ml) were used as positive and negative controls, respectively. The a-synuclein PFF:binder mix was incubated at 37°C, 5% CO2 for 1 h and then added to the cells for 72 h. Cells were then fixed with 4% PFA and imaged by confocal microscopy (Leica, Stellaris 5, 63x oil objective). The area of a-synuclein- tRFP aggregates was calculated per cell.

[0674] For all the tested binding molecules, a concentration-dependent decrease in a-synuclein-tRFP aggregates in cells treated with complexes of binding molecule with PFF was observed. An increasing binder to PFF ratio led to decreased a- synuclein-tRFP aggregate area after 72 h treatment (Figure 41).

[0675] EXAMPLE 24

[0676] Characterization of functional effects

[0677] This example describes the functional effects of BA201, BA202 and BA203 generated and produced in Example 20. The potency of the molecules with respect to the mediation of uptake of a-synuclein PFF into THP-1 cells was evaluated.

[0678] An in vitro uptake assay was used to investigate whether BA201, BA202 and BA203 could induce uptake of a-synuclein PFF 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). BA201, BA202 and BA203 were incubated with Alexa488- labelled a-synuclein PFF for 30 min at RT (final concentrations 25-0.1 nM for binding molecule and 50 nM for a-synuclein PFF). 200,000 THP-1 cells were added to the wells of a 96-well plate (Corning) and pelleted by centrifugation at 300 x g 5 min at RT, resuspended with the binder / a-synuclein PFF complexes and incubated at 37°C, 5% CO2 for 60-120 min. Cells were washed in PBS before data acquisition using a BD FACS Lyric flow cytometer. Data was evaluated using FCS Express 4 Flow Research Edition software (De Novo Software). EC50 values were calculated using non-linear regression with the sigmoidal 4PL equation on GraphPad Prism.

[0679] The results indicate that all three binding molecules induced the uptake of a- synuclein PFF by THP-1 cells in a concentration-dependent manner (Figure 42). The calculated EC50 values are listed in Table 26.

[0680] Table 26: Uptake of a-synuclein pre-formed fibrils in THP-1 cells (Mean+SD, n=2) EXAMPLE 25

[0681] Plasma and brain exposure in hTfRl-KI mice

[0682] This example describes the plasma and brain exposure profile in mice expressing the human transferrin receptor 1 (hTfR-KI mice; see Example 7 and Figure 13) after a single dose of either BA201, BA202 or BA203, generated and produced in Example 20.

[0683] Administration of antibody and blood collection: Each binding molecule was administered intravenously (i.v.) at a dose of 40 nmol / kg via the tail vein to 4 months old female and male hTfR-KI mice (three to five mice per antibody). To establish the full plasma PK profile, blood samples were collected at 5 min, 4 h, 24 h, 72 h, 168 h, 14 days, 21 days, 28 days and 35 days after i.v. injections. To establish the brain to plasma exposure relationship, blood and brain were collected at 5 min, 4 h, 24 h, 72 h, 168 h and 14 days after i.v. injections. All mice received the anti-CD4 antibody GK1.5 ( BioXcel I) at 7 days and 1 day prior to test item administration, in order to prevent the formation of anti-drug antibodies during the course of the study. In a separate study, brains were collected at 48 h after i.v. injection for immunohistological evaluation of exposure. Blood was collected into Microvette EDTA tubes and put on wet ice immediately after collection. The samples were centrifuged at 2400 x g for 10 min at 4 °C shortly after collection (within 30 min). Plasma was collected and stored at -80 °C until bioanalysis.

[0684] Brain collection: At termination, animals were deeply anaesthetized with isoflurane and perfused with ice-cold PBS and brains were extracted. The left-brain hemispheres were snap-frozen on dry ice and stored at -80°C until bioanalysis.

[0685] Determination of antibody concentrations in plasma: Concentrations of BA201, BA202 and BA203 were determined in EDTA plasma samples from different time points using an electrochemoluminescence assay ("MSD"; Meso Scale Discovery). Briefly, free binding sites on MSD standard 96-well plates were blocked by incubation with 1 % Blocker A (MSD Blocker A in lxPBS-Tween 200.1%) for 1 h at RT with shaking. The plates were coated with biotinylated anti-idiotypic pAb ABBV- 0805 (Cat. no. PR-1812275, AbbVie) for 1 h at RT with shaking. Washing with lxPBS- Tween 200.1% was performed before coating and before each subsequent incubation step. Standard and plasma samples were added and incubated for 1 h at RT with shaking. The plates were then incubated with Sulfo-tagged anti-idiotypic pAb ABBV-0805 (Cat. no. PR-1812276, AbbVie) for 1 h at RT with shaking. Read buffer T (MSD 2x) was added and the plates were read using an MSD sector imager. The signal strength was correlated to the amount of binding molecule in the samples.

[0686] Brain homogenization and determination of concentration of binding molecules in brain: Brains were homogenized in 1:5 (weight:vol) TBS pH 7.6 using a FastPrep homogenizer (MP Biomedicals). Each sample was then resuspended in TBS pH 7.6 containing 0.5% Triton X100 to a final 1:10 (weight:vol) ratio, vortexed 10 s before centrifuging at 16,000 x g for 1 h at RT. Concentrations of BA201, BA202 and BA203 were determined in brain samples using the MSD-based method described above for plasma samples.

[0687] Immunohistochemistry: Exposure of BA201, BA202 and BA203 in the brain at 48 h was determined by immunohistochemistry. Immunohistochemistry was performed on 20 pm-thick cryosections of hemibrains fixed in paraformaldehyde 4% in PBS and cryoprotected in OCT (Thermo Fisher Scientific). Sections were permeabilized and blocked in PBS containing 0.1% Triton-XlOO (Sigma, X100) and 5% donkey serum (Nordic Biosite, PSB-TOQIOH-IO) for 1 h at room temperature. Sections were then incubated with rabbit anti-NeuN (1:1000, Abeam, abl77487) and Alexa647-donkey anti-human IgG (4 pg / ml, Jackson Immunoresearch, 709-605-149) in PBS-Tween-20 0.1% (Medicago, 09-9410-100) with 5% BSA (Sigma, A7284) overnight at 4°C. Sections were washed 3 times with PBS-Tween-20 0.1% under gentle shaking and incubated with Alexa488-donkey anti-rabbit IgG (4 pg / ml, ThermoFisher, A32790) and Alexa647-goat / donkey anti-human IgG in PBS-Tween-20 0.1% with 5% BSA for 1.5 h at room temperature. Sections were then washed 3 times with PBS-Tween-20 0.1%, mounted using Fluoromount-G (Invitrogen, 00-4958- 02), and imaged using a SlideScanner (Leica) equipped with a lOx air objective, and a confocal microscope (Leica, Stella ris 5) equipped with a 63x oil objective.

[0688] Results: The plasma and brain exposure profiles of BA201, BA202 and BA203 were evaluated after a single i.v. bolus injection into hTfR-KI mice. The profiles of brain to plasma concentration ratios as a function of time are shown in Figure 43, and the brain exposure based on area under the curve up to 168 h is shown in Figure 44. Brain exposure was higher for the bispecific binding molecules BA202 and BA203 as compared to that for the BA201 control (Figure 43, Table 27). The brain to plasma ratio based on AUC0-168h was more than 7 times higher for BA202 compared to the BA201 control. Furthermore, the plasma clearance was higher and AUCinf lower for bispecific binding molecules BA202 and BA203 compared to the BA201 control. Pharmacokinetic parameters in plasma are shown in Table 28.

[0689] Table 27: Brain and plasma AUC0-168h

[0690] Table 28: Plasma PK parameters

[0691] Representative immunohistochemistry pictures of brain cortex sections of mice injected with BA201, BA202 and BA203 are shown in Figure 45. In plasma, BA201 was detected at higher concentrations than BA202 and BA203.

[0692] EXAMPLE 26

[0693] Immunotoxicity assessment

[0694] This example describes the evaluation of immunotoxicity of the bispecific binding molecules generated and produced in Example 20 in cellular assays for antibody-dependent cellular cytotoxicity (ADCC) and in a human blood loop system.

[0695] ADCC measurements: To investigate the effector function of bispecific binding molecules, 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 BA201 antibody, i.e. lacking an hTfRl-binding scFv, as negative control and the monoclonal antibody rituximab (MabThera; Roche) as positive control. Target cells 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.

[0696] The results are shown in Figure 46. 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 molecules BA202 and BA203 (Figure 46A), which was similar to the negative control antibody BA201 (Figure 46B).

[0697] Human blood loop: Blood from six healthy human volunteers (above 50 years of age) was used to investigate if and to what extent the test molecule BA202 induced cytokine release, complement activation and / or cell activation in freshly collected, circulating blood. The assessment was performed using an ex vivo blood loop test system (ID. Flow; Immuneed). The binding molecules 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 yielding a cytokine release which is manageable 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.

[0698] At all concentrations of BA202 that were 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 BA202 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, BA202 did not have 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 BA202 on cytokine release, complement activation or cell activation in circulating human blood at any of the concentrations tested.

[0699] EXAMPLE 27

[0700] Target engagement in vivo

[0701] This example describes target engagement of bispecific binding molecules generated and produced in Example 20 in brain, following intravenous dosing in AAV-A53TxhTfRl-KI mice.

[0702] At 12-13 weeks of age, hTfR-KI heterozygous mice (Example 7) underwent stereotactic surgery and received a unilateral infusion into the substantia nigra of an AAV vector expressing human a-synuclein with A53T mutation (AAV-A53T), a control AAV vector (AAV-Null), or sham surgery. Eight weeks after surgery, animals were dosed i.v. with BA201, BA202 or BA203 (40 nmol / kg) or vehicle. Live bleeds were performed at three timepoints (2 h before dosing; 15 min and 4 h post-dosing) to collect in-life plasma samples. Two days after i.v. injection, endpoint procedures were performed, allowing for the timely collection of CSF biofluids, plasma samples and brain tissues.

[0703] Preliminary data indicate that the i.v. injected bispecific binding molecules BA202 and BA203 bind to intracellular, aggregated a-synuclein in the parenchyma of the brains of hTfR-KI mice.

[0704] EXAMPLE 28

[0705] Design and production of further bispecific binding molecules

[0706] Design of constructs: Analogously to Example 20, further bispecific binding molecules BA205, BA206, BA207 and BA008 were designed as knob-into-hole variants of the alpha synuclein-specific antibody BA201, with an 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 tested molecules are listed in Table 29. Table 29: Amino acid sequences of bispecific binding molecules

[0707] Expression from transient transfection: The tested molecules were transiently produced in CHO cells and purified with protein A affinity purification and preparative SEC. The fractions containing monomeric protein were collected and brought to 8.8 mg / ml final concentration in PBS, pH 7.4. Protein purity, as defined by SEC-HPLC, was >95 % monomer content for all constructs.

[0708] EXAMPLE 29 SPR analysis of binding of further bispecific binding molecules to hTfRl

[0709] Binding of the purified bispecific binding molecules produced in Example 28 to human TfRl was evaluated using SPR on a Biacore 8K instrument (Cytiva) as described in Example 21 for BA202, BA203 and BA204. The calculated KD values are given in Table 30 below. Representative sensorgrams are shown in Figure 47.

[0710] Table 30: SPR analysis of binding to hTfRl

[0711] Furthermore, binding of bispecific binding molecules BA205, BA206, BA207 and BA208 to a-synuclein monomers was analyzed by SPR and KD values were determined. Briefly, the analysis was performed as follows: a CM5 chip was prepared according to manufacturer's instructions (Cytiva, cat. no. 29234600) with 10 pg / ml of each of the binding molecules BA205, BA206, BA207 and BA208 immobilized on the surface. For each cycle, a-synuclein monomers was injected using a 5-fold dilution in five steps starting at 1500 nM, 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 Glycin-HCI, pH 1.7 (Cytiva, cat. no. 29-2152-81). The binding data was fitted using a 1:1 interaction model. All four bispecific binding molecules had a similar affinity for the a-synuclein monomer. The calculated ka, kd and KD values are shown in Table 31. Representative sensorgrams are shown in Figure 48.

[0712] Table 31: SPR analysis of binding to a-synuclein monomers

[0713] Finally, binding of bispecific binding molecules BA205, BA206, BA207 and BA208 to a-synuclein protofibrils (HNE-PF) was analyzed by SPR as described in Example 21 for BA202, BA203 and BA204, and KD values were determined. All four binding molecules demonstrated binding to a-synuclein HNE-PF at a similar affinity. The calculated ka, kd and KD values are shown in Table 32. Representative sensorgrams are shown in Figure 49.

[0714] Table 32: SPR analysis of binding to a-synuclein HNE-PF

[0715] EXAMPLE 30

[0716] Analysis of serum antibody reactivity against bispecific binding molecules

[0717] The additional bispecific binding molecules designed and produced according to Example 28 were analyzed for response by pre-existing anti-drug antibodies (PE ADA) in serum using a bridging assay. Materials and methods

[0718] 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).

[0719] Bridging assay: A bridging assay was set up as follows to detect potential PE ADA with reactivity to the test compounds BA205, BA206, BA207 and BA208 in comparison with the standard antibody BA201 (Example 1) and the bispecific binding molecules BA202 and BA203 (Example 20). One part of the respective protein was biotinylated using a standard biotinylation kit (A39257, Thermo Scientific), and another part was labelled with SULFO™ tag according to the manufacturers' instructions (R31AA-2, Mesoscale). Serum from 107 donors to a final concentration of 2 % was incubated with sulfo-tagged and biotinylated protein (0.5 pg / ml each, final concentration) in 1% Blocker A (R93BA-4, Mesoscale) for 2 h at room temperature with 900 rpm shaking to allow for complexes to form. 25 pl of the mix was added to the wells of 1% Blocker A pre-blocked and washed MSD Gold 96- well small spot streptavidin plates (L45SA-1, Mesoscale). The plates were incubated for 1 h at room temperature with 900 rpm shaking to allow for the biotinylated antibody to bind to streptavidin on the plate. The plates were washed and 150 pl MSD Read Buffer T (2x) (R92TC-1, Mesoscale) was added. The plates were immediately read using a Meso Sector S 600 (Mesocale). Electrochemiluminescence (ECL) counts were normalized based on the response for an anti-idiotypic antibody towards BA201, included as standard, and plotted for all individuals and assays.

[0720] Procedure to calculate the cutpoint for the assay: The sample population was analyzed for statistical outliers by ROUT analysis in GraphPad Prism (Q=l%; allowing for 1% of false positive identified outliers). The sample population minus statistical outliers made up the pseudo-negative population and was used to calculate the assay cutpoint. In case of low PE ADA prevalence, the cutpoint can be directly calculated from the screening assay: cutpoint = mean (screening population with outliers removed) + 1.645 x STDEV (of screening population with outliers removed), allowing for a 5% false positive rate. This type of cutpoint calculation was performed on BA201, BA205, BA206, BA207 and BA208. For BA202 and BA203, having a higher PE ADA prevalence, the cutpoint for BA201 was used. Results

[0721] For bispecific binding molecules BA205, BA206, BA207 and BA208, the response frequencies above the cutpoint in the bridging assay were all approximately 20%, whereas bispecific binding molecules BA202 and BA203 exhibited values of 96% and 86% above the cutpoint, respectively. The results were plotted and are shown in Figure 50, wherein the median response for each tested molecule is shown by a black horizontal bar, and the average cutpoint value for the additional binding molecules BA205, BA206, BA207 and BA208 is indicated as a common cutpoint in the graph.

[0722] In summary, the data show that the reactivity from serum is reduced for BA205, BA206, BA207 and BA208, as compared to BA202 and BA203. In other words, the response detected against BA202 and BA203 in serum could be decreased by the introduction of one or two point mutations in the hTfRl binding module comprised in the bispecific binding molecule. The four mutated variants gave responses in the same range as the response observed against the comparator antibody BA201.

[0723] EXAMPLE 31

[0724] In vivo efficacy in AAV-A53T x hTfR-KI mouse model

[0725] This example describes the in vivo efficacy of the bispecific binding molecule BA202, designed and produced according to Example 20, in AAV-A53T x hTfR-KI mice.

[0726] Materials and methods

[0727] AAV-A53T x hTfR-KI mice were unilaterally infused with an AAV1 / 2 vector expressing human a-synuclein (aSyn) with the A53T missense mutation under a chicken beta actin (CBA) promoter hybridized with the cytomegalovirus (CMV) immediate early enhancer sequence.

[0728] Stereotaxic injection of AAV1 / 2-A53T (5.1 x 1012vg / ml) was performed as follows: a beveled injection needle (30 G) connected to a 5 pl Hamilton Neuros syringe mounted on a digitally guided infusion unit (Digital Lab Standard, Harvard Apparatus) and pump (Pump 11, Elite Nanomite, Harvard Apparatus) was lowered to the desired DV level. Using an infusion speed of 0.2 pl / min, 2 pl of AAV solution was infused unilaterally to the right (ipsilateral) substantia nigra. After completion of the infusion, the needle was left in place for an additional 5 min prior to withdrawal. The incision was sutured and disinfected.

[0729] At 7 and 13 days post surgery, mice received 1 mg of the anti-mouse CD4 antibody GK1.5 (BioXcell) in order to deplete CD4+ T cells and consequently to prevent any generation of anti-drug antibodies. Starting at 14 days post surgery, animals received i.v. injections of either 10 mg / kg BA202 or PBS once weekly for 8 consecutive weeks.

[0730] 48 h after dosing of the 8th injection, the animals were terminally anesthetized with pentobarbital (180 mg / kg, i.p.). Next, the animals were transcardially perfused with 0.01M PBS (IX) followed by tissue fixation with 4% PFA in 0.01M phosphate buffer (PB). Brain samples were collected and post-fixed in tubes containing 20 ml of 4% PFA in 0.01M PB for 72 h at 4°C. After post-fixation, the samples were transferred into tubes containing 0.5% PFA in 0.01M PB and thereafter to 15% sucrose / PBS and stored at 4°C until they were sunk to the bottom of the tube to ensure cryoprotection, and subseqently embedded in OCT medium for cryosectioning in coronal orientation, frozen in dry ice-cooled isopentane and stored in an ultra deep freezer (set at -80 °C).

[0731] The brain region covering the AAV-injected area was sectioned coronally at 10 pm thickness on a Leica CM1950 or a Thermo Scientific NX70 cryotome. The analysis in this study is focused on substantia nigra. To verify successful inoculation and neurodegeneration in substantia nigra, the following histological labeling experiment was executed.

[0732] All steps were executed in Dulbecco's phosphate buffered saline pH 7.5 (PBS) at room temperature unless noted otherwise. Cryo-sections were air-dried for 45 min and then washed in PBS for 10 min.

[0733] For antigen de-masking, sections were treated with Proteinase K for 20 min at 37°C and washed 2 x 5 minutes each in PBS. Unspecific binding sites were blocked with 10% normal donkey serum in 0.1% Tergitol in PBS for 60 min in a damp chamber and sections were washed 3 x 5 min each in PBS. Sections were incubated with primary antibodies in 1% normal donkey serum overnight at 4 °C in a damp chamber: rat anti-ha-Syn monoclonal [15G7] antibody (Enzo Life Sciences, ALX-804- 258), 1:10, rabbit anti-pSerl29 a-Syn polyclonal antibody [EP1536Y] antibody (Abeam, ab51253), 1:1000 and sheep anti-tyrosine hydroxylase (TH) polyclonal antibody (Novus Biologica Is, NB300-110), 1:1000. Following a PBS wash, sections were incubated with secondary antibodies in 1% normal donkey serum for 60 min in a damp chamber (light protected): donkey anti-rat IgG H+L Alexa Fluor 647- conjugated (Abeam, abl50155), 1:500, donkey anti-rabbit IgG H+L Alexa Fluor 750- conjugated (Abeam, abl75728), 1:500 and donkey anti-goat IgG H+L DyLight 550- conjugated, (Abeam, ab96936), 1:500. After washing, sections were incubated with DAPI working solution for 15 min (light protected) and subsequently washed in PBS, destilled water and covered with Mowiol and coverslips (light protected) using a Leica CV5030 coverslipper.

[0734] Whole slide scans of the stained sections were recorded at a lOx magnification on a Zeiss automatic microscope AxioScan Z1 with high aperture lenses, equipped with a Zeiss Axiocam 506 mono and a Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 3.7 software.

[0735] Image analysis and quantification was done with Image Pro 10 (Media Cybernetics). Statistical analysis was performed in GraphPad Prism (v. 10.4.2) using unpaired t-test to compare BA202 treatment with PBS control.

[0736] Results

[0737] The immunofluorescence analysis revealed a significant ipsilateral neurodegeneration in the substantia nigra of inoculated mice. A representative image of TH+ neurons is shown in Figure 51. As shown in Figure 52, treatment with BA202 resulted in a significantly higher TH object density staining compared to PBS- treated controls (p value < 0.05), demonstrating that BA202 causes neuronal rescue.

[0738] ITEMIZED LISTING OF EMBODIMENTS

[0739] 1. A bispecific binding molecule, comprising

[0740] - a first moiety Ml, which is an a-synuclein protofibril binding moiety comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, said VH region in Ml comprising the amino acid sequence SEQ ID NO:447:

[0741] QVQLQESGPG LVKPSETLSL TCTVSGFSLT SYGVHWIRQP PGKGLEWSGV IWRGGSTDYS AAFMSRLTI S KDTSKNQVSL KLSSVTAADT AVYYCAKLLR SVGGFADWGQ GTMVTVSS said VL region in Ml comprising the amino acid sequence SEQ ID NO:448:

[0742] DIVMTQSPLS LPVTPGEPAS I SCRSSQTIV HNNGNTYLEW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCFQGSHVP FTFGQGTKLE IK and

[0743] - a second moiety M2, which is a human transferrin receptor 1 (hTfRl) binding moiety comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, in which said antigen-binding 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:85.

[0744] 2. 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:85.

[0745] 3. Bispecific binding molecule according to item 1 or 2, in which said antigen-binding surface of M2 is composed of three complementarity-determining regions (CDRs) from said VH region and three CDRs from said VL region, and in which said CDRs comprise the following:

[0746] VHCDR1: X1X2NMX3 (SEQ ID NO:1), wherein

[0747] XI is selected from D and A;

[0748] X2 is selected from Y and A; and X3 is selected from D and A;

[0749] VHCDR2: X4INPX5X6X7TTSX8X9X10KFKG (SEQ ID N0:2), 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;

[0750] X9 is selected from N and S; and X10 is selected from E and Q;

[0751] VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID N0:4), wherein Xll 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;

[0752] VLCDR2: X15ASTRES (SEQ ID N0:5) wherein X15 is selected from W and A; and

[0753] VLCDR3: QQX16X17X18X19PX20T (SEQ ID N0:6) wherein X16 is selected from X17 is selected from X18 is selected from X19 is selected from X20 is selected from

[0754] 4. Bispecific binding molecule according to item 3, said antigen-binding surface of

[0755] M2 further comprising

[0756] VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:3) wherein

[0757] X21 is selected from Y and A;

[0758] X22 is selected from Y and A;

[0759] X23 is selected from Y and A;

[0760] X24 is selected from D and A; and

[0761] X25 is selected from Y and A.

[0762] 5. Bispecific binding molecule according to any one of items 3-4, in which said

[0763] VHCDR2 in moiety M2 is:

[0764] VHCDR2: X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO:7), wherein

[0765] X4 is selected from D and A;

[0766] X5 is selected from D and A; X6 is selected from Y and A;

[0767] X7 is selected from D and A; and

[0768] X8 is selected from Y and A.

[0769] 6. Bispecific binding molecule according to any one of items 3-5, in which said

[0770] VLCDR1 in moiety M2 is:

[0771] VLCDR1: KSSQSLLX11STNQKNX14LA (SEQ ID NO:8), wherein

[0772] Xll is selected from Y and A; and X14 is selected from Y and A.

[0773] 7. Bispecific binding molecule according to any one of items 3-6, in which said

[0774] VLCDR3 in moiety M2 is:

[0775] VLCDR3: QQX16FIX19PRT (SEQ ID NO:9) wherein

[0776] X16 is selected from Y and A;

[0777] X19 is selected from Y and A.

[0778] 8. Bispecific binding molecule according to any one of items 3-7, in which the amino acid sequence of said VHCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:10 and 16-18.

[0779] 9. Bispecific binding molecule according to any one of items 3-8, in which the amino acid sequence of said VHCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:11, 19-23 and 34, for example selected from the group consisting of SEQ ID NO:ll and 19-23.

[0780] 10. Bispecific binding molecule according to any one of items 3, 5-9, in which the amino acid sequence of said VHCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:12, 24-28 and 35, for example selected from the group consisting of SEQ ID NO:12 and 24-28.

[0781] 11. Bispecific binding molecule according to any one of items 3-10, in which the amino acid sequence of said VLCDR1 in moiety M2 is selected from the group consisting of SEQ ID NO:13, 29, 30 and 36, for example selected from the group consisting of SEQ ID NO:13, 29 and 30. 12. Bispecific binding molecule according to any one of items 3-11, in which the amino acid sequence of said VLCDR2 in moiety M2 is selected from the group consisting of SEQ ID NO:14 and 31.

[0782] 13. Bispecific binding molecule according to any one of items 3-12, in which the amino acid sequence of said VLCDR3 in moiety M2 is selected from the group consisting of SEQ ID NO:15, 32, 33 and 37, for example selected from the group consisting of SEQ ID NO:15, 32 and 33.

[0783] 14. Bispecific binding molecule according to any one of items 3-13, in which the amino acid sequences of the six CDRs in moiety M2 are the following:

[0784] VHCDR1: DYNMD (SEQ ID NQ:10),

[0785] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0786] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12),

[0787] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0788] VLCDR2: WASTRES (SEQ ID NO:14), and

[0789] VLCDR3: QQYFIYPRT (SEQ ID NO:15).

[0790] 15. Bispecific binding molecule according to any one of items 3-13, in which the amino acid sequences of the six CDRs in moiety M2 are the following:

[0791] VHCDR1: DYNMD (SEQ ID NQ:10),

[0792] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0793] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12),

[0794] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0795] VLCDR2: WASTRES (SEQ ID NO:14), and

[0796] VLCDR3: QQYFIYPRT (SEQ ID NO:15).

[0797] 16. Bispecific binding molecule according to item 3, 5-13, in which the amino acid sequences of the six CDRs in moiety M2 are the following:

[0798] VHCDR1: DYNMD (SEQ ID NQ:10),

[0799] VHCDR2: DINPNYDTTSYSQKFKG (SEQ ID NO:34),

[0800] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:35),

[0801] VLCDR1: KSSQSLLYSSNRKNYLA (SEQ ID NO:36),

[0802] VLCDR2: WASTRES (SEQ ID NO:14), and

[0803] VLCDR3: QQYYNYPYT (SEQ ID NO:37). 17. Bispecific binding molecule according to any preceding item, wherein said VH region in moiety M2 comprises or consists of an amino acid sequence selected from

[0804] (i) the group consisting of SEQ ID NO:44-57, 65 and 67, for example the group consisting of SEQ ID NO:44-57, for example the group consisting of SEQ ID NO:44 and 50; and

[0805] (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).

[0806] 18. Bispecific binding molecule according to any preceding item, wherein said VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0807] (i) the group consisting of SEQ ID NO:58-64, 66 and 68, for example the group consisting of SEQ ID NO:58-64; and

[0808] (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).

[0809] 19. Bispecific binding molecule according to any one of items 17-18, wherein said VH region in moiety M2 is as defined in item 17 and said VL region in moiety M2 is as defined in item 18.

[0810] 20. Bispecific binding molecule according to item 19, in which said VH region in moiety M2 comprises SEQ ID NO:44 and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58-64.

[0811] 21. Bispecific binding molecule according to item 19, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44-57 and said VL region in moiety M2 comprises SEQ ID NO:58. 22. Bispecific binding molecule according to any one of items 20-21, in which said VH region in moiety M2 comprises SEQ ID NO:44 and said VL region in moiety M2 comprises SEQ ID NO:58.

[0812] 23. Bispecific binding molecule according to item21, in which said VH region in moiety M2 comprises SEQ ID NQ:50 and said VL region in moiety M2 comprises SEQ ID NO:58.

[0813] 24. Bispecific binding molecule according to any one of items 1-16, which comprises one first cysteine residue in said VH region in moiety M2 and one second cysteine residue in said VL region in moiety M2, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL regions.

[0814] 25. Bispecific binding molecule according to item 24, 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.

[0815] 26. Bispecific binding molecule according to item 24 or 25, 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.

[0816] 27. Bispecific binding molecule according to any one of items 24-26, 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.

[0817] 28. Bispecific binding molecule according to any one of items 24-27, wherein said VH region in moiety M2 comprises or consists of an amino acid sequence selected from (i) the group consisting of SEQ ID NO:88-103, for example the group consisting of SEQ ID NO:88-101, for example the group consisting of SEQ ID NO:88 and 94; and

[0818] (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.

[0819] 29. Bispecific binding molecule according to any one of items 24-28, wherein said VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0820] (i) the group consisting of SEQ ID NQ:105-113, for example the group consisting of SEQ ID NQ:105-lll; and

[0821] (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.

[0822] 30. Bispecific binding molecule according to any one of items 28-29, wherein said VH region in moiety M2 is as defined in item 28 and said VL region in moiety M2 is as defined in item 29.

[0823] 31. Bispecific binding molecule according to item 30, in which said VH region in moiety M2 comprises SEQ ID NO:88 and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105-lll.

[0824] 32. Bispecific binding molecule according to item 30, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NQ:88-101 and said VL region in moiety M2 comprises SEQ ID NQ:105. 33. Bispecific binding molecule according to any one of items 31-32, in which said VH region in moiety M2 comprises SEQ ID NO:88 and said VL region in moiety M2 comprises SEQ ID NO:105.

[0825] 34. Bispecific binding molecule according to item 30, in which said VH region in moiety M2 comprises SEQ ID NO:94 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0826] 35. Bispecific binding molecule according to any one of items 24-27, wherein said VH domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0827] (i) the group consisting of SEQ ID NO:457-467, for example the group consisting of SEQ ID NO:457-462, for example the group consisting of SEQ ID NO:457-458; and

[0828] (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.

[0829] 36. Bispecific binding molecule according to item 35, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from

[0830] (i) the group consisting of SEQ ID NQ:105-113, for example the group consisting of SEQ ID NQ:105-lll; and

[0831] (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.

[0832] 37. Bispecific binding molecule according to item 36, in which said VH domain in moiety M2 comprises SEQ ID NO:457 and said VL domain in moiety M2 comprises a sequence selected from SEQ ID NQ:105-lll. 38. Bispecific binding molecule according to item 36, in which said VH domain in moiety M2 comprises a sequence selected from SEQ ID NO:457-458 and said VL domain in moiety M2 comprises SEQ ID NO:105.

[0833] 39. Bispecific binding molecule according to any one of items 37-38, in which said VH domain in moiety M2 comprises SEQ ID NO:457 and said VL domain in moiety M2 comprises SEQ ID NQ:105.

[0834] 40. Bispecific binding molecule according to any one of items 37-38, in which said VH domain in moiety M2 comprises SEQ ID NO:458 and said VL domain in moiety M2 comprises SEQ ID NQ:105.

[0835] 41. Bispecific binding molecule according to any one of items 1-16, wherein said VH / VL pair in moiety M2 comprises from two to four histidine residues, and wherein the affinity of the binding molecule for hTfRl is higher at pH 7.4 than at pH 5.5.

[0836] 42. Bispecific binding molecule according to item 41, wherein said VH region in moiety M2 comprises from one to four histidine residues, such as from one to three histidine residues or from two to four histidine residues, such as from one to two histidine residues, from two to three histidine residues or from three to four histidine residues, such as comprising one, two, three or four histidine residues, subject to the proviso that the total number of histidine residues in the VH / VL pair in moiety M2 is from two to four.

[0837] 43. Bispecific binding molecule according to item 41 or 42, wherein said VL region in moiety M2 comprises from zero to three histidine residues, such as from zero to two histidine residues or from one to three histidine residues, such as from zero to one histidine residues, from one to two histidine residues or from two to three histidine residues, such as comprising zero, one, two or three histidine residues, subject to the proviso that the total number of histidine residues in the VH / VL pair in moiety M2 is from two to four. 44. Bispecific binding molecule according to any one of items 41-43, wherein said VH region in moiety M2 comprises two histidine residues and said VL region in moiety M2 comprises zero histidine residues.

[0838] 45. Bispecific binding molecule according to any one of items 41-43, wherein said VH region in moiety M2 comprises three histidine residues and said VL region in moiety M2 comprises zero histidine residues.

[0839] 46. Bispecific binding molecule according to any one of items 41-43, wherein said VH region in moiety M2 comprises two histidine residues and said VL region in moiety M2 comprises one histidine residue.

[0840] 47. Bispecific binding molecule according to any one of items 41-46, wherein said VH region in moiety M2 comprises or consists of an amino acid sequence selected from

[0841] (i) the group consisting of SEQ ID NO:44, 50, 129-145, 163-178, 195-211, 229- 245, 263-279, 297-312, 329-344 and 442, for example the group consisting of SEQ ID NO: 129-145, 163-178, 195-211, 229-245, 263-279, 297-312, 329-344 and 442, for example the group consisting of SEQ ID NO:129-142, 163-175, 195-208, 229-242, 263-276, 297-309, 329-341 and 442, for example the group consisting of SEQ ID NO:229-242, 263-276 and 297-309 or the group consisting of SEQ ID NO:129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329 and 335, for example the group consisting of SEQ ID NO:229, 235, 263, 269, 297 and 303; and

[0842] (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 any histidine residue in the sequence defined in (i) is present.

[0843] 48. Bispecific binding molecule according to any one of items 41-47, wherein said VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0844] (i) the group consisting of SEQ ID NO:58, 361-370, 381-390, 401-409 and 419- 427, for example the group consisting of SEQ ID NQ:361-370, 381-390, 401-409 and 419-427, for example the group consisting of SEQ ID NO:361-367, 381-387, 401-406 and 419-424, for example the group consisting of SEQ ID NO:419-424 or the group consisting of SEQ ID NO:361, 381, 401 and 419, for example SEQ ID NO:419; and

[0845] (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 any histidine residue in the sequence defined in (i) is present.

[0846] 49. Bispecific binding molecule according to any one of items 47-48, wherein said VH region in moiety M2 is as defined in item 47 and said VL region in moiety M2 is as defined in item 48.

[0847] 50. Bispecific binding molecule according to item 49, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329, 335 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58, 361-367, 381-387, 401-406 and 419-424, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0848] 51. Bispecific binding molecule according to item 49, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129-142, 163- 175, 195-208, 229-242, 263-276, 297-309, 329-341 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58 and 419-424, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0849] 52. Bispecific binding molecule according to item 50, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129-142, 163- 175, 195-208, 229-242, 263-276, 297-309, 329-341 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58, 361, 381, 401 and 419, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58. 53. Bispecific binding molecule according to any one of items 50-51, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329, 335 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58 and 419- 424, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0850] 54. Bispecific binding molecule according to item 50, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329, 335 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:58, 361, 381, 401 and 419, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0851] 55. Bispecific binding molecule according to item 54, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 229, 263, 297, 329 and 442, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO: 58, 361, 381, 401 and 419, with the proviso that the VH region does not comprise SEQ ID NO:44 or 50 when the VL region comprises SEQ ID NO:58.

[0852] 56. Bispecific binding molecule according to item 55, wherein the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:58; b) a VH region comprising SEQ ID NO:263 and a VL region comprising SEQ ID NO:58; c) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:361; d) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:381; e) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NQ:401; f) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:419; g) a VH region comprising SEQ ID NO:297 and a VL region comprising SEQ ID NO:419; and h) a VH region comprising SEQ ID NO:442 and a VL region comprising SEQ ID NO:58. 57. Bispecific binding molecule according to item 56, wherein the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:229 and a VL region comprising SEQ ID NO:58; b) a VH region comprising SEQ ID NO:263 and a VL region comprising SEQ ID NO:58; and c) a VH region comprising SEQ ID NO:297 and a VL region comprising SEQ ID NO:419.

[0853] 58. Bispecific binding molecule according to item 57, in which said VH region in moiety M2 comprises SEQ ID NO:229 and said VL region in moiety M2 comprises SEQ ID NO:58.

[0854] 59. Bispecific binding molecule according to item 57, in which said VH region in moiety M2 comprises SEQ ID NO:263 and said VL region in moiety M2 comprises SEQ ID NO:58.

[0855] 60. Bispecific binding molecule according to item 57, in which said VH region in moiety M2 comprises SEQ ID NO:297 and said VL region in moiety M2 comprises SEQ ID NO:419.

[0856] 61. Bispecific binding molecule according to any one of items 1-16, 24-27 and 41-46, wherein said VH region in moiety M2 comprises or consists of an amino acid sequence selected from

[0857] (i) the group consisting of SEQ ID NO:88, 94, 146-162, 179-194, 212-228, 246- 262, 280-296, 313-328, 345-360 and 443, for example the group consisting of SEQ ID NO:146-162, 179-194, 212-228, 246-262, 280-296, 313-328, 345-360 and 443, for example the group consisting of SEQ ID NO:146-159, 179-191, 212-225, 246-259, 280-293, 313-325, 345-357 and 443, for example the group consisting of SEQ ID NO:246-259, 280-293 and 313-325 or the group consisting of SEQ ID NO:146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345 and 351, for example the group consisting of SEQ ID NO:246, 252, 280, 286, 313 and 319; and; 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), provided that any histidine residue in the sequence defined in (i) is present, and provided that the sequence comprises a cysteine residue at position 44.

[0858] 62. Bispecific binding molecule according to any one of items 1-16, 24-27, 41-46 and 61, wherein said VL region in moiety M2 comprises or consists of an amino acid sequence selected from

[0859] (i) the group consisting of SEQ ID NO:105, 371-380, 391-400, 410-418 and 428-436, for example the group consisting of SEQ ID NO:371-380, 391-400, 410-418 and 428-436, for example the group consisting of SEQ ID NO:371-377, 391-397, 410- 415 and 428-433, for example the group consisting of SEQ ID NO:428-433 or the group consisting of SEQ ID NO:371, 391, 410 and 428, for example SEQ ID NO:428; and

[0860] (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), provided that any histidine residue in the sequence defined in (i) is present, and provided that the sequence comprises a cysteine residue at position 106.

[0861] 63. Bispecific binding molecule according to any one of items 61-62, wherein said VH region in moiety M2 is as defined in item 61 and said VL region in moiety M2 is as defined in item 62.

[0862] 64. Bispecific binding molecule according to item 63, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345, 351 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105. 65. Bispecific binding molecule according to item 63, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146-159, 179- 191, 212-225, 246-259, 280-293, 313-328, 345-360 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO:105 and 428-433, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0863] 66. Bispecific binding molecule according to item 63, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146-159, 179- 191, 212-225, 246-259, 280-293, 313-325, 345-357 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371, 391, 410 and 428, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0864] 67. Bispecific binding molecule according to any one of items 64-65, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345, 351 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105 and 428- 433, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0865] 68. Bispecific binding molecule according to item 64, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 152, 179, 212, 218, 246, 252, 280, 286, 313, 319, 345, 351 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NQ:105, 371, 391, 410 and 428, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0866] 69. Bispecific binding molecule according to item 68, in which said VH region in moiety M2 comprises a sequence selected from SEQ ID NO:88, 94, 146, 179, 212, 246, 280, 313, 345 and 443, and said VL region in moiety M2 comprises a sequence selected from SEQ ID NO: 105, 371, 391, 410 and 428, with the proviso that the VH region does not comprise SEQ ID NO:88 or 94 when the VL region comprises SEQ ID NQ:105.

[0867] 70. Bispecific binding molecule according to item 69, wherein the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NQ:280 and a VL region comprising SEQ ID NQ:105; c) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:371; d) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:391; e) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:410; f) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:428; g) a VH region comprising SEQ ID NO:313 and a VL region comprising SEQ ID NO:428; and h) a VH region comprising SEQ ID NO:443 and a VL region comprising SEQ ID NQ:105.

[0868] 71. Bispecific binding molecule according to item 70, wherein the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NQ:280 and a VL region comprising SEQ ID NQ:105; and c) a VH region comprising SEQ ID NO:313 and a VL region comprising SEQ ID NO:428.

[0869] 72. Bispecific binding molecule according to item 71, wherein said VH region in moiety M2 comprises SEQ ID NO:246 and said VL region in moiety M2 comprises SEQ ID NQ:105.

[0870] 73. Bispecific binding molecule according to item 71, wherein said VH region in moiety M2 comprises SEQ ID NQ:280 and said VL region in moiety M2 comprises SEQ ID NQ:105. 74. Bispecific binding molecule according to item 71, wherein said VH region in moiety M2 comprises SEQ ID NO:313 and said VL region in moiety M2 comprises SEQ ID NO:428.

[0871] 75. Bispecific binding molecule according to any preceding item, in which the VH / VL pair in moiety M2 forms part of an antibody construct.

[0872] 76. Bispecific binding molecule according to item 75, 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.

[0873] 77. Bispecific binding molecule according to item 76, wherein the VH / VL pair of the moiety M2 forms part of an scFv, in which the VH and VL regions are coupled together by a peptide scFv linker.

[0874] 78. Bispecific binding molecule according to item 77, wherein said scFv linker is attached to the N-terminal amino acid residue of the VH region and to the C- terminal amino acid residue of the VL region.

[0875] 79. Bispecific binding molecule according to item 77, wherein said scFv linker is attached to the C-terminal amino acid residue of the VH region and to the N- terminal amino acid residue of the VL region.

[0876] 80. Bispecific binding molecule according to any one of items 77-79, 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:455).

[0877] 81. 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. 82. Bispecific binding molecule according to item 81, 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 region of M2 or to the N-terminal amino acid residue of the VL region of M2.

[0878] 83. Bispecific binding molecule according to any one of items 81-82, wherein said at least one peptide linker between Ml and M2 is a flexible linker.

[0879] 84. Bispecific binding molecule according to item 83, wherein said flexible linker(s) comprise(s) glycine, serine, alanine and / or threonine residues.

[0880] 85. Bispecific binding molecule according to item 84, 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.

[0881] 86. Bispecific binding molecule according to any one of items 81-85, 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.

[0882] 87. 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.

[0883] 88. Bispecific binding molecule according to item 87, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of an amino acid sequence selected from SEQ ID NO:124-128 and 452-454. 89. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NO:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:124.

[0884] 90. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:125.

[0885] 91. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:126.

[0886] 92. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:127.

[0887] 93. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:128.

[0888] 94. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:452.

[0889] 95. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:453.

[0890] 96. Bispecific binding molecule according to item 88, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:454.

[0891] 97. Binding molecule which is an a-synuclein protofibril binding antibody or a fragment thereof, comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, said VH region comprising the amino acid sequence SEQ ID NO:447:

[0892] QVQLQESGPG LVKPSETLSL TCTVSGFSLT SYGVHWIRQP PGKGLEWSGV IWRGGSTDYS AAFMSRLTI S KDTSKNQVSL KLSSVTAADT AVYYCAKLLR SVGGFADWGQ GTMVTVSS said VL region comprising the amino acid sequence SEQ ID NO:448:

[0893] DIVMTQSPLS LPVTPGEPAS I SCRSSQTIV HNNGNTYLEW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCFQGSHVP FTFGQGTKLE IK .

[0894] 98. Binding molecule according to item 97, which comprises an antibody heavy chain represented by SEQ ID NO:449 and an antibody light chain represented by SEQ ID NQ:450.

[0895] 99. Bispecific binding molecule or binding molecule according to any preceding item, which has a higher affinity for a-synuclein protofibrils than for a-synuclein monomers. 100. Bispecific binding molecule or binding molecule according to item 99, which has at least 2x higher affinity for a-synuclein protofibrils than for a-synuclein 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, such as at least lOOOOx higher, such as at least 25000x higher, such as at least 50000x higher, such as at least 75000x higher, such as at least lOOOOOx higher affinity.

[0896] 101. Bispecific binding molecule or binding molecule according to any preceding item, which has a binding affinity for a-synuclein 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 50 and 150 pM, such as between 75 and 125 pM, as determined by surface plasmon resonance.

[0897] 102. Pharmaceutical composition, comprising a bispecific binding molecule or binding molecule according to any preceding item and a pharmaceutically acceptable carrier or excipient.

[0898] 103. Bispecific binding molecule or binding molecule according to any one of items 1-101 or a composition according to item 102 for use in treatment, such as for use in therapeutic treatment and / or for use in prophylactic treatment.

[0899] 104. Bispecific binding molecule or binding molecule according to any one of items 1-101 or a composition according to item 102 for use in diagnosis in vivo and / or prognosis in vivo.

[0900] 105. Bispecific binding molecule, binding molecule or composition for use according to any one of items 103-104, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a neurodegenerative disorder associated with a-synuclein aggregation, for example a disorder selected from the group consisting of Parkinson's disease (PD), multiple system atrophy (MSA) and Lewy body dementia (LBD), including dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).

[0901] 106. Bispecific binding molecule, binding molecule or composition for use according to item 105, wherein said neurodegenerative disorder is Parkinson's disease (PD).

[0902] 107. Bispecific binding molecule, binding molecule or composition for use according to item 105, wherein said neurodegenerative disorder is multiple system atrophy (MSA).

[0903] 108. 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 or binding molecule according to any one of items 1-101 or a composition according to item 102.

[0904] 109. Method according to item 108, wherein said neurodegenerative disorder is a disorder associated with a-synuclein aggregation, for example a disorder selected from the group consisting of Parkinson's disease (PD), multiple system atrophy (MSA) and Lewy body dementia (LBD), including dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).

[0905] 110. Method according to item 109, wherein said neurodegenerative disorder is Parkinson's disease (PD).

[0906] 111. Method according to item 109, wherein said neurodegenerative disorder is multiple system atrophy (MSA).

Claims

1. CLAIMS1. A bispecific binding molecule, comprising- a first moiety Ml, which is an a-synuclein protofibril binding moiety comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, said VH region in Ml comprising the amino acid sequence SEQ ID NO:447:QVQLQESGPG LVKPSETLSL TCTVSGFSLT SYGVHWIRQP PGKGLEWSGV IWRGGSTDYS AAFMSRLTI S KDTSKNQVSL KLSSVTAADT AVYYCAKLLR SVGGFADWGQ GTMVTVSS said VL region in Ml comprising the amino acid sequence SEQ ID NO:448:DIVMTQSPLS LPVTPGEPAS I SCRSSQTIV HNNGNTYLEW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCFQGSHVP FTFGQGTKLE IK and- a second moiety M2, which is a human transferrin receptor 1 (hTfRl) binding moiety comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), said VH and VL regions forming a VH / VL pair comprising an antigen-binding surface, in which said antigen-binding 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:85, for example 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:85.

2. Bispecific binding molecule according to claim 1, in which said antigen-binding surface of M2 is composed of three complementarity-determining regions (CDRs) from said VH region and three CDRs from said VL region, and in which said CDRs comprise the following:VHCDR1: X1X2NMX3 (SEQ ID NO:1), whereinXI is selected from D and A;X2 is selected from Y and A; andX3 is selected from D and A;VHCDR2: X4INPX5X6X7TTSX8X9X10KFKG (SEQ ID NO:2), whereinX4 is selected from D and A; X5 is selected from D, N and A; X6 is selected from Y and A; X7 is selected from D and A; X8 is selected from Y and A;X9 is selected from N and S; and X10 is selected from E and Q;VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID NO:4), whereinXll is selected from Y and A; X12 is selected from T and S;X13 is selected from Q and R; and X14 is selected from Y and A;VLCDR2: X15ASTRES (SEQ ID NO:5) wherein X15 is selected from W and A; andVLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:6) wherein X16 is selected from X17 is selected from X18 is selected from X19 is selected from X20 is selected fromoptionally further comprisingVHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:3) wherein X21 is selected from Y and A; X22 is selected from Y and A; X23 is selected from Y and A;X24 is selected from D and A; and X25 is selected from Y and A.

3. Bispecific binding molecule according to claim 2, in which the amino acid sequences of the six CDRs in moiety M2 are the following:VHCDR1: DYNMD (SEQ ID NQ:10),VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12),VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),VLCDR2: WASTRES (SEQ ID NO:14), andVLCDR3: QQYFIYPRT (SEQ ID NO:15).

4. Bispecific binding molecule according to claim 2, in which the amino acid sequences of the six CDRs in moiety M2 are the following:141VHCDR1: DYNMD (SEQ ID NQ:10),VHCDR2: DINPDADTTSYNEKFKG (SEQ ID N0:21),VHCDR3: GGYSGSSYYHPMDY (SEQ ID N0:12),VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID N0:13),VLCDR2: WASTRES (SEQ ID N0:14), andVLCDR3: QQYFIYPRT (SEQ ID N0:15).

5. Bispecific binding molecule according to any preceding claim, which comprises one first cysteine residue in said VH region in moiety M2 and one second cysteine residue in said VL region in moiety M2, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL regions, 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.

6. Bispecific binding molecule according to claim 5, wherein said VH region in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NQ:88-103, for example the group consisting of SEQ ID NQ:88-101, for example the group consisting of SEQ ID NO:88 and 94; 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.

7. Bispecific binding molecule according to any one of claims 5-6, wherein said VL region in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NQ:105-113, for example the group consisting of SEQ ID NQ:105-lll; 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.

8. Bispecific binding molecule according to any one of claims 5-7, wherein said VH region in moiety M2 comprises SEQ ID NO:88 and said VL region in moiety M2 comprises SEQ ID NO:105.

9. Bispecific binding molecule according to any one of claims 5-7, wherein said VH region in moiety M2 comprises SEQ ID NO:94 and said VL region in moiety M2 comprises SEQ ID NQ:105.

10. Bispecific binding molecule according to claim 5, 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:457-467, for example the group consisting of SEQ ID NO:457-462, for example the group consisting of SEQ ID NO:457-458; 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.

11. Bispecific binding molecule according to claim 10, wherein said VL domain in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NQ:105-113, for example the group consisting of SEQ ID NQ:105-lll; 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.

12. Bispecific binding molecule according to any one of claims 10-11, in which said VH domain in moiety M2 comprises SEQ ID NO:457 and said VL domain in moiety M2 comprises SEQ ID NO:105.

13. Bispecific binding molecule according to any one of claims 10-11, in which said VH domain in moiety M2 comprises SEQ ID NO:458 and said VL domain in moiety M2 comprises SEQ ID NQ:105.

14. Bispecific binding molecule according to any preceding claim, wherein said VH / VL pair in moiety M2 comprises from two to four histidine residues, and wherein the affinity of the binding molecule for hTfRl is higher at pH 7.4 than at pH 5.5.

15. Bispecific binding molecule according to claim 14, wherein said VH region in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NO:44, 50, 129-145, 163-178, 195-211, 229- 245, 263-279, 297-312, 329-344 and 442, for example the group consisting of SEQ ID NO: 129-145, 163-178, 195-211, 229-245, 263-279, 297-312, 329-344 and 442, for example the group consisting of SEQ ID NO:129-142, 163-175, 195-208, 229-242, 263-276, 297-309, 329-341 and 442, for example the group consisting of SEQ ID NO:229-242, 263-276 and 297-309 or the group consisting of SEQ ID NO:129, 135, 163, 195, 201, 229, 235, 263, 269, 297, 303, 329 and 335, for example the group consisting of SEQ ID NO:229, 235, 263, 269, 297 and 303; 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 any histidine residue in the sequence defined in (i) is present.

16. Bispecific binding molecule according to any one of claims 14-15, wherein said VL region in moiety M2 comprises or consists of an amino acid sequence selected from(i) the group consisting of SEQ ID NO:58, 361-370, 381-390, 401-409 and 419- 427, for example the group consisting of SEQ ID NQ:361-370, 381-390, 401-409 and144419-427, for example the group consisting of SEQ ID NO:361-367, 381-387, 401-406 and 419-424, for example the group consisting of SEQ ID NO:419-424 or the group consisting of SEQ ID NO:361, 381, 401 and 419, for example SEQ ID NO:419; 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 any histidine residue in the sequence defined in (i) is present.

17. Bispecific binding molecule according to any one of claims 14-16, wherein the VH region in moiety M2 and the VL region in moiety M2 are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NQ:280 and a VL region comprising SEQ ID NQ:105; c) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:371; d) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:391; e) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NQ:410; f) a VH region comprising SEQ ID NO:246 and a VL region comprising SEQ ID NO:428; g) a VH region comprising SEQ ID NO:313 and a VL region comprising SEQ ID NO:428; and h) a VH region comprising SEQ ID NO:443 and a VL region comprising SEQ ID NQ:105.

18. 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.

19. Bispecific binding molecule according to claim 18, in which the amino acid sequence of said Ml antibody light chain comprises or consists of SEQ ID NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy145 chain with linked M2 scFv comprises or consists of an amino acid sequence selected from SEQ ID NO:124-128 and 452-454.

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:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:124.

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 NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:125.

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 NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:126.

23. 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 NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:127.

24. 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 NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:128.14625. 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:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:452.

26. 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 NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:453.

27. 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 NQ:450, the amino acid sequence of said Ml antibody hole heavy chain comprises or consists of SEQ ID NO:451, and the amino acid sequence of said Ml antibody knob heavy chain with linked M2 scFv comprises or consists of SEQ ID NO:454.

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

29. Bispecific binding molecule according to any one of claims 1-27 or a pharmaceutical composition according to claim 28 for use in treatment, such as for use in therapeutic treatment and / or for use in prophylactic treatment.

30. Bispecific binding molecule according to any one of claims 1-27 or a pharmaceutical composition according to claim 28 for use in diagnosis in vivo and / or prognosis in vivo.

31. Bispecific binding molecule or pharmaceutical composition for use according to any one of claims 29-30, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a neurodegenerative disorder associated with a- synuclein aggregation, for example a disorder selected from the group consisting of147Parkinson's disease (RD), multiple system atrophy (MSA) and Lewy body dementia (LBD), including dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).

32. Bispecific binding molecule, binding molecule or composition for use according to claim 31, wherein said neurodegenerative disorder is Parkinson's disease (PD).

33. Bispecific binding molecule, binding molecule or composition for use according to claim 31, wherein said neurodegenerative disorder is multiple system atrophy (MSA).

34. 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 or binding molecule according to any one of claims 1-27 or a composition according to claim 28.

35. Method according to claim 34, wherein said neurodegenerative disorder is a disorder associated with a-synuclein aggregation, for example a disorder selected from the group consisting of Parkinson's disease (PD), multiple system atrophy (MSA) and Lewy body dementia (LBD), including dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD).

36. Method according to claim 35, wherein said neurodegenerative disorder is Parkinson's disease (PD).

37. Method according to claim 35, wherein said neurodegenerative disorder is multiple system atrophy (MSA).

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