PH sensitive binding molecule

Abstract

The present disclosure relates to a pH sensitive binding molecule, for example an antibody or antigen-binding fragment thereof, which binds to the protease-like domain of human transferrin receptor 1 (hTfR1), and to therapeutic and diagnostic uses thereof.

Description

[0001] PH SENSITIVE BINDING MOLECULE

[0002] Field

[0003] The present disclosure relates to a pH sensitive binding molecule, for example an antibody or antigen-binding fragment thereof, which binds to the protease-like domain of human transferrin receptor 1 (hTfRl), and to therapeutic and diagnostic uses thereof.

[0004] 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.

[0005] 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.

[0006] 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).

[0007] 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.

[0008] 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).

[0009] 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.

[0010] It has been reported that hTfRl apical binders can induce both acute clinical signs and decreased 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.

[0011] 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.

[0012] Single-chain Fv (scFv) domains of antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are normally linked by a flexible polypeptide linker to promote the assembly of the VL and VH domains. scFvs have the advantages of being smaller and thus more susceptible to genetic manipulation and engineering, as compared to full size antibody molecules and other, larger, types of antibody fragments. Nevertheless, the practical use of scFvs has long been limited due to low homogeneity, in turn caused by a propensity for aggregation mediated by inter-chain VH-VL interactions. Because of relatively weak interactions between its VH and VL domains, the scFv structure is in an equilibrium state between an open form, in which the two domains are dissociated, and a closed form, in which the two domains are associated through inter-domain interactions formed by surfaces matching the VL and VH together. The dynamics between the open and closed forms are more prominent in an scFv than in a Fab fragment, due to a lack in an scFv of CL and CHI regions that stabilize the structure within the Fv part. If the open state of an scFv accumulates, this could lead to interchain VH-VL interactions, resulting in the formation of dimers and oligomers (Arndt et al (1998) Biochemistry 37:12918-12926). The formation of these dimers or oligomers, in turn, may increase the avidity effect as they contain more than one binding site. Also, larger oligomers or aggregates likely lead to precipitation of the constructs. Due to a lack of general methods to overcome this problem, therapeutic scFv have not been in focus since their development around 1990 (Bird et al (1988) Science 242:423-426).

[0013] Against this background, the development of general methods to suppress the aggregation tendency of scFv and to stabilize the interaction between the VL and VH domains within an scFv has been necessary for these next-generation antibody formats to be successful. One such approach is the design and generation of disulfide-stabilized Fv fragments (denoted "dsFv"). This method addresses the problems of instability and aggregation frequently associated with scFv:s (see above). The VH and VL domains in dsFv are connected by an interdomain disulfide bond (Weatherill et al (2012) Protein Engineering, Design & Selection 25(7):321-329; Yamauchi et al (2019) Molecules 24(14):2620). Different locations for the engineered introduction of pairs of VL:VH interface cysteines have been described, most commonly in the context of Fv fragments (see e.g. Glockshuber et al (1990) Biochemistry 29:1362-1367; Brinkmann et al (1993) Proc. Natl. Acad. Sci. USA 92:7538-7542). To generate one such molecule, one amino acid each in the framework regions of the VH (typically at Kabat position 44) and VL (typically at Kabat position 100) domains are mutated to cysteine. These introduced cysteines then form a stable interchain disulfide bond when the VL and VH domains come into proximity with each other. The resulting dsFv (interchain disulfide bond, no linker peptide) or scdsFv (linker peptide and interchain disulfide bond) can be fused to other antibody domains and produced in various expression systems. Disulfide stabilized Fv's or scFv's may be able to solve problems that are frequently associated with Fv's or scFv's; they become very stable and, in most instances, exhibit full antigen binding activity.

[0014] Transport of a biopharmaceutical drug through the BBB via the hTfRl is dependent on transcytosis. After having been formed at the endothelial cell surface, the complex of binding molecule and receptor undergoes internalization by endocytosis, leading to formation of trafficking vesicles inside the cell. Because there is a pH differential between the extracellular environment at physiological pH 7.4 and the more acidic endosomes, researchers have proposed using hTfRl binding molecules with a pH sensitive affinity in order to achieve more efficient transcytosis (reviewed in Klaus and Deshmukh (2021), J Biomed Sci 28:11).

[0015] Sade et al, in PLoS ONE 9(4):e96340 (2014) and in WO2012143379, investigate the properties of a known TfRl antibody and finds that its affinity for TfRl is pH dependent. Tillotson et al, in PLoS ONE 10(12):e0145820 (2015) and in US10233252, take a more deliberate approach and actively engineer and screen TfRl antibodies to introduce pH dependent binding. The more recent WO2021205358 discloses a diverse set of antibody constructs that exhibit a ratio of KD value at pH 5 / KD value at pH 7.4 which is in the range from 1.1 to 115.

[0016] There remains a need in the field for pH sensitive antibodies and other binding molecules, which do not exhibit the drawbacks and risks associated with hitherto known binding molecules and which have a binding affinity for hTfRl which is different at different pH values.

[0017] Disclosure of the invention

[0018] It is an object of the disclosure to address this need, by providing a hTfRl binding molecule which utilizes a different binding site on hTfRl than the naturally occurring ligands.

[0019] One such object is to provide a hTfRl binding molecule which utilizes a different binding site on hTfRl than transferrin. Another such object is to provide a hTfRl binding molecule which utilizes a different binding site on hTfRl than ferritin.

[0020] Yet another such object is to provide a hTfRl binding molecule which utilizes a different binding site on hTfRl than HFE (homeostatic iron regulator).

[0021] A related object of the disclosure is to provide a hTfRl binding molecule which interacts with hTfRl in a way which minimizes the interference with hTfRl itself and / or its normal function.

[0022] Another object of the disclosure is to provide a hTfRl binding molecule suitable for use as a fusion partner in constructs arranged for transport through the BBB.

[0023] A related object of the disclosure is to provide a hTfRl binding molecule which exhibits an improved stability, e.g. in the form of storage stability and / or resistance against multimerization, as compared to other hTfRl binding molecules.

[0024] 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.

[0025] 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.

[0026] 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 improve the half life in vivo and / or cytoplasmic exposure of the binding molecule.

[0027] One or more of these objects, and other objects that are evident to the skilled person from the teachings herein, are met by the various aspects of the disclosure.

[0028] Thus, in a first aspect, the present disclosure provides a human transferrin receptor 1 (hTfRl) binding molecule, which - is 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,

[0029] - comprises 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, which antigen-binding surface provides the binding molecule with the capacity to bind selectively to said epitope; and

[0030] - comprises from two to four histidine residues in said VH / VL pair, and wherein the affinity of the binding molecule for hTfRl is higher at pH 7.4 than at pH 5.5.

[0031] In one embodiment, the VH region of said VH / VL pair 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 is from two to four.

[0032] In one embodiment, the VL region of said VH / VL pair 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 is from two to four.

[0033] In one embodiment, said VH region comprises two histidine residues and said VL region comprises zero histidine residues.

[0034] In another embodiment, said VH region comprises three histidine residues and said VL region comprises zero histidine residues.

[0035] In yet another embodiment, said VH region comprises two histidine residues and said VL region comprises one histidine residue.

[0036] In one embodiment of the binding molecule of the first aspect of the disclosure, 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.

[0037] 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.

[0038] In one embodiment, said first KD value is no more than 1 x 10-6M, such as no more than 1 x 10-7M, such as no more than 1 x IO-8M.

[0039] Without wishing to be bound by theory, the binding 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 hTfRl.

[0040] Also without wishing to be bound by theory, the provision of from two to four histidine residues in the VH / VL pair of the binding molecule is contemplated to provide a pH sensitive affinity of the binding molecule for its hTfRl target. The differential binding at neutral and acidic pH is in turn contemplated to enhance release of the binding molecule when in an endosome, thus escaping hTfRl- associated trafficking and accumulating more readily in the cell.

[0041] In a specific embodiment, the epitope or binding site for the hTfRl binding molecule of the disclosure 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 molecule of the disclosure 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 molecules 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 12, this embodiment of the epitope for the binding molecules identified and disclosed herein ensures binding that does not interfere with the natural hTfRl ligands transferrin and ferritin.

[0042] 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 binding molecule is a conformational epitope.

[0043] The binding molecule comprises 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 molecule with the capacity to bind selectively to said epitope. Said VH / VL pair may for example be provided in a full- length traditional antibody, or 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 specific embodiment, the VH / VL pair forms part of an scFv. 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 Ig domain with an antigen-binding surface. As such, 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.

[0044] With respect to the antigen-binding surface of the VH / VL pair, it may suitably be composed of three complementarity-determining regions (CDRs) from said VH region and three CDRs from said VL region. In one embodiment, said CDRs comprise the following:

[0045] VHCDR1: X1X2NMX3 (SEQ ID NO:1), wherein

[0046] XI is selected from D and A;

[0047] X2 is selected from Y and A; and

[0048] X3 is selected from D and A;

[0049] 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; X9 is selected from N and S; and X10 is selected from E and Q;

[0050] 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;

[0051] VLCDR2: X15ASTRES (SEQ ID NO:5) wherein X15 is selected from W and A; and

[0052] VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:6) wherein

[0053] X16 is selected from

[0054] X17 is selected from

[0055] X18 is selected from

[0056] X19 is selected from

[0057] X20 is selected from

[0058] In one embodiment, the antigen-binding surface of the VH / VL pair further comprises: VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:3) wherein

[0059] 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.

[0060] In an alternative embodiment, the antigen-binding surface of the VH / VL pair further comprises:

[0061] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:35)

[0062] As defined herein, embodiments of the binding molecule of the first aspect of the disclosure that comprise a VH / VL pair 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 generated and characterized as described in Examples 1-9. It is contemplated that the specific sequence information provided for the generated antibodies 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 as SEQ ID NO:l-6.

[0063] In one embodiment, said VHCDR2 is:

[0064] VHCDR2: X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO:7), wherein

[0065] X4 is selected from D and A; X5 is selected from D and A; X6 is selected from Y and A;

[0066] X7 is selected from D and A; and X8 is selected from Y and A.

[0067] In one embodiment, said VLCDR1 is:

[0068] VLCDR1: KSSQSLLX11STNQKNX14LA (SEQ ID NO:8), wherein

[0069] Xll is selected from Y and A; and X14 is selected from Y and A.

[0070] In one embodiment, said VLCDR3 is: VLCDR3: QQX16FIX19PRT (SEQ ID NO:9) wherein

[0071] X16 is selected from Y and A;

[0072] X19 is selected from Y and A.

[0073] In one embodiment, the amino acid sequence of said VHCDR1 is selected from the group consisting of SEQ ID NO:10 and 16-18.

[0074] In one embodiment, the amino acid sequence of said VHCDR2 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.

[0075] In one embodiment, the amino acid sequence of said VHCDR3 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.

[0076] In one embodiment, the amino acid sequence of said VLCDR1 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.

[0077] In one embodiment, the amino acid sequence of said VLCDR2 is selected from the group consisting of SEQ ID NO:14 and 31.

[0078] In one embodiment, the amino acid sequence of said VLCDR3 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.

[0079] In some embodiments, the CDR sequences can be freely combined among the options listed above. Such embodiments for example include, but are not limited to, those combinations exemplified in Example 9 for alanine substituted variants of the h26D3 embodiment of the binding molecule of the disclosure.

[0080] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0081] VHCDR1: DYNMD (SEQ ID NQ:10),

[0082] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0083] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0084] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0085] VLCDR2: WASTRES (SEQ ID NO:14)

[0086] VLCDR3: QQYFIYPRT (SEQ ID NO:15) In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0087] VHCDR1: DYNMD (SEQ ID NQ:10),

[0088] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0089] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0090] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0091] VLCDR2: WASTRES (SEQ ID NO:14)

[0092] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0093] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0094] VHCDR1: DYNMD (SEQ ID NQ:10),

[0095] VHCDR2: DINPNYDTTSYSQKFKG (SEQ ID NO:34),

[0096] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:35)

[0097] VLCDR1: KSSQSLLYSSNRKNYLA (SEQ ID NO:36),

[0098] VLCDR2: WASTRES (SEQ ID NO:14)

[0099] VLCDR3: QQYYNYPYT (SEQ ID NO:37)

[0100] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0101] VHCDR1: NYWLG (SEQ ID NO:38),

[0102] VHCDR2: DIFPGSDNTYYNEKFKG (SEQ ID NO:39),

[0103] VHCDR3: SGNFYAMDY (SEQ ID NQ:40)

[0104] VLCDR1: SASSSVNYMN (SEQ ID NO:41),

[0105] VLCDR2: DTSKLAS (SEQ ID NO:42)

[0106] VLCDR3: FQGSGYPFT (SEQ ID NO:43)

[0107] In one embodiment, said CDRs comprise the following:

[0108] 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;

[0109] VHCDR2: X4IX26PX5X6X7X27TSX8X9X10KFKG (SEQ ID NQ:450), wherein

[0110] X4 is selected from D and A; X26 is selected from N and H; X5 is selected from D, N and A;

[0111] X6 is selected from Y, H and A;

[0112] X7 is selected from D and A;

[0113] X27 is selected from T and H;

[0114] X8 is selected from Y and A;

[0115] X9 is selected from N and S; and X10 is selected from E and Q;

[0116] VHCDR3: GGX21X28GSX29X22X23HPMX24X25 (SEQ ID NO:451) wherein

[0117] X21 is selected from Y and A;

[0118] X28 is selected from S and H;

[0119] X29 is selected from S and H;

[0120] X22 is selected from Y, H and A;

[0121] X23 is selected from Y, H and A;

[0122] X24 is selected from D and A; and

[0123] X25 is selected from Y and A.

[0124] VLCDR1: KSSQSLLX11X30X12NX13KNX14LA (SEQ ID NO:452), wherein

[0125] Xll is selected from Y and A;

[0126] X30 is selected from S and H;

[0127] X12 is selected from T, H and S;

[0128] X13 is selected from Q and R; and

[0129] X14 is selected from Y, H and A;

[0130] VLCDR2: X15ASTRES (SEQ ID NO:5) wherein

[0131] X15 is selected from W and A; and

[0132] VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:453) wherein X16 is selected from X17 is selected from X18 is selected from X19 is selected from X20 is selected from

[0133] In one embodiment, the amino acid sequence of said VHCDR1 is selected from the group consisting of SEQ ID NQ:10 and 16-18. In one embodiment, the amino acid sequence of said VHCDR2 is selected from the group consisting of SEQ ID NO:11, 19-23, 34 and 454-456, for example selected from the group consisting of SEQ ID NO:454-456.

[0134] In one embodiment, the amino acid sequence of said VHCDR3 is selected from the group consisting of SEQ ID NO:12, 24-28, 35 and 457-460, for example selected from the group consisting of SEQ ID NQ:457-460.

[0135] In one embodiment, the amino acid sequence of said VLCDR1 is selected from the group consisting of SEQ ID NO:13, 29, 30, 36 and 461-463, for example selected from the group consisting of SEQ ID NO:461-463.

[0136] In one embodiment, the amino acid sequence of said VLCDR2 is selected from the group consisting of SEQ ID NO:14 and 31.

[0137] In one embodiment, the amino acid sequence of said VLCDR3 is selected from the group consisting of SEQ ID NO:15, 32, 33, 37 and 464, for example being SEQ ID NO:464.

[0138] In some embodiments, the CDR sequences can be freely combined among the options listed above. Such embodiments for example include, but are not limited to, those combinations exemplified in Example 9 for alanine substituted variants of the h26D3 embodiment of the binding molecule of the disclosure.

[0139] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0140] VHCDR1: DYNMD (SEQ ID NQ:10),

[0141] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0142] VHCDR3: GGYHGSSYYHPMDY (SEQ ID NO:457)

[0143] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0144] VLCDR2: WASTRES (SEQ ID NO:14)

[0145] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0146] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0147] VHCDR1: DYNMD (SEQ ID NQ:10),

[0148] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0149] VHCDR3: GGYHGSSYYHPMDY (SEQ ID NO:457)

[0150] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0151] VLCDR2: WASTRES (SEQ ID NO:14)

[0152] VLCDR3: QQYFIYPRT (SEQ ID NO:15) In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0153] VHCDR1: DYNMD (SEQ ID NQ:10),

[0154] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0155] VHCDR3: GGYSGSSHYHPMDY (SEQ ID NO:459)

[0156] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0157] VLCDR2: WASTRES (SEQ ID NO:14)

[0158] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0159] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0160] VHCDR1: DYNMD (SEQ ID NQ:10),

[0161] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0162] VHCDR3: GGYSGSSHYHPMDY (SEQ ID NO:459)

[0163] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0164] VLCDR2: WASTRES (SEQ ID NO:14)

[0165] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0166] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0167] VHCDR1: DYNMD (SEQ ID NQ:10),

[0168] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0169] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0170] VLCDR1: KSSQSLLYHTNQKNYLA (SEQ ID NO:461),

[0171] VLCDR2: WASTRES (SEQ ID NO:14)

[0172] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0173] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0174] VHCDR1: DYNMD (SEQ ID NQ:10),

[0175] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0176] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0177] VLCDR1: KSSQSLLYHTNQKNYLA (SEQ ID NO:461),

[0178] VLCDR2: WASTRES (SEQ ID NO:14)

[0179] VLCDR3: QQYFIYPRT (SEQ ID NO:15)

[0180] In a specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following: VHCDR1: DYNMD (SEQ ID NQ:10),

[0181] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0182] VHCDR3: GGYSGSSYYHPMDY (SEQ ID N0:12)

[0183] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID N0:13),

[0184] VLCDR2: WASTRES (SEQ ID N0:14)

[0185] VLCDR3: QQYFIHPRT (SEQ ID NO:464)

[0186] In another specific embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the amino acid sequences of the six CDRs are the following:

[0187] VHCDR1: DYNMD (SEQ ID NQ:10),

[0188] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0189] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12)

[0190] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0191] VLCDR2: WASTRES (SEQ ID NO:14)

[0192] VLCDR3: QQYFIHPRT (SEQ ID NO:464)

[0193] In one embodiment, CDR sequences in an antigen-binding interface comprised in a binding molecule of the disclosure are as 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).

[0194] In one embodiment of a binding molecule of the disclosure, said VH region comprises or consists of an amino acid sequence selected from

[0195] (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

[0196] (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. In one embodiment of a binding molecule of the disclosure, said VL region comprises or consists of an amino acid sequence selected from

[0197] (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 NO: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

[0198] (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.

[0199] In a particular such embodiment, the VH region and VL region 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.

[0200] In one embodiment, said VH region 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 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.

[0201] In one embodiment, said VH region 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 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.

[0202] In one embodiment, said VH region 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 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.

[0203] In one embodiment, said VH region 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 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.

[0204] In one embodiment, said VH region 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 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.

[0205] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 229, 263, 297, 329 and 442, and said VL region 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.

[0206] In one embodiment, the VH region and the VL region 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.

[0207] In one embodiment, the VH region and the VL region 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.

[0208] In a specific embodiment of the binding molecule of the disclosure, said VH region comprises SEQ ID NO:229 and said VL region comprises SEQ ID NO:58.

[0209] In another specific embodiment, said VH region comprises SEQ ID NO:263 and said VL region comprises SEQ ID NO:58.

[0210] In yet another specific embodiment, said VH region comprises SEQ ID NO:297 and said VL region comprises SEQ ID NO:419.

[0211] In still another specific embodiment, said VH region comprises SEQ ID NO:442 and said VL region comprises SEQ ID NO:58.

[0212] In some embodiments of the binding molecule of the disclosure, it comprises one first cysteine residue in said VH region and one second cysteine residue in said VL region, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL regions.

[0213] Without wishing to be bound by theory, the provision of the first and second cysteine residues in the VH and VL regions, 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.

[0214] 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. The hTfRl binding molecule according to this embodiment of the present disclosure is engineered with a disulfide bridge in order to stabilize the VL / VH or VH / VL forms. This is shown to be beneficial towards producing constructs that are stable and only bind in a monomeric form to transferrin receptor. Data shows that the presence of only small amounts of dimeric forms of binding molecules leads to avidity binding to the transferrin receptor. Also, without the stabilizing disulfide, the disclosed constructs may be produced in a dimeric or oligomeric form and be unstable over time under various conditions. Thus, by introducing a disulfide bond between the VL / VH or VH / VL domains, the hTfRl binding molecules according to this embodiment may both be more stably produced and in addition prevent avidity binding to the transferrin receptor.

[0215] In one embodiment, said first cysteine (in the 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.

[0216] In one embodiment, said second cysteine (in the 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.

[0217] In one exemplary embodiment, said first cysteine residue is located at VH position 44 and said second cysteine residue is located at VL position 100, as determined by reference to the Kabat numbering scheme.

[0218] In one embodiment of a binding molecule of the disclosure, said VH region comprises or consists of an amino acid sequence selected from

[0219] (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

[0220] (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.

[0221] In one embodiment of a binding molecule of the disclosure, said VL region comprises or consists of an amino acid sequence selected from

[0222] (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

[0223] (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.

[0224] In a particular such embodiment, the VH region and VL region 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 positions 106.

[0225] In one embodiment, said VH region 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 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.

[0226] In one embodiment, said VH region 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 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.

[0227] In one embodiment, said VH region 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 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.

[0228] In one embodiment, said VH region 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 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.

[0229] In one embodiment, said VH region 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 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.

[0230] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:88, 94, 146, 179, 212, 246, 280, 313, 345 and 443, and said VL region 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. In one embodiment, the VH region and the VL region 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 NO:105; b) a VH region comprising SEQ ID NO: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.

[0231] In one embodiment, the VH region and the VL region 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.

[0232] In a specific embodiment, said VH region comprises SEQ ID NO:246 and said VL region comprises SEQ ID NQ:105.

[0233] In another specific embodiment, said VH region comprises SEQ ID NQ:280 and said VL region comprises SEQ ID NQ:105.

[0234] In yet another specific embodiment, said VH region comprises SEQ ID NO:313 and said VL region comprises SEQ ID NO:428.

[0235] In still another specific embodiment, said VH region comprises SEQ ID NO:443 and said VL region comprises SEQ ID NQ:105.

[0236] In one embodiment of a binding molecule of the disclosure, said VH region comprises or consists of an amino acid sequence selected from

[0237] (i) the group consisting of SEQ ID NO:487-534, for example the group consisting of SEQ ID NQ:487-510, for example the group consisting of SEQ ID NO:487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507 and 509, for example the group consisting of SEQ ID NO:487, 491, 495, 499, 503 and 507, or the group consisting of SEQ ID NO:489, 493, 497, 501, 505 and 509; or the group consisting of SEQ ID NO:511-534, for example the group consisting of SEQ ID NO:511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531 and 533, for example the group consisting of SEQ ID NO:511, 515, 519, 523, 527 and 531 or the group consisting of SEQ ID NO:513, 517, 521, 525, 529 and 533; and

[0238] (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.

[0239] In one such embodiment of a binding molecule of the disclosure, said VL region comprises or consists of an amino acid sequence selected from

[0240] (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

[0241] (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.

[0242] In a particular such embodiment, the VH region and VL region are both as defined immediately above, i.e. a VH comprising or consisting of a sequence selected from SEQ ID NO:487-534 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. In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487-534, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0243] In one embodiment, said VH region comprises a sequence selected from SEQ ID NQ:487-510, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0244] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507 and 509, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410- 415 and 428-433.

[0245] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487, 491, 495, 499, 503 and 507, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0246] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:489, 493, 497, 501, 505 and 509, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0247] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511-534, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0248] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531 and 533, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410- 415 and 428-433.

[0249] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511, 515, 519, 523, 527 and 531, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0250] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:513, 517, 521, 525, 529 and 533, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371-377, 391-397, 410-415 and 428-433.

[0251] In one embodiment, said VH region comprises a sequence selected from SEQ

[0252] ID NO:487-534, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433. In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487-510, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0253] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507 and 509, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0254] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487, 491, 495, 499, 503 and 507, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0255] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:489, 493, 497, 501, 505 and 509, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0256] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511-534, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0257] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531 and 533, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0258] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511, 515, 519, 523, 527 and 531, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0259] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:513, 517, 521, 525, 529 and 533, and said VL region comprises a sequence selected from SEQ ID NQ:105 and 428-433.

[0260] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487-534, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0261] In one embodiment, said VH region comprises a sequence selected from SEQ ID NQ:487-510, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0262] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507 and 509, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428. In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:487, 491, 495, 499, 503 and 507, and said VL region comprises a sequence selected from SEQ ID NO:105, 371 and 428.

[0263] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:489, 493, 497, 501, 505 and 509, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0264] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511-534, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0265] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531 and 533, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0266] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:511, 515, 519, 523, 527 and 531, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0267] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:513, 517, 521, 525, 529 and 533, and said VL region comprises a sequence selected from SEQ ID NQ:105, 371 and 428.

[0268] In one embodiment, said VH region comprises a sequence selected from SEQ ID NO:535-536, and said VL region comprises a sequence selected from SEQ ID NO:371 and 428.

[0269] In one embodiment, the VH region and the VL region are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:487 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NO:495 and a VL region comprising SEQ ID NQ:105; c) a VH region comprising SEQ ID NO:511 and a VL region comprising SEQ ID NQ:105; d) a VH region comprising SEQ ID NO:519 and a VL region comprising SEQ ID NQ:105; e) a VH region comprising SEQ ID NO:487 and a VL region comprising SEQ ID NQ:106; f) a VH region comprising SEQ ID NO:495 and a VL region comprising SEQ ID NQ:106; g) a VH region comprising SEQ ID NO:511 and a VL region comprising SEQ ID NQ:106; h) a VH region comprising SEQ ID NO:519 and a VL region comprising SEQ ID NQ:106; i) a VH region comprising SEQ ID NO:535 and a VL region comprising SEQ ID NO:371; j) a VH region comprising SEQ ID NO:535 and a VL region comprising SEQ ID NO:428; k) a VH region comprising SEQ ID NO:536 and a VL region comprising SEQ ID NO:371; and l) a VH region comprising SEQ ID NO:536 and a VL region comprising SEQ ID NO:428.

[0270] In one embodiment, the VH region and the VL region are represented by one of the following VH / VL combinations: a) a VH region comprising SEQ ID NO:487 and a VL region comprising SEQ ID NQ:105; b) a VH region comprising SEQ ID NO:495 and a VL region comprising SEQ ID NQ:105; c) a VH region comprising SEQ ID NO:511 and a VL region comprising SEQ ID NQ:105; and d) a VH region comprising SEQ ID NO:519 and a VL region comprising SEQ ID NQ:105.

[0271] In a specific embodiment, said VH region comprises SEQ ID NO:487 and said VL region comprises SEQ ID NQ:105.

[0272] In another specific embodiment, said VH region comprises SEQ ID NO:495 and said VL region comprises SEQ ID NQ:105.

[0273] In yet another specific embodiment, said VH region comprises SEQ ID NO:511 and said VL region comprises SEQ ID NQ:105.

[0274] In still another specific embodiment, said VH region comprises SEQ ID NO:519 and said VL region comprises SEQ ID NQ:105.

[0275] In certain embodiments, the VH and VL sequences, when present in the 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.

[0276] In one embodiment of a binding molecule of the disclosure comprising a VH / VL pair, said VH / VL pair forms part of an antibody construct.

[0277] In one such embodiment, said antibody construct has more than one binding specificity. It may for example be bispecific or trispecific, or have more than three binding specificities. In a particular embodiment, the binding molecule is bispecific, for example a bispecific antibody construct.

[0278] In one embodiment of a binding molecule of the disclosure comprising a VH / VL pair, the VH / VL pair is present in an antibody fragment selected from the group consisting of a Fab fragment, a single chain Fab (scFab) fragment, an Fv fragment and a single chain (scFv) fragment. In a particular embodiment of such a binding molecule, said antibody fragment is an scFv.

[0279] In an important embodiment of the binding molecule disclosed herein, said antibody construct further comprises an antibody or an antigen binding fragment thereof, in addition to the VH / VL pair providing selective binding to hTfRl. In one such embodiment, this additional antibody or fragment thereof is capable of selective binding to a target present in the brain of a mammal. In some embodiments, said target is selected from the group consisting of amyloid-p peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, TAR DNA-binding protein 43 (TDP-43) or derivatives or fragments thereof, triggering receptor expressed on myeloid cells 2 (TREM2), beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, Tau, phosphorylated Tau or fragments thereof, apolipoprotein E4, CD20, prion protein, leucine rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, amyloid-p precursor protein, p75 neurotrophin receptor, neuregulin and caspase 6. In a more specific embodiment, said target is selected from the group consisting of amyloid-p peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, TAR DNA-binding protein 43 (TDP-43) or derivatives or fragments thereof, triggering receptor expressed on myeloid cells 2 (TREM2), Tau, phosphorylated Tau or fragments thereof and apolipoprotein E4. In an even more specific embodiment, said target is selected from the group consisting of amyloid-p peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof and TAR DNA-binding protein 43 (TDP-43) or derivatives or fragments thereof.

[0280] In one embodiment, said antibody or antigen binding fragment thereof capable of selective binding to a target present in the brain of a mammal is an anti- AP antibody, for example an antibody selected from the group consisting of lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06 and KHK6640.

[0281] In another embodiment, said antibody or antigen binding fragment thereof capable of selective binding to a target present in the brain of a mammal is an anti- alpha-synuclein antibody, for example an antibody selected from the group consisting of prasinezumab, UCB7853, Lu AF82422, TAK-341 and exidavnemab. Affinity for a target

[0282] 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 hTfRl bound by the VH / VL pair of the binding molecule as defined above), refer to a property of a binding molecule, such as a property of an antibody or antigen-binding fragment thereof or of a bi- or multispecific construct incorporating such an antibody or antigen-binding fragment thereof, which may be tested for example by ELISA, by surface plasmon resonance (SPR) or by bio-layer interferometry (BLI). The skilled person is aware of these methods and others.

[0283] According to the disclosure, the binding molecule has an affinity for its target which is different at different pH values. Specifically, the 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.

[0284] For example, the binding affinity for a target, 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 X or a molecule comprising 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 for X of the binding molecule. 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. As used herein, the term "EC50" refers to the half maximal effective concentration of binding molecule which induces a response halfway between the baseline and maximum after a specified exposure time.

[0285] Additionally or alternatively, 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 target X in a fluid sample is measured by detecting interference in an expected signal output. In principle, a known target or epitope-bearing substance is used to coat a multi-well plate. In parallel, a binding molecule with putative affinity for X is added and incubated with a solution containing target at varied concentrations. Following standard blocking and washing steps, samples containing the mixture of said binding molecule and the target are added to the well. Labeled detection antibody with affinity for the binding molecule is then applied for detection using relevant substrates (for example TMB). In principle, if there is a high concentration of target in the fluid sample, a significant reduction in signal output will be observed. In contrast, if there is very little target 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 target.

[0286] 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 target is required to interfere with the binding of the detection antibody to the known target coated on the plate, as compared to a higher IC50 value. Thus, a lower IC50 value typically corresponds to a higher affinity.

[0287] The binding affinity of a binding molecule may also be tested by surface plasmon resonance (SPR). For example, the affinity may be tested in an experiment in which target 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 for X of the binding molecule. 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 target 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.

[0288] 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 (target or epitope X) immobilized on the biosensor tip surface and an analyte (such as a binding molecule with a putative 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.

[0289] The skilled person is aware of the above mentioned and other methods for measuring the affinity of a binding molecule for a target or epitope X, either qualitatively or quantitatively or both.

[0290] Stability of a binding molecule of the disclosure

[0291] The introduction of cysteine residues and the resulting formation of a disulfide bridge in some embodiments of the 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 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 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 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.

[0292] Pharmaceutical compositions

[0293] In a second aspect, the disclosure provides a pharmaceutical composition comprising a binding molecule as described herein and at least one pharmaceutically acceptable excipient or carrier.

[0294] Techniques for formulating polypeptides such as antibodies and their derivatives 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.

[0295] 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.

[0296] 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.

[0297] Methods of prevention, treatment, diagnosis, prognosis and detection

[0298] The binding molecule according to the present disclosure may be useful as a therapeutic, prophylactic, diagnostic and / or prognostic agent.

[0299] Hence, in a further aspect of the disclosure, there is provided a binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a medicament.

[0300] In yet another aspect of the disclosure, there is provided a binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a diagnostic agent.

[0301] In yet another aspect of the disclosure, there is provided a binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a prognostic agent.

[0302] Also provided are methods of preventing disease, treating disease or diagnosing disease or assessing disease prognosis, wherein a binding molecule as disclosed herein is administered to a subject in need thereof, typically a human subject.

[0303] Also provided is the use of the disclosed binding molecule for the manufacture of compositions (such as medicaments) for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of any one of the listed diseases.

[0304] Thus, in one embodiment, there is provided the binding molecule, or pharmaceutical composition comprising it, for use in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of a neurodegenerative disorder, for example a disorder selected from Alzheimer's disease and other disorders associated with A|3 protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), the Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy. In a more specific embodiment, said disorder is selected from Alzheimer's disease and other disorders associated with A|3 protein aggregation, Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, Parkinson's disease (PD), Parkinson's disease dementia (PDD) and the Lewy body variant of Alzheimer's disease.

[0305] In an even more specific embodiment, said disorder is selected from Alzheimer's disease and other disorders associated with A|3 protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), in particular Alzheimer's disease.

[0306] In an alternative embodiment, there is provided the binding molecule, or pharmaceutical composition comprising it, for use in in the therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of another disorder, for example a disorder selected from brain cancer, multiple sclerosis and lysosomal storage diseases.

[0307] In another aspect, there is provided a method of therapeutic treatment, prophylactic treatment, diagnosis and / or prognosis of a disorder as listed above, said method comprising administering to said mammal an amount, such as a therapeutically effective amount, of a binding molecule as defined herein, or pharmaceutical composition comprising it. reference

[0308] Various publications are cited in the present application, each of which is incorporated by reference herein in its entirety.

[0309] Brief description of the figures

[0310] Figure 1 shows the results of a binding screen of the indicated IgG antibodies from the immunization described in Example 1 towards human (hTfRl), cyno (cTfRl) and mouse (mTfRl) TfRl in crude hybridoma supernatants by biolayer interferometry (BLI).

[0311] Figure 2 shows the result of the BLI binding analysis described in Example 2 for the indicated Fab fragments of mouse antibodies 24B4, 26D3 and 37D10 as well as for a Fab fragment of control antibody 8D3. Figure 3 shows mapping of antibody binding epitopes to the protease-like domain of hTfRl as described in Example 2, by selective antibody binding to ELISA plates coated with either human, mouse or one of three different chimeric human / mouse TfRl receptors. Antibodies 24B4, 26D3 and 37D10 bind to hTfRl (A) but not to mTfRl (B). In addition, 24B4, 26D3 and 37D10 also bind to h / m protease like domain chimera (D), but not to any of the plates coated with the other chimeric receptors (C and E).

[0312] 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.

[0313] Figure 5 shows the result of carrying out the epitope binding assay as described in Example 2, showing the degree of competition between antibodies for simultaneous binding to hTfRl. Binding of (A) antibody 26D3, (B) antibody 24B4 and (C) control antibody 15G11-1 to preformed complexes of hTfRl and either of the indicated antibodies. Binding responses for all antibodies are normalized to the binding response measured to free hTfRl (no competing antibody).

[0314] Figure 6 shows binding by the indicated binders to hTfRl on the surface of cells, studied as described in Example 2. The Y axes of both diagrams show the mean fluorescence intensity when staining cells with (A) whole antibodies and (B) Fab fragments of the indicated binders. No background staining is detected with the negative isotype control IgG (A) or the non-related Fab fragment, Lyl28 (B).

[0315] Figure 7 shows the result of competition analysis of indicated binders with ferritin and transferrin as described in Example 3. The diagrams show (A) MFI of the indicated binders binding to 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.

[0316] Figure 8 is a collection of sensorgrams showing the result of SPR analysis of original 26D3 and 26D3 humanized as described in Example 4 (h26D3) in Fab formats when binding to hTfRl and cTfRl as indicated. Figure 9 shows the result of BLI and ELISA binding studies carried out on mouse and humanized versions of 26D3 in an scFv format as described in Example 4. (A) Sensorgrams obtained by BLI measurement of binding of the indicated constructs to hTfRl. (B) Binding responses from ELISA measurement of binding of the indicated constructs to coated TfRl.

[0317] Figure 10 are depictions of the x-ray structure of the complex of h26D3-Fab and hTfRl, determined as described in Example 5. The chain names as used in the coordinate files are indicated. (A) Refined structure showing overall folds of three independent complexes in the asymmetric unit. (B) Example of electron density (2m|Fo|-D|Fc|) contoured at the 1 G level. The protein chains are drawn in cartoon representation while sugar moieties are shown in stick representation.

[0318] Figure 11 is a ribbon representation of the h26D3-Fab human TfRl complex determined with x-ray crystallography as described in Example 5. h26D3-Fab is depicted in dark gray and hTfRl in white. The binding interface (epitope / paratope) is encircled.

[0319] 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.

[0320] Figure 13 illustrates the work on generating and characterizing an hTfRl-KI mouse model as described in Example 6. (A) Schematic illustration of the transgenic hTfRl-KI mouse construct. The extracellular domain of human TFRC was inserted in the murine Tfrc gene by homologous recombination. (B) Quantitative reverse transcription PCR (RT-qPCR) analysis of mouse Tfrc and human TFRC gene expression in brain (N=3 / genotype). hTfRl-KI mice (grey circles) express human TFRC and mouse Tfrc in total brain homogenate, WT littermates only express mouse Tfrc (white). (C) Western blot analysis for hTfRl, total TfRl, and 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).

[0321] Figure 14 shows the results of in vivo brain and plasma exposure analysis of various indicated hTfRl binding molecules in hTfRl-KI transgenic mice as described in Example 7. (A) Brain exposure 24 h after i.v. administration of the indicated hTfRl binders. (B) Plasma exposure 24 h after i.v. administration of the indicated hTfRl binders. (C) Brai Plasma ratio 24 h after i.v. administration of the indicated hTfRl binders. The negative control is denoted "158", and the positive control "15G11-1".

[0322] Figure 15 shows the results of in vivo brain exposure analysis of various indicated hTfRl binding molecules in hTfRl-KI mice by immunohistochemistry as described in Example 8. Cortical brain capillary staining observed for several binding molecules, including h26D3. Reference hTfRl-binder "15G11-1" and non-TfRl binder "158" were used as positive and negative control, respectively.

[0323] Figure 16 shows BLI sensorgrams for the indicated alanine variants of h26D3 as described in Example 9. Each variant showed a different kinetic profile, illustrating the possibility to generate variants with different affinities against human TfRl with specific mutations in the CDR regions of the heavy or light chain.

[0324] Figure 17 shows representative SPR sensorgrams of the interaction between the indicated alanine variants of h26D3 with hTfRl and cTfRl, measured as described in Example 9.

[0325] Figure 18 shows the results of indirect ELISA analysis of the binding of the indicated alanine variants of h26D3 with hTfRl and cTfRl, measured as described in Example 9.

[0326] Figure 19 shows SPR sensorgrams of the interaction between the indicated alanine variants of h26D3, studied as scFv building blocks within a bispecific protein format as described in Example 9.

[0327] Figure 20 shows chromatograms from preparative SEC of (A) h26D3-HC6_DS and (B) h26D3-HC6, carried out as described in Example 10.

[0328] Figure 21 shows chromatograms from analytical SEC of the indicated scFv proteins after formulation and short-term storage at -80 °C, as described in Example 11.

[0329] Figure 22 shows chromatograms from analytical SEC analysis of the indicated scFv proteins kept at -80 °C (TO) and then at 40 °C for 1, 2 and 4 weeks as indicated, carried out as described in Example 12.

[0330] Figure 23 is a series of bar diagrams showing the percentage of monomeric scFv, as measured by analytical SEC, in samples of the respective indicated scFv molecule subjected to the thermal stability evaluation described in Example 12. Figure 24 shows chromatograms from analytical SEC analysis of the indicated scFv proteins kept at -80 °C (TO) and then at 40 °C for 1, 2 and 4 weeks as indicated, carried out as described in Example 12. The asterisk (*) in Figure 24C highlights a shift in retention time for h26D3-LCl_DS which occurred due to drift in the chromatography equipment. The shift was also seen for a standard size control (not shown) injected on the same column, and is unrelated to the analyzed sample.

[0331] Figure 25 demonstrates the results of the ELISA experiment described in Example 13, showing (A) hTfRl binding of a stabilized binding molecule of the disclosure after 48 h incubation in mouse serum at 37 °C and 4 °C in three separate experiments, (B) binding curves obtained from the binding molecule incubated in serum, in comparison to incubation in PBS, and (C) the ratio of binding activity at 37 °C to the binding activity at 4 °C in serum or PBS as indicated.

[0332] Figure 26 shows representative SPR sensorgrams of the interaction between the indicated variants of h26D3 with hTfRl, measured as described in Example 15.

[0333] Figure 27 shows chromatograms from analytical SEC on samples of the bispecific binding molecules BA301 (A), BA302 (B), BA303 (C), BA304 (D) and BA305 (E), as described in Example 16.

[0334] Figure 28 shows SPR sensorgrams of binding to hTfRl for the indicated bispecific binding molecules, measured as described in Example 16. The measured sensorgrams are shown in black and the fitted curves are shown in grey.

[0335] Figure 29 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.

[0336] Figure 30 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.

[0337] Figure 31 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.

[0338] Figure 32 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 33 shows the results of competitive ELISA at pH 6 and pH 7.4, carried out as described in Example 18.

[0339] Figure 34 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.

[0340] Figure 35 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.

[0341] Figure 36 shows the results of analytical SEC of S102-DS scFv samples subjected to temperature hold as described in Example 19, 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.

[0342] Figure 37 shows chromatograms from analytical SEC of the indicated bispecific binding molecules, measured as described in Example 20. The X-axis shows relative mAU of the respective samples.

[0343] Figure 38 shows SPR sensorgrams from measurement of binding to hTfRl for the bispecific binding molecules BA306 (A) and BA307 (B). The measured sensorgrams are shown in black and the fitted curves are shown in grey.

[0344] Figure 39 shows the results of the ADCC assay described in Example 20, showing fold induction for (A) the positive control Rituximab with BA308 and BA306, and (B) BA309 and BA307, measured over unstimulated cells as a function of the concentration of the indicated test construct and controls.

[0345] Figure 40 shows binding of test constructs BA306 (A) and BA307 (B) to K562 cells tested as described in Example 20, showing median fluorescence intensity (MFI) of binding to the surface of TfRl expressing K562 cells.

[0346] Figure 41 shows the result of the competitive transferrin inhibition assay described in Example 20, in which K562 cells were incubated with a constant concentration of fluorescently labelled transferrin and a dilution series of the indicated test compounds.

[0347] Figure 42 shows stacked chromatograms from analytical SEC of the indicated scFv samples collected as TO or after temperature hold at 40 °C for 1, 2 or 4 weeks respectively, as described in Example 22. The relative absorbance mAU (220 nm) is shown on the X-axis of respective graph (A-F).

[0348] Figure 43 shows representative SPR sensorgrams of the interaction between the indicated scFv variants and hTfRl, as described in Example 24.

[0349] Figure 44 shows representative SPR sensorgrams of the interaction between the indicated scFv variants and cTfRl, as described in Example 24.

[0350] Figure 45 shows the results from screening and competition ELISA with serum from 107 donors against h26D3-HC6 scFv variants, as described in Example 25: (A) PE-ADA screening ELISA with 21 serum samples against TfRl binding scFv variants, as indicated; (B) Screening ELISA with serum from 107 donors against six selected variants show a reduced response frequency for all six variants compared to "h26D3-HC6_DS, VL-first"; (C) ELISA with 107 serum samples against h26D3- HC6_DS, VL-first, with competition (HC6 competition) och without competition (HC6 screen) (dotted line represents the assay cut-off point in the competition setting); (D) Among the serum samples from 107 donors, a majority of the samples render a response level above the assay cut-off point (horisontal line) in the ELISA setting without competition.

[0351] Examples

[0352] 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.

[0353] 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.

[0354] EXAMPLE 1

[0355] Identification of binders of human TfRl by immunization and screening Immunization and hybridoma screening

[0356] 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.

[0357] 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.

[0358] Three days before fusion, the final intraperitoneal booster injection was given to the mice in absence of adjuvant. At sacrifice, mice were anesthetized with isoflurane. Intact spleens were collected by opening the abdominal cavity and dissected. Briefly, a single cell suspension of the spleen from an immunized mouse was prepared and mixed with Sp2 / 0 cells at a 3:1 ratio. The cells were fused using PEG and the cells were added to a bottle of ClonaCell™-HY Medium D (STEMCELL Technologies). 60-70 pl per well was then dispensed into 96-well plates. After 6-7 days, 150 pl HAT-medium was added to each well in the semi solid 96-well plates. The day after, 120 pl of supernatant was discarded from each well and 100 pl fresh HAT-medium was added. The next day, 100 pl of the supernatant of each well was taken and transferred to a storage plate and tested for presence of antibodies against mouse TfRl using indirect ELISA on nickel-coated plates according to the protocol below. A repeated screen of the hybridoma plates was performed by adding 120 pl HAT-medium on day 12 and by 3 days later transferring 25 pl supernatant to ELISA plates to screen for reactivity against mouse TfRl (both screens referred to as "primary screen"). Clones that were positive towards mouse TfRl with OD>0.2 were transferred to 24-well plates, cultured for at least 3 days, and subjected to a secondary screen for reactivity towards murine, human and cynomolgus TfRl in solution using biolayer interferometry (BLI) (referred to as "secondary screen"). Whereas binding of both hTfRl and cynomolgus TfRl was indicated, only very weak or no binding was detected for mTfRl in the secondary screen. Supernatants from 24-well plates were also screened for binding towards His-tagged hTfRl as well as lack of binding towards His-tagged amyloid-p precursor protein (APP; negative control) using both direct coated TfRl plates and nickel- coated plates as described below. Binding towards cynomolgus TfRl (cTfRl) was also analyzed using direct TfRl coat. Notably, ELISA responses (OD450 values) were very low for mTfRl compared to hTfRl and cTfRl, indicating weaker binding to mTfRl compared to the binding to hTfRl and cTfRl for all positive clones. Selected clones were diluted using limiting dilution assays (LDA) to reach monoclonality. Reactivity against mouse TfRl and human TfRl were re-tested by ELISA on monoclonal cultures following LDA and expansion.

[0359] Indirect ELISA screening

[0360] 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.

[0361] 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).

[0362] Examples of clones considered to be positive in binding mouse TfRl and human TfRl are shown in Table 1. These clones were also confirmed to bind both His-tagged hTfR and cTfR by ELISA, and to lack binding to His-tagged APP (negative control). Selected clones were further characterized in various assays.

[0363] Table 1: Examples of identified clones from hybridoma screening

[0364] Biolayer interferometry measurements

[0365] 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.

[0366] 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.

[0367] Sequencing of selected clones Clones of interest were cryopreserved and sequenced by whole transcriptome shotgun sequencing. Among the sequenced hybridoma clones were clones denoted 26D3, 24B4 and 37D10. The amino acid sequences obtained for the respective heavy chain variable (VH) and light chain variable (VL) regions of these antibodies are given in Table 2 below:

[0368] Table 2: 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 3 below.

[0369] Table 3: CDR sequences of primary antibodies In accordance with some embodiments of the present disclosure, the VH and

[0370] 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 4.

[0371] Table 4: Disulfide stabilized variants of primary antibodies

[0372] EXAMPLE 2

[0373] In vitro binding to human and cynomolgus TfRl and epitope screen A more detailed binding analysis by BLI was performed on purified, selected antibodies. Binding of Fab fragments from the murine antibodies 26D3, 24B4 and 37D10 to human TfRl and cynomolgus TfRl was investigated. For example, the BLI instrument Octet Red384 was used to measure binding between immobilized TfRl and the tested Fab fragments in solution. Antibody binding to TfRl was measured with TfRl complexed to the human transferrin ligand (Tf). Tf / TfRl-complexes were formed on streptavidin biosensors by first loading sensors with biotinylated human holo-transferrin followed by a complex-formation step by capturing either hTfRl or cTfRl on the sensors. Final complex density on the sensors was similar for both hTfRl and cTfRl. Antibody binding to TfRl was measured during an association phase of 120 s and a dissociation phase of 300 s. Figure 2 shows sensorgrams for 15 nM of each of 24B4-Fab, 26D3-Fab and 37D10-Fab, as well as for a Fab derived from the known TfRl binding antibody 8D3 (Boado et al (2009), Biotechnol Bioeng 102:1251-1258). The data indicate a similar binding profile against human and cTfRl for both 24B4-Fab and 26D3-Fab, and cross-reactive binding to both species is also detected for 37D10-Fab, while no significant binding of 8D3-Fab against human or cynomolgus TfRl was detected. Importantly, the experiment shows that 24B4-Fab, 26D3-Fab and 37D10-Fab all bind to TfRl when the natural ligand transferrin is in complex with TfRl.

[0374] Next, an ELISA experiment showed that antibodies 26D3, 24B4 and 37D10 bind to the protease-like domain of TfRl. In the ELISA experiment, human, mouse or three different chimeric TfRl receptors were used to coat ELISA plates (Figure 3). The ELISA protocol was slightly modified as follows from the indirect ELISA described in Example 1. Briefly, ELISA plates were coated with the following His-tagged antigens at 1 pg / ml: ectodomain of human TfRl (HislO-hTfRl; SEQ ID NO: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.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] EXAMPLE 3

[0379] Competition for hTfRl binding with ferritin and transferrin The unique binding to hTfRl of the binders according to the disclosure, binding to the protease-like domain of hTfRl and identified as described in Example 1, was evaluated for competition with natural TfRl ligands ferritin (Ft) and transferrin (Tf). In order to test ferritin competition with antibody, the human monocytic cell line THP-1 (Sigma / ECACC) was used. Binding of the scFv-Fc format (see Example 4 below) and control antibody (M-A712) to hTfRl on the THP-1 cell surface was confirmed, as shown in Figure 7A. For evaluating the competition between ferritin and the disclosed binders, cells were incubated with serially diluted test binders along with ferritin from human liver (BioRad, 4420-4804) for 1 h at 4 °C. After incubation, ferritin that had bound to hTfRl on the cell surface was captured using a primary sheep antibody against human liver ferritin (BioRad, AHP2179G) and analyzed using flow cytometry. The results are displayed in Figure 7B, and show that the 26D3 scFv-Fc does not compete with ferritin on the cell surface, whereas the control antibody anti-CD71, clone M-A712, known to bind to the same epitope on hTfRl as Ft (Maier et al (2016), Mol Ther Nucleic Acids 5:e321) clearly competes with Ft binding. Also for the identified 26D3 hTfRl binder, the impact on Ft binding is much less, illustrating that 26D3 has a different epitope on hTfRl than the binding site for Ft (Figure 7B).

[0380] 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.

[0381] 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.

[0382] EXAMPLE 4

[0383] Humanization of hTfRl binder 26D3

[0384] The Fab sequence of mouse antibody 26D3, identified and characterized as described in Examples 1-3, was analyzed and an in silica model of the 26D3 Fab 3D structure was generated using Bioluminate Software (Schrodinger). This murine Fab model was used as input for humanization. In this process, the CDRs of the VH and VL 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.

[0385] 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).

[0386] 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, WBR100633) according to the manufacturer's instruction. The h26D3 and 26D3 Fabs were injected over the chip using a 2-fold dilution series in five steps starting at 25 nM. The interaction was measured using the single cycle kinetics method with a contact time of 120 s at a flow rate of 30 pl / ml followed by a dissociation time of 600 s. Regeneration of the surface between cycles was done by injecting 3M MgCL The binding data were fitted to a 1:1 interaction model. The Fabs were diluted in HBS-EP+ (Cytiva, WBR100669). Experiments were performed at 25°C. The data confirm that the humanized variant of 26D3, i.e. h26D3, retained binding capacity for human and cynomolgus TfRl (Figure 8). The kinetic parameters obtained in the experiment are given in Table 5 below. Table 5: SPR analysis of murine and humanized 26D3 Fabs vs. hTfRl and cTfRl

[0387] 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 1.

[0388] EXAMPLE 5 Crystallization and structure determination of h26D3-Fab in complex with hTfRl This example describes crystallization of a complex between h26D3-Fab and hTfRl and determination of the binding interface. Ectodomain of human TfRl (SEQ ID NO: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 2OOpg 26 / 600 (Cytiva). The buffers used and purification of the humanized Fab were as described in Example 4.

[0389] The formation of a complex between humanized h26D3-Fab and hTfRl was done by mixing of the two components at a molar ratio of 1:1 in lx dPBS and incubation at room temperature for 1 h. Subsequently, the complex was purified using size exclusion chromatography on HiLoad Superdex 200pg 26 / 600 (Cytiva) as described in Example 4.

[0390] 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.

[0391] X-ray data collection and refinement were performed as follows. Data was collected to 3.87 A at Diamond Light Source beamline 104. The beamline was equipped with a DECTRIS Eiger2 XE 16M detector. The data set was integrated using XDS (Kabsch (2010), Acta Crystallogr D Biol Crystallogr 66:125-132) with STARANISO anisotropic scaling (Tickle et al (2018), Global Phasing Ltd) and diffracted to 3.87 A along the c* direction of the reciprocal lattice, and to 4.82 A in the a* / b* plane. Three complexes were found in the asymmetric unit. The structure was refined using the Buster refinement software and model building was carried out in Coot. Data collection and refinement parameters and statistics are given in Table 6 below. Table 6: X-ray diffraction data collection and refinement statistics

[0392] The final, refined structure of the complexes showing the overall folds is depicted in Figure 10. As shown in Figure 10A, there were three independent complexes in the asymmetric unit. The chain names as used in the coordinate files are indicated. Figure 10B shows an example of the electron density contoured at the interface between hTfRl and heavy / light chain of h26D3-Fab. The protein chains are drawn in cartoon representation while sugar moieties are shown in stick representation. The binding interface interaction between h26D3 and human TfRl was extracted from the X-ray structure and described in the following to provide information about the precise binding of h26D3 to human TfRl. The binding interface between hTfRl and h26D3-Fab is depicted in Figures 10 and 11, and interaction was observed between the amino acid residues indicated in Table ?. Table 7: Amino acid residues involved i interaction between h26D3 and hTfRl

[0393] Table 7 describes the key reside from both sides involved in the epitope / paratope interface as determined from the crystal structure. Additional residues in the vicinity are also likely to be important for the binding between h26D3 and hTfRl. In addition, as described in Example 9 below, several positions outside the observed binding interaction show important participation in binding of h26D3 to hTfRl.

[0394] In Table 8 below, the amino acids of human TfRl that are involved in the respective interactions with h26D3, Ft and Tf are listed. Notably, no amino acids involved in the binding of h26D3 form part of any of the binding interfaces for the endogenous ligands. This illustrates that the binders of the present disclosure, as exemplified by h26D3, bind to hTfRl outside the binding sites used by Ft and Tf. Table 8: Amino acid residues in hTfRl which interact with the respective ligand

[0395] * Montemiglio et al (2019), Nat Commun 10:1121

[0396] # 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.

[0397] EXAMPLE 6

[0398] 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.

[0399] EXAMPLE 7

[0400] Brain uptake of hTfRl binding constructs in vivo

[0401] To evaluate hTfRl-mediated brain uptake in vivo, monovalent Fc-scFv constructs (see Example 4) were produced for four different binding proteins. A known binder to hTfRl, 15G11-1, was used as a control (Yu et al (2014), supra). This hTfRl binder has been described to be active in vivo and is used as a positive reference control for brain uptake. In addition, a construct containing a non-hTfRl scFv binder based on the anti-amyloid 0 antibody mAbl58 was designed and included as a negative control in the form of an Fc fusion construct (Fc-scFvl58, also referred to as simply "158" here and in the figures). The different Fc-scFv constructs were injected intravenously (i.v.) into hTfRl knock-in (hTfRl-KI) mice produced as described in Example 6 (n=4 per construct) at equimolar doses of 30 nmol / kg (corresponding to approximately 2.3 mg / kg). Plasma and brain exposure was assessed 24 h after dose.

[0402] The animals were anaesthetized using isoflurane and terminal blood samples were collected from the orbital plexus into BD Microtainer K2EDTA tubes. The samples were inverted and centrifuged at 2400 x g for 10 min at 4 °C. Plasma was extracted and transferred to Eppendorf tubes and frozen at -80 °C. Immediately following blood sampling, the abdomen of the animals was cut open and a cannula (21 G) was inserted into the left ventricle of the heart. A small cut was made in the right atrium and transcardial perfusion was performed with a minimum of 50 ml of cold PBS. Following perfusion, brains were extracted and the olfactory bulbs removed. The brains were separated into left and right hemispheres and cerebellum was removed from the left hemisphere, after which the left hemisphere was weighed and snap frozen on dry ice and stored at -80 °C until further preparation and analysis of the concentrations of injected constructs using a Meso Scale Discovery (MSD) based assay. The right hemispheres were placed in 4 % formaldehyde and stored at 4 °C for 24 h, after which they were rinsed in cold PBS, transferred to cold 30 % sucrose solution prepared in PBS and stored at 4 °C for further immunohistochemistry (IHC) processing (see Example 8 below).

[0403] 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.

[0404] 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.

[0405] 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.

[0406] EXAMPLE 8

[0407] Immunohistochemistry data on brain exposure

[0408] In vivo engagement of hTfRl by the Fc-scFv construct was studied further using a qualitative immunohistochemistry (IHC) analysis. In brief, coronal brain sections at a thickness of 20 pm were obtained from PBS-perfused brain hemispheres of the mice described in Example 7 using a cryostat (Microm NX50 CryoStar, Epredia). The sections were collected on Superfrost plus slides (Menzel- Glaser, #J1800AMNZ) and air-dried prior to IHC. The brain sections were washed with PBS (pH 7.4) for 15 min and incubated in blocking buffer (5 % BSA, 0.25 % Triton-X in PBS) for 2 h at room temperature. To visualize i.v. dosed constructs, brain sections were incubated with a secondary goat anti-human IgG (heavy and light chain specific) conjugated to Alexa Fluor 488 (Invitrogen, #A11013) for 120 min at room temperature followed by 3x15 min wash in PBS. Slides were mounted with Fluoromount-G (Invitrogen, #00-4958-02) for imaging analysis. Confocal images from cerebral cortex were captured using a Leica Stellaris 5 confocal system equipped with a HC PL APO 40x / 1.25 GLYC motCORR CS2 objective (Leica, #11506423).

[0409] Distinct IHC immunofluorescence signals were observed in brain capillaries with positive reference module 15G11-1, while a minimal IHC signal was detected in brain sections from mice injected with negative control 158 (Figure 15). Brain capillary IHC signal was observed for the two test constructs h26D3 and 37D10, of which h26D3 showed the strongest immunofluorescence signal, comparable to the positive control 15G11-1. Taken together, the MSD (Example 7) and IHC (this Example) analyses demonstrate that the hTfRl binders of the disclosure in a scFv format exhibit an increased brain exposure in hTfRl-KI mice. EXAMPLE 9

[0410] Generation of affinity variants and affinity determinations

[0411] 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 9 below provides a summary of the specific mutations in each of the alanine variants.

[0412] Table 9: Alanine substitution variants of VH and VL of h26D3

[0413] 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 10 below provides a summary of the specific mutations in each of the alanine variants that were selected.

[0414] 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.

[0415] 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 11 below.

[0416] Table 11: SPR analysis of variant h26D3 Fabs vs. hTfRl and cTfRl For the indirect ELISA, half area 96-well plates (Corning, #3690) were coated with 1 pg / ml recombinant ectodomain of hTfRl (SEQ ID NO: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.

[0417] 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.

[0418] Finally, selected variants were reformatted to scFv and used in the context of the bispecific binding molecule format disclosed in WO2022 / 258841. Bispecific binding molecules comprising scFv modules constructed from h26D3 and selected alanine mutants were expressed in ExpiCHO cells as described above. Filtered supernatants were applied to a MabSelect SuRe column (Cytiva) which was subsequently washed with DPBS pH 7.4. Expressed binding molecules were eluted by application of 0.7 % HAc pH 2.5, followed by immediate neutralization of the sample to pH 7.5. Purified samples were polished further by subjecting them to size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex 200; Cytiva) in DPBS pH 7.4. The purified constructs were concentrated using centrifugal concentrators Amicon Ultra (30 MWCO, Millipore). Each purified expressed construct was characterized using SDS-PAGE, size-exclusion chromatography (Superdex 200 Increase 3.2 / 300; Cytiva) and UV protein determination. Binding to hTfRl was evaluated using SPR as described above with adjustments of the concentration interval depending on the variant. As shown in Figure 19 and in Table 12 below, the different tested variants exhibited a range of affinities for the hTfRl target. Table 12: SPR analysis of variant h26D3 scFv in bispecific format vs. hTfRl

[0419] EXAMPLE 10

[0420] Design, production and preparative SEC of disulfide-stabilized hTfRl binding molecules

[0421] A panel of hTfRl binding molecules in the scFv format were designed, produced and purified. The designed hTfRl binding scFv molecules are listed in Table 13.

[0422] Table 13: hTfRl binding scFv molecules and their amino acid sequences

[0423] 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.

[0424] The test items were produced as Hise tagged scFv constructs with the Hise tag spaced from the remainder of the scFv by a flexible (646)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.

[0425] 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.

[0426] EXAMPLE 11

[0427] Analytical SEC of disulfide-stabilized hTfRl binding molecules Following three freeze / thaw cycles between room temperature and -80 °C, 1 pg of each scFv variant produced in Example 10 was injected to a SEC column (Waters BioSuite 250 UHR SEC 4 pm, 4.6 x 300 mm). Analyses were done with a running buffer of 0.2 M potassium phosphate, 0.25 M KCI, pH 6.2 at a flow rate of 0.35 ml / min.

[0428] The results of the analytical SEC experiment are shown in Figure 21 and Table 14. The monomeric form of all scFv samples have a retention time of 11 min (Figure 21A-H). In scFv molecules lacking the DS mutations, additional peaks, corresponding to multimerized forms of scFv are detected (Figure 21A-D). In all samples with DS mutations, 100 % of the respective molecule migrate at 11 min as monomeric scFv (Figure 21E-H). The percentage distribution of integrated peak areas from the analytical SEC samples are listed in Table 14. Again, for the four samples with stabilizing DS mutations, 100 % of injected proteins are detected in the monomer peak, whereas additional peaks of multimeric forms are detected for corresponding samples without DS mutations.

[0429] 5

[0430] Table 14: Distribution of peak areas from SEC chromatograms

[0431] EXAMPLE 12

[0432] 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 10-11, purified and stored in PBS, were subjected to temperature hold for one, two or four weeks at temperatures 4 °C, 40 °C and frozen at -80 °C, except for h26D3-HC6_DS, VL-first and h26D3-LCl, VL-first, which were held frozen at -70 °C and -75 °C respectively. At 5 each timepoint, samples of each variant from each temperature were analyzed by HPLC-SEC as described in Example 11. At the initiation of the study, frozen samples were thawed and analyzed, and are denoted TO.

[0433] 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 10. However, analytical SEC revealed that all samples contain both monomer (retention at approximately 11 min) and dimer (retention at approximately 10 min) forms already at the initial time point TO (Figure 22A-D). The share of dimers is the most significant for h26D3-HC6 (Figure 22A) and h26D3-HC6, VL-first (Figure 22B), while 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.

[0434] 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.

[0435] 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.

[0436] As can be seen in Table 15 below, the high stability of the scFv variants with DS mutations as compared to corresponding variants without DS mutations was also seen for samples kept at 4 °C or frozen. Table 15: Percentage of monomer forms of scFv samples

[0437] EXAMPLE 13

[0438] 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:

[0439] 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).

[0440] EXAMPLE 14

[0441] Dynamic light scattering analysis of disulfide-stabilized hTfRl binding molecules Dynamic light scattering (DLS) analysis of scFv variants h26D3-HC6_DS, VL- first; h26D3-HC6 and h26D3-HC6_DS (see Example 10) was performed at 25 °C or 20 °C using an Uncle instrument (Unchained Labs). Average hydrodynamic diameter and polydispersity index (PDI) were calculated from analyses run in triplicates h26D3-HC6_DS, VL-first) or duplicates (h26D3-HC6 and h26D3-HC6_DS). Samples were diluted to 1 mg / ml in PBS prior to analysis.

[0442] The results are shown in Table 16. The scFv variant without DS mutations (h26D3-HC6) exhibits a larger average hydrodynamic diameter than either of the two variants with DS mutations (h26D3-HC6_DS, VL-first and h26D3-HC6_DS). The results are expected from the high monomeric content for DS stabilized scFv as shown by analytical SEC (Figure 21 and Table 14), and agree with other reported DLS analyses of scFv (Morioka et al (2019), Molecules 24(14):2620). The observed PDI values are around 0.1 in all variants, indicating monodisperse (PDI < 0.1) or a low degree of polydispersity (PDI 0.1-0.2).

[0443] Table 16: Dynamic light scattering analysis of scFv variants

[0444] EXAMPLE 15

[0445] Surface plasmon resonance analysis of disulfide-stabilized hTfRl binding molecules

[0446] Binding to hTfRl of six different purified scFv variants from Example 10 was evaluated by surface plasmon resonance (SPR) using Biacore 8K (Cytiva). 30 pg / ml of DYKDDDDK Tag mAb FG4R (Thermo Fisher Scientific, MAI-91878) in 10 mM acetate buffer, pH 4.5 was immobilized on a CM5 sensor chip (Cytiva, WBR100399) using the amine coupling kit type 2 (Cytiva, WBR100633) according to the manufacturer's instruction, resulting in immobilization levels of approximately 10000 RU. hTfRl was captured via an N-terminal DYKDDDDK tag. To this end, the receptor was injected over the chip with a contact time of 60 s at a flow rate of 10 pl / min. Each of the six scFv variants h26D3-HC6; h26D3-HC6, VL-first; h26D3-LCl; h26D3-HC6_DS; h26D3- HC6_DS, VL-first and h26D3-LCl_DS, VL-first were injected over the chip using a 3- fold dilution series in five steps starting at 700 nM or 400 nM. Interaction was measured using the single cycle kinetics method with a contact time of 120 s at a flow rate of 30 pl / min followed by a dissociation time of 600 s. Regeneration of the surface between cycles was done by injecting 10 mM glycine-HCI pH 1.7 with a contact time of 30 s and a flow rate of 30 p.l / min . The binding modules were diluted in HBS-EP+ (Cytiva, WBR100669). Experiments were performed at 25 °C.

[0447] 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

[0448] 5 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.

[0449] 10 EXAMPLE 16

[0450] Design, production and characterization of bispecific binding molecules with stabilized hTfRl binding scFv domain

[0451] A number of different bispecific binding molecules were designed as knob- into-hole antibody constructs containing an hTfRl binding scFv with stabilizing DS

[0452] 15 mutations linked to the C-terminal amino acid residue of the knob heavy chain of the antibody. The tested molecules are listed in Table 17, with reference to the amino acid sequences of the individual polypeptide chains making up the respective molecule.

[0453] 20 Table 17: Amino acid sequences of tested bispecific binding molecules

[0454] The tested molecules were expressed in Chinese hamster ovary cells (ExpiCHO; Thermo Fisher Scientific) according to the manufacturer's instructions. The harvested supernatant was purified by affinity chromatography, followed by

[0455] 25 preparative size-exclusion chromatography (SEC) and buffer exchange into phosphate buffered saline (PBS) solution. Purified molecules were characterized using SEC and UV protein determination. Bispecific binding molecules were successfully produced and purified to a final concentration of 3.5 mg / ml.

[0456] Samples of the purified bispecific binding molecules were analyzed by analytical size exclusion chromatography using a 1260 Infinity II LC system (Agilent) equipped with a TSKG3000SWXL column (7.8 x 300 mm, 5 pm particle size; Tosoh Bioscience) coupled to an in-line filter. Samples were analyzed by injecting 10 pg of sample at a flow rate of 0.5 ml / min of 0.2M sodium phosphate at pH 7.0. The percentage monomer elution peak area was quantified using Agilent OpenLab Data Analysis version 2.5.

[0457] The results from aSEC are shown in Table 18 and Figure 27, and demonstrate that bispecific binding molecules comprising any one of the tested, disulfide- stabilized hTfRl binding scFv domains were recovered with a high percentage of monomeric content and a very low content of species of higher molecular weight (HMW), in agreement with the results seen in Examples 11 and 12 above.

[0458] Table 18: Analytical HPLC SEC of bispecific binding molecules

[0459] Binding of the purified bispecific binding molecules to human TfRl was evaluated using SPR on a Biacore 8K instrument (Cytiva). 30 pg / ml of DYKDDDDK Tag Monoclonal Antibody (FG4R) (ThermoFisher Scientific, #14-6681-80) was immobilized on a CM5 sensor chip (Cytiva, #BR100399) using the amine coupling kit type 2 (Cytiva, #BR100633) according to the manufacturer's instruction. hTfRl (SEQ ID NO:86) containing FLAG tag was captured on the chip via injection of a 5 pg / ml solution for 60 s at 10 pl / min flow rate. The tested molecules were injected over the chip using a 3-fold dilution series in five steps starting at 100, 500 or 1500 nM of hTfRl. The interaction for each molecule was measured in 3 or 4 replicates 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. The binding data was fitted to a 1:1 interaction model. The bispecific binding molecules were diluted in HBS-EP+ (Cytiva, WBR100669). Experiments were performed at 25 °C. Results are shown in Figure 28A-E and Table 19, and confirm maintained binding for respective molecules.

[0460] Table 19: Affinity to hTfRl of tested bispecific binding molecules by SPR

[0461] EXAMPLE 17

[0462] Generation of pH sensitive mutants

[0463] This example describes the design, production and purification of pH sensitive variants of the hTfRl binding molecules described in the previous examples. Such variants are examples of pH dependent binding molecules according to the present disclosure. 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.

[0464] 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 20.

[0465] Table 20: Produced and purified Fab constructs

[0466] _ . VH sequence VL sequence

[0467] Designation(SEQ|DN0:) (SEQ|DN0:)

[0468] N52H 129 58

[0469] S105H 263 58

[0470] T33H 44 381 wt 44 58

[0471] HC6 50 58

[0472] S32H 44 361

[0473] S102H 229 58

[0474] T57H 195 58

[0475] Y100H 44 419

[0476] Y107H 329 58

[0477] Y38H 44 401

[0478] Y55H 163 58

[0479] Y106H 297 58

[0480] S32H x N52H 129 361

[0481] S32HxS102H 229 361

[0482] S32HxY55H 163 361

[0483] S32HxY106H 297 361

[0484] S32HxY107H 329 361

[0485] T33H x N52H 129 381

[0486] T33HxS102H 229 381

[0487] T33HxY55H 163 381

[0488] T33HxY106H 297 381

[0489] T33HxY107H 329 381

[0490] Y38H x N52H 129 401

[0491] Y38H x S102H 229 401

[0492] Y38HxY55H 163 401

[0493] Y38HxY106H 297 401

[0494] Y38HxY107H 329 401

[0495] Y100H x N52H 129 419

[0496] Y100HxS102H 229 419

[0497] Y100HxY55H 163 419

[0498] Y100HxY106H 297 419

[0499] Y100HxY107H 329 419 EXAMPLE 18

[0500] Affinity determination using SPR, BLI and competitive ELISA

[0501] This example describes the binding of molecules generated and produced in Example 17 to human TfRl by SPR, BLI and competitive ELISA.

[0502] 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.

[0503] 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.

[0504] 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 21. Representative sensorgrams are shown in Figure 29 for binding of single mutants to hTfRl, in Figure 30 for binding of single mutants to cTfRl, and in Figure 31 for binding of double mutants to hTfRl. Table 21: Affinities by SPR of pH sensitive variants for immobilized hTfRl and cTfRl . KDVS. hTfRl KDVS. cTfRl

[0505] Designation(M) (M)

[0506] N52H 1.68e-7 3.30e-5

[0507] S105H 1.30e-6 9.70e-8

[0508] T33H 6.83e-9 1.38e-8 wt 4.81e-9 3.14e-9

[0509] S32H 5.55e-9 3.72e-9

[0510] S102H 7.77e-8 1.44e-7

[0511] T57H 1.32e-5 4.01e-6

[0512] Y100H 7.02e-9 2.00e-8

[0513] Y107H 6.20e-9 3.19e-8

[0514] Y38H 1. Ole-7 1.61e-7

[0515] Y55H 1.15e-6 5.81e-7

[0516] Y106H 3.82e-9 2.16e-8

[0517] S32H x N52H 2.57e-7

[0518] S32HxS102H 8.75e-8

[0519] S32HxY55H 3.58e-7

[0520] S32HxY106H 2.28e-8

[0521] S32HxY107H 2.91e-8

[0522] T33H x N52H Poor fit

[0523] T33HxS102H 2.14e-7

[0524] T33HxY55H 1.99e-7

[0525] T33HxY106H 1.09e-7

[0526] T33HxY107H 7.08e-8

[0527] Y38H x N52H 8.65e-9

[0528] Y38H x S102H 8.01e-9

[0529] Y38HxY55H 1.07e-8

[0530] Y38HxY106H 2.12e-7

[0531] Y38HxY107H 1.76e-7

[0532] Y100H x N52H Poor fit

[0533] Y100HxS102H Poor fit

[0534] Y100HxY55H Poor fit

[0535] Y100HxY106H 1.34e-7

[0536] Y100HxY107H 4.95e-8

[0537] 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.

[0538] 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 22. Representative sensorgrams are shown in Figure 32.

[0539] Table 22: Determination of affinity pH sensitivity by BLI

[0540] KDVS. hTfRl KDVS. hTfRl „ .

[0541] Ratio

[0542] Designation at pH 7.4 at pH 5.5 .u_ „

[0543] , . .. , . .. KDat pH5.5 / KDat pH7.4

[0544] (nM) (nM) wt 23 26 1.1

[0545] HC6 280 450 1.6

[0546] S102H 68 690 10

[0547] Y106H 28 120 4.2

[0548] Y38H 150 230 1.5

[0549] Y100H 36 89 2.5

[0550] S32H x Y55H 1000 2200 2.2

[0551] S32H x S102H 68 420 6.2

[0552] S32H x Y106H 32 120 3.6

[0553] S32H x Y107H 33 140 4.2

[0554] T33H x S102H 760 ND1

[0555] T33H x Y106H 170 330 1.9

[0556] T33H x Y107H 110 340 3.3

[0557] Y100H x Y106H 270 ND1

[0558] Y100H x Y107H 74 240 3.3

[0559] 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).

[0560] The results are shown in Figure 33 and Table 23, 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.

[0561] Table 23: IC50 for hTfRl at different pH values

[0562] , , , IC50 at pH 7.4 IC50 at pH 6

[0563] Test molecule . ... . ...

[0564] (nM) (nM)

[0565] Wt 6.16 4.73

[0566] HC6 838 211

[0567] S102H 584 ND

[0568] Y100H x Y106H 950 ND

[0569] EXAMPLE 19

[0570] Stability assessment of pH sensitive module in an scFv format

[0571] 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 10-16. 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.

[0572] 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 34.

[0573] 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 35 and show that the recovered molecule is stable, exhibiting an intact monomeric state over freeze / thawing cycles.

[0574] 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 36 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 36C).

[0575] EXAMPLE 20

[0576] Design and testing of bispecific binding molecules with a pH sensitive hTfRl binding domain

[0577] Bispecific binding molecules denoted BA306 and BA307 were designed as knob-into-hole antibody constructs containing the hTfRl-binding scFv S102H-DS of Example 19 linked to the C-terminal amino acid residue of the knob heavy chain of the antibody. As controls, the corresponding standard antibodies, respectively denoted BA308 and BA309, were used. The amino acid sequences of the tested molecules are listed in Table 24.

[0578] Table 24: Amino acid sequences of tested bispecific binding and control molec u I e s

[0579] Bispecific binding molecules BA306 and BA307 were expressed and purified as described for constructs BA301-BA305 in Example 16. The purified molecules were characterized using SEC and UV protein determination as described in Example 16. In agreement with Example 19, the protein purity as defined by SEC-HPLC exhibited a high monomer content and only trace residuals of HMW species could be observed. The results are shown in Table 25 and in Figure 37. Table 25: Analytical HPLC SEC of bispecific binding molecules

[0580] The purified bispecific binding molecules were analyzed for binding to hTfRl by SPR as described in Example 16. The results confirm binding of BA306 and BA307 to hTfRl, as shown in Figure 38 and in Table 26 below.

[0581] Table 26: Affinity to hTfRl of tested bispecific binding molecules by SPR

[0582] Safety assessment (ADCC): To investigate the respective effector functions of BA306 and BA307, a commercial antibody-dependent cellular cytotoxicity (ADCC) assay was run (Promega; #G7018). The effector cells stably express FcyRllla receptor, V158 (high affinity) variant, and an NFAT response element driving expression of firefly luciferase as a measure of ADCC activity. When an Fc part is bound to FcyR, the activation signal for the effector cells is triggered, leading to killing of those target cells that are coated with antibodies on their surface. Ramos cells (Sigma, #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 effectontarget of 6:1, with and without serially diluted test construct.

[0583] BA308 and BA309, i.e. corresponding regular IgGl antibodies without hTfRl binding scFv, and commercially available rituximab were used as negative controls and positive control respectively. Target cells with test constructs were plated in a 96 well assay plate (Costar, #3917), mixed with effector cells and incubated for 18 h at 37°C with 5% CO2. When an Fc-containing protein forms a bridge between target and effectors (through the interaction between Fc and FcyR), it leads to luciferase activity. After 18 h incubation, Bio-Gio luciferase reagent (Promega, #G7941) was added and the luciferase signal was quantitated in a SPARK plate reader (Tecan). The results are shown in Figure 39. Rituximab is known to be a strong inducer of ADCC, and this was verified in the assay setup. Neither of the tested bispecific constructs BA306 and BA307, nor any one of the antibodies BA308 and BA309, induced any ADCC signal.

[0584] On-cell binding: Next, K562 cells (Sigma / ECACC, #CB_89121407) were used in a cell binding experiment. The cells were washed twice in PBS and then resuspended in staining buffer consisting of D-PBS (Gibco, #14190-144) with 1% BSA (Sigma- Aldrich, #A7030-100G) and 0.1% sodium azide (G-Biosciences, #766-299). Fc-block (BD human Fc-Block #564220) was added, and the cells were incubated for 15 min at 4°C to exclude unspecific binding to cells via Fc receptors. Cells were then seeded in a 96 well V-bottom plate (Costar, #3897) and incubated with serially diluted test constructs in staining buffer for 1 h at 4°C. Cells were washed three times with staining buffer to remove unbound test construct and then fixed for 15 min at room temperature with 4% methanol-free PFA (Thermo Fisher Scientific, #J61899.AP). After fixation, cells were washed three times with staining buffer. To detect bound test construct, cells were incubated with PE-conjugated anti-human IgG Fc-specific antibody (eBioscience, #12-4998-82) for 30 min at 4°C. Cells were washed three times prior to acquisition on a flow cytometer (BD Lyric). Samples were analyzed using the FlowJo software (BD) and the median fluorescence intensity of bound test construct was plotted against concentration. The results are shown in Figure 40 and demonstrate that BA306 and BA307 both bind to hTfRl on the surface of the cells.

[0585] Taken together, the ADCC and cell binding experiments show that the pH dependent construct of the disclosure binds to the cell surface via hTfRl but that the Fc part does not engage Fcyllla receptors in an amount sufficient to cause ADCC.

[0586] Transferrin inhibition of binding to hTfRl: The physiological role of hTfRl is to transport iron, in a complex with transferrin, into organs via receptor mediated transcytosis. Since this is the major pathway of iron uptake it is important to know what impact TfR targeting binding molecules have on this process. Therefore, K562 cells (Sigma / ECACC, #CB_89121407) were used in a transferrin competition assay. Fc receptors were blocked (BD human Fc-Block #564220) for 15 min at 4°C to exclude binding to Fc receptors. Cells were then washed with staining buffer, seeded in a 96 well V-bottom plate (Costar, #3897) and incubated with a constant concentration of 10 pg / ml Alexa Fluor-647 labelled transferrin from human serum (Invitrogen, #T23366, labelled in house) and serially diluted test constructs BA306 and BA307, along with the respective corresponding standard antibody BA308 or BA309, or unlabeled holo-transferrin as positive control in staining buffer for 30 min at 4°C. Cells were washed twice with staining buffer to remove unbound transferrin and binding molecules, and were subsequently acquired on a flow cytometer (BD Lyric). Samples were analyzed using FlowJo software (BD) and the mean fluorescence intensity of bound AF647-transferrin was plotted against the concentration of binding molecule. The results are shown in Figure 41 and demonstrate that unlabeled holo-transferrin competed with AF647-labeled transferrin in a dosedependent manner, whereas neither of the bispecific binding molecules BA306 (A) and BA307 (B) compete with transferrin binding to hTfRl on the cell surface at any concentration tested.

[0587] EXAMPLE 21

[0588] Design, production and SEC analysis of additional stabilized hTfRl binding molecules In the single-chain variable fragment (scFv) format, hydrophobic patches within the former V / C interface of the full-length antibody become exposed (Nieba et al (1997), Prot Eng 10(4):435-444). These newly accessible hydrophobic areas, particularly within the VH domain, can serve as binding sites for preexisting antidrug antibodies (PE-ADA) when the scFv, or construct comprising it, is administered to a subject (Holland et al (2013), J Clin Immunol 33:1192-1203). Previous work has shown that mutation of certain hydrophobic residues within the former V / C interface of the VH domain to more hydrophilic residues leads to a reduction of such potential PE-ADA reactivity (Johansson et al (2023), mAbs 15(1) :2215887).

[0589] Eleven variants of the disulfide-stabilized VH domain of h26D3-HC6 were designed, containing either single or double substitutions of hydrophobic amino acid residues to more hydrophilic and / or less bulky amino acids residues. The variants are listed in Table 27. All VH domain variants were expressed in the form of scFv domains having the VL first orientation. A flexible (648)4 linker connects the VL domain to the VH domain within each scFv. For purification purposes, a Hise tag and (648)4 linker was added to the N-terminus of the VL domain (combined tag sequence given by SEQ ID NO:122).

[0590] These eleven scFv variants were produced in CHO cells and purified with IMAC plus preparative SEC as described in Example 10. The SEC fractions containing monomeric scFv were collected, sterile filtered and brought to 1 mg / ml final concentration in PBS, pH 7.4. The recovered molecules were analysed by CE-SDS, reducing and non-reducing, by loading 2.5 pg of respective molecule on a LabChip (GXII Touch™ HT Chip, PerkinElmer). Samples were prepared with the ProteinEXact Assay Reagent Kit (PerkinElmer). Instrument Assay used: LabChip GXII Touch HT Protein Characterization System (Perkin Elmer). Sample treatment: Before loading, samples were treated with DTT for reduced conditions, and both non-reduced and reduced samples were heated at 70 °C for 10 min. The results showed bands of expected size and high purity for all molecules.

[0591] Each scFv variant produced was then injected onto an SEC column and analyzed as described in Example 11. The results of the analytical SEC experiment are shown in Table 28 and demonstrate that all variants of the molecule were isolated with a high monomer content, similar to the DS-stabilized scFv molecules produced in Example 11. Table 28: Results from aSEC of hTfRl binding scFv variants

[0592] Based on the successful introduction of single and double mutations into the

[0593] VH domain of stabilized h26D3 HC6 as evidenced above, the same mutations may also be introduced in any pH sensitive VH domain of the disclosure, in parallel to the histidine residues already introduced. As a non-limiting listing of exemplary such domains, introducing a subset of the eleven mutation patterns into a number of different VH domains of the pH sensitive, stabilized, humanized hTfRl binding molecules disclosed herein, gives rise to VH domains having the sequences SEQ ID NO:487-534. These VH domains are also individual embodiments of the disclosure, as discussed in the general description of the invention above.

[0594] EXAMPLE 22

[0595] Thermal stability of additional variants of hTfRl binding molecules

[0596] From the production and analysis of hTfR binding molecules in Example 21, six variant scFv:s (SEQ ID NO:476-481) were selected and subjected to a thermal stability study. Monomer stability of scFv samples was evaluated by HPLC SEC analysis. The scFv molecules, purified and stored in PBS, were subjected to temperature hold for one, two or four weeks at temperatures 4°C, 25°C, 40°C and frozen at -75°C. At each timepoint, samples of each variant from each temperature were analyzed by HPLC-SEC as described in Example 21. At the initiation of the study, frozen samples were thawed and analyzed, and denoted "TO". In addition, for each of the six molecules a sample was subjected to freeze-thaw stress. Molecules were subjected to 3x freeze-thaw cycles. Freezing was done at -75 °C for at least 16 h and thawing was done at ambient temperature until complete thawing of the sample (up to 60 min). Samples are denoted FT_3x and were analyzed immediately after the third freeze-thaw cycle completion.

[0597] All six scFv molecules were stable monomers at the tested conditions. The results for the six selected scFv molecules from samples collected as TO or after temperature hold at 40 °C for 1-4 weeks respectively are shown as stacked chromatograms in Figure 42A-F. As seen in Figure 42, the main peak for all samples has a retention time at ~11 min, as expected for a monomeric scFv. All samples have a high monomer content at all tested conditions, demonstrating that the stabilized DS design is maintained in all six variants.

[0598] EXAMPLE 23

[0599] Immunogenicity in silica of additional variants of hTfRl binding molecules

[0600] The six scFv sequences analyzed in Example 22 (SEQ ID NO:476-481) and the variant "h26D3 HC6 DS, VL-first" (SEQ ID NO:118; Example 10) were subjected to an in silica analysis of immunogenicity risk, based on identification of potential T cell epitopes in the respective amino acid sequence. The assessment was done using iTope-AI (Abzena Ltd., Cambridge, UK). In addition, the sequences were analysed for homology to known T cell epitopes, previously identified by ex vivo EpiScreen™ analysis of other protein sequences. Homology scores against known T cell epitopes (Bryson et al (2010), BioDrugs 24(1) :l-8) are shown as TCED in Table 29.

[0601] The immunogenicity assessment of the respective protein sequence was performed using overlapping 9mer peptides, tested against 46 MHC class II allotypes used in the iTope-AI platform. Individual peptides, together spanning the whole sequence, were given a binding score from 0 to 3 for each allotype, and those scores were added together to provide a "Position Risk Score". The "Total Score" for the respective test protein in Table 29 was calculated by adding the "Position Risk Scores" obtained for all individual peptides. The highest "Position Risk Score" for each sequence was denoted "Hotspot Max" as also shown in Table 29.

[0602] Taken together, all seven tested protein sequences show very similar results, indicating that no novel risk sites of concern were identified in any of the assessed variants of the TfR binding molecule.

[0603] EXAMPLE 24

[0604] SPR analysis of binding of additional scFv variants to hTfRl and cTfRl

[0605] 5 The 11 scFv molecules produced and purified as described in Example 21 and the variant "h26D3 HC6 DS, VL-first" (SEQ ID NO:118; Example 10) were evaluated for binding to human and cynomolgus TfRl using SPR in a Biacore 8K instrument (Cytiva). For this, 30 pg / ml of DYKDDDDK Tag Monoclonal Antibody (FG4R) (ThermoFisher SCIENTIFIC. #14-6681-80) was immobilized on a CM5 sensor chip

[0606] 10 (Cytiva. #BR100399) using the amine coupling kit type 2 (Cytiva. #BR100633) according to the manufacturer's instruction. hTfRl (SEQ ID NO:86) or cTfRl (SEQ ID NO:87), each containing a FLAG tag, was captured on the chip via injection of a 5 pg / ml solution for 60 s at 10 pl / min flow rate. Each scFv variant was injected over the chip using a 3-fold dilution series in five steps starting at 700 nM (hTfRl) or 1500

[0607] 15 nM (cTfRl). The interaction was measured using the single cycle kinetics method with a contact time of 120 s at a flow rate of 30 pl / min followed by a dissociation time of 600 s. Regeneration of the surface between cycles was done by injecting 10 mM glycine-HCI, pH 1.7. The binding data was fitted to a 1:1 interaction model. The scFv variants were diluted in HBS-EP+ (Cytiva. #BR100669). Experiments were

[0608] 20 performed at 25 °C. As expected, the binding to TfR was similar for all analyzed molecules. The results for binding to hTfR are shown in Figure 43A-L, and to cTfR in Figure 44A-L. The calculated KD values for binding to hTfR and cTfR are given below in Tables 30 and 31, respectively. Table 30: Affinity constants for binding to hTfRl

[0609] Table 31: Affinity constants for binding to cTfRl

[0610] 5 Next, in order to compare binding parameters with more certainty, a followup SPR analysis with additional replicates (N=4), was conducted for six selected molecules (SEQ ID NO:476-481) and the variant "h26D3 HC6 DS, VL-first". The selected molecules were produced as described above. As shown in Table 32 below, the measured affinity KD values are very similar between all selected variants.

[0611] 10 Table 32: Affinity constants for binding to hTfRl measured in SPR with 4 replicates

[0612] EXAMPLE 25

[0613] PE-ADA assay in serum

[0614] 5 To investigate the degree of reactivity from pre-existing anti-drug antibodies among non-exposed individuals to a set of scFv molecules, a representative subset of 21 human serum samples was used to screen for binding response to 11 new scFv variants (SEQ ID NO:476-486) produced as described in Example 21. The response was measured using a direct ELISA as described below. Results from the direct ELISA

[0615] 10 screen show that the reactivity frequences differ between the screened variants, and that the response frequency is reduced for some of the investigated variants in line with previous findings (Johansson et al (2023), mAbs 15(1):2215887). The binding reactivity is shown in Figure 45A.

[0616] From the results of this initial ELISA screen, six variants (SEQ ID NO:476-481)

[0617] 15 were selected and subjected to additional ELISA measurements of PE-ADA reactivity in a larger set of 107 human serum samples, as described below. As seen in Figure 45B, all six variants have a reduced reactivity compared to "h26D3-HC6_DS, VL-first" (SEQ ID NO:118).

[0618] To further evaluate the observed PE-ADA reactivity, a competition ELISA

[0619] 20 assay was conducted, as described below. The result is shown in Figure 45C-D and verify that the reactivity is directed to "h26D3-HC6_DS, VL-first" and could be largely blocked by competition with "h26D3-HC6_DS, VL-first" in solution.

[0620] 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

[0621] 25 Stockholm, Sweden).

[0622] Screening of PE-ADA response by direct ELISA: 96-well half-area ELISA plates (Corning. #3690) were coated with 50 pl per well of 3.4 nM scFv variant in PBS for 2 h at RT with shaking at 600-900 rpm. Two wells per plate were instead coated with anti-human IgG F(ab')2 (Jacksonlmmuno; #109-005-097) to serve as positive control wells. For blocking, plates were emptied and tapped dry on paper towels prior to addition of 150 pl per well of blocking solution (1% BSA, 0.01% Tween20 in PBS). Plates were incubated with blocking solution for at least 1 h at RT. shaking at 600- 900 rpm. Plates were then washed 4 times with 150 pl per well of ELISA wash buffer with an automated plate washer (Tecan). Plates were tapped dry on paper towels and 25 pl of LowCross buffer (Candor Bioscience; #100-050) were added to each well. Then, 25 pl of 1:20 diluted serum samples in LowCross Buffer were added in duplicates to reach a final serum dilution of 1:40. Rituximab diluted in LowCross buffer was added to positive control wells. Samples were incubated for 25 min at RT without shaking. Plates were washed 4 times as above prior to addition of 50 pl per well of HRP-F(ab')2 goat anti-human IgG Fey specific polyclonal antibody (Jacksonlmmuno #109-036-008) diluted 1:5000 in LowCross buffer for 1 h at RT without shaking. Plates were washed 4 times as above prior to addition of 50 pl per well of TMB (Neogen; #331177). The colorimetric signal was allowed to develop for 5 min prior to stopping with 50 pl per well of sulfuric acid (Honeywell; #35354-lL). Plates were read immediately after addition of sulfuric acid on a plate reader at 450nM (Tecan).

[0623] Confirmatory competition ELISA assay: The competition ELISA was performed analogous to the direct ELISA screening assay described above, with the following adaptation: during blocking of plates, serum diluted 1:20 in LowCross buffer was either mixed with an equal volume of LowCross buffer or with an equal volume of 3400 nM "h26D3-HC6_DS, VL-first" scFv, resulting in a final serum dilution of 1:40 and 500x excess scFv compared to coating. The samples were pre-incubated in 96- well PP plate for 1 h, with shaking at 900 rpm. After blocking and washing, duplicates of either serum only or serum with soluble scFv were added to the plate and incubated for 25 min at RT without shaking. From here, the protocol for screening was followed.

[0624] Calculation of assay cut-off points: In order to determine the assay cut-off point, technical outliers with a %CV larger than 20 were first removed. For this, the mean of the sample duplicates was calculated, followed by calculation of the standard deviation. %CV was calculated as %CV = STDEV / MEAN*100. The sample population minus technical outliers was then analyzed for statistical outliers by ROUT analysis in GraphPad Prism (Q=l%; allowing for 1% of false positive identified outliers). The sample population minus technical and statistical outliers made up the pseudo-negative population and was used to calculate the assay cut-off point. In case of high PE-ADA prevalence, the cut-off point was based on the competition assay: cut-off point = mean (of pseudo negative population) + 2.33 x STDEV (of pseudo negative population), allowing for a 1% false positive rate.

[0625] In case of low PE-ADA prevalence, the cut-off point can be directly calculated from the screening assay: cut-off point = mean (screening population with outliers removed) + 1.645 x STDEV (of screening population with outliers removed), allowing for a 5% false positive rate.

[0626] ITEMIZED LISTING OF EMBODIMENTS

[0627] 1. A human transferrin receptor 1 (hTfRl) binding molecule, which

[0628] - is 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,

[0629] - comprises 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, which antigen-binding surface provides the binding molecule with the capacity to bind selectively to said epitope; and

[0630] - comprises from two to four histidine residues in said VH / VL pair, and wherein the affinity of the binding molecule for hTfRl is higher at pH 7.4 than at pH 5.5.

[0631] 2. Binding molecule according to item 1, wherein said VH region 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 is from two to four.

[0632] 3. Binding molecule according to item 1 or 2, wherein said VL region 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 is from two to four.

[0633] 4. Binding molecule according to any preceding item, wherein said VH region comprises two histidine residues and said VL region comprises zero histidine residues. 5. Binding molecule according to any one of items 1-3, wherein said VH region comprises three histidine residues and said VL region comprises zero histidine residues.

[0634] 6. Binding molecule according to any one of items 1-3, wherein said VH region comprises two histidine residues and said VL region comprises one histidine residue.

[0635] 7. Binding molecule according to any preceding item, said epitope comprising or consisting of the amino acid residues 150, 151, 154, 158, 159, 161, 163 and 385 in SEQ ID NO:85.

[0636] 8. Binding molecule according to any preceding item, in which said antigen-binding surface is composed of three complementarity-determining regions (CDRs) from said VH region and three CDRs from said VL region, and wherein said CDRs comprise the following:

[0637] VHCDR1: X1X2NMX3 (SEQ ID NO:1), wherein

[0638] XI is selected from D and A;

[0639] X2 is selected from Y and A; and

[0640] X3 is selected from D and A;

[0641] 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; X9 is selected from N and S; and X10 is selected from E and Q;

[0642] VLCDR1: KSSQSLLX11SX12NX13KNX14LA (SEQ ID NO:4), wherein Xll is selected from Y and A;

[0643] X12 is selected from T and S;

[0644] 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; and

[0645] VLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:6) wherein X16 is selected from

[0646] X17 is selected from

[0647] X18 is selected from

[0648] X19 is selected from X20 is selected from

[0649] 9. Binding molecule according to item 8, said antigen-binding surface further comprising

[0650] VHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:3) wherein X21 is selected from Y and A;

[0651] X22 is selected from Y and A;

[0652] X23 is selected from Y and A;

[0653] X24 is selected from D and A; and X25 is selected from Y and A.

[0654] 10. Binding molecule according to any one of items 8-9, in which said VHCDR2 is:

[0655] VHCDR2: X4INPX5X6X7TTSX8NEKFKG (SEQ ID NO:7), wherein

[0656] X4 is selected from D and A;

[0657] X5 is selected from D and A;

[0658] X6 is selected from Y and A;

[0659] X7 is selected from D and A; and X8 is selected from Y and A.

[0660] 11. Binding molecule according to any one of items 8-10, in which said VLCDR1 is:

[0661] VLCDR1: KSSQSLLX11STNQKNX14LA (SEQ ID NO:8), wherein Xll is selected from Y and A; and X14 is selected from Y and A.

[0662] 12. Binding molecule according to any one of items 8-11, in which said VLCDR3 is:

[0663] VLCDR3: QQX16FIX19PRT (SEQ ID NO:9) wherein X16 is selected from Y and A; and

[0664] X19 is selected from Y and A.

[0665] 13. Binding molecule according to any one of items 8-12, in which the amino acid sequence of said VHCDR1 is selected from the group consisting of SEQ ID NO:10 and 16-18.

[0666] 14. Binding molecule according to any one of items 8-13, in which the amino acid sequence of said VHCDR2 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.

[0667] 15. Binding molecule according to any one of items 8, 10-14, in which the amino acid sequence of said VHCDR3 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.

[0668] 16. Binding molecule according to any one of items 8-15, in which the amino acid sequence of said VLCDR1 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

[0669] 30.

[0670] 17. Binding molecule according to any one of items 8-16, in which the amino acid sequence of said VLCDR2 is selected from the group consisting of SEQ ID NO:14 and

[0671] 31.

[0672] 18. Binding molecule according to any one of items 8-17, in which the amino acid sequence of said VLCDR3 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.

[0673] 19. Binding molecule according to any one of items 8-18, in which the amino acid sequences of the six CDRs are the following: VHCDR1: DYNMD (SEQ ID NQ:10),

[0674] VHCDR2: DINPDYDTTSYNEKFKG (SEQ ID NO:11),

[0675] VHCDR3: GGYSGSSYYHPMDY (SEQ ID N0:12),

[0676] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID N0:13),

[0677] VLCDR2: WASTRES (SEQ ID N0:14), and

[0678] VLCDR3: QQYFIYPRT (SEQ ID N0:15).

[0679] 20. Binding molecule according to any one of items 8-18, in which the amino acid sequences of the six CDRs are the following:

[0680] VHCDR1: DYNMD (SEQ ID NQ:10),

[0681] VHCDR2: DINPDADTTSYNEKFKG (SEQ ID NO:21),

[0682] VHCDR3: GGYSGSSYYHPMDY (SEQ ID NO:12),

[0683] VLCDR1: KSSQSLLYSTNQKNYLA (SEQ ID NO:13),

[0684] VLCDR2: WASTRES (SEQ ID NO:14), and

[0685] VLCDR3: QQYFIYPRT (SEQ ID NO:15).

[0686] 21. Binding molecule according to any one of items 8, 10-18, in which the amino acid sequences of the six CDRs are the following:

[0687] VHCDR1: DYNMD (SEQ ID NQ:10),

[0688] VHCDR2: DINPNYDTTSYSQKFKG (SEQ ID NO:34),

[0689] VHCDR3: SEAGNYYWYFDV (SEQ ID NO:35),

[0690] VLCDR1: KSSQSLLYSSNRKNYLA (SEQ ID NO:36),

[0691] VLCDR2: WASTRES (SEQ ID NO:14), and

[0692] VLCDR3: QQYYNYPYT (SEQ ID NO:37).

[0693] 22. Binding molecule according to any preceding item, wherein said VH region comprises or consists of an amino acid sequence selected from

[0694] (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.

[0695] 23. Binding molecule according to any preceding item, wherein said VL region comprises or consists of an amino acid sequence selected from

[0696] (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 NO: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

[0697] (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.

[0698] 24. Binding molecule according to any one of items 22-23, wherein said VH region is as defined in item 22 and said VL region is as defined in item 23.

[0699] 25. Binding molecule according to item 24, in which said VH region 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 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.

[0700] 26. Binding molecule according to item 24, in which said VH region 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 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.

[0701] 27. Binding molecule according to item 25, in which said VH region 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 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.

[0702] 28. Binding molecule according to any one of items 25-26, in which said VH region 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 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.

[0703] 29. Binding molecule according to item 25, in which said VH region 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 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.

[0704] 30. Binding molecule according to item 29, in which said VH region comprises a sequence selected from SEQ ID NO:44, 50, 129, 135, 163, 195, 229, 263, 297, 329 and 442, and said VL region 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.

[0705] 31. Binding molecule according to item 30, wherein the VH region and the VL region 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.

[0706] 32. Binding molecule according to item 31, wherein the VH region and the VL region 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.

[0707] 33. Binding molecule according to item 32, in which said VH region comprises SEQ ID NO:229 and said VL region comprises SEQ ID NO:58.

[0708] 34. Binding molecule according to item 32, in which said VH region comprises SEQ ID NO:263 and said VL region comprises SEQ ID NO:58.

[0709] 35. Binding molecule according to item 32, in which said VH region comprises SEQ ID NO:297 and said VL region comprises SEQ ID NO:419.

[0710] 36. Binding molecule according to any one of items 1-21, which comprises one first cysteine residue in said VH region and one second cysteine residue in said VL region, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL regions.

[0711] 37. Binding molecule according to item 36, wherein said first cysteine residue is located at an amino acid position selected from VH position 39-49, such as selected from VH position 41-47, such as selected from VH position 43-45, such as at VH position 44, all as determined by reference to the Kabat numbering scheme. 38. Binding molecule according to item 36 or 37, wherein said second cysteine residue is located at an amino acid position selected from VL position 95-105, such as selected from VL position 97-103, such as selected from VL position 99-101, such as at VL position 100, all as determined by reference to the Kabat numbering scheme.

[0712] 39. Binding molecule according to any one of items 36-38, wherein said first cysteine residue is located at VH position 44 and said second cysteine residue is located at VL position 100, as determined by reference to the Kabat numbering scheme.

[0713] 40. Binding molecule according to any one of items 36-39, wherein said VH region comprises or consists of an amino acid sequence selected from

[0714] (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

[0715] (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.

[0716] 41. Binding molecule according to any one of items 36-40, wherein said VL region comprises or consists of an amino acid sequence selected from

[0717] (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

[0718] (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.

[0719] 42. Binding molecule according to any one of items 40-41, wherein said VH region is as defined in item 40 and said VL region is as defined in item 41.

[0720] 43. Binding molecule according to item 42, in which said VH region 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 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.

[0721] 44. Binding molecule according to item 42, in which said VH region 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 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.

[0722] 45. Binding molecule according to item 42, in which said VH region 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 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. 46. Binding molecule according to any one of items 43-44, in which said VH region 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 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.

[0723] 47. Binding molecule according to item 43, in which said VH region 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 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.

[0724] 48. Binding molecule according to item 47, in which said VH region comprises a sequence selected from SEQ ID NO:88, 94, 146, 179, 212, 246, 280, 313, 345 and 443, and said VL region 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.

[0725] 49. Binding molecule according to item 48, wherein the VH region and the VL region 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.

[0726] 50. Binding molecule according to item 49, wherein the VH region and the VL region 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 NO:105; b) a VH region comprising SEQ ID NO: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.

[0727] 51. Binding molecule according to item 50, in which said VH region comprises SEQ ID NO:246 and said VL region comprises SEQ ID NQ:105.

[0728] 52. Binding molecule according to item 50, in which said VH region comprises SEQ ID NQ:280 and said VL region comprises SEQ ID NQ:105.

[0729] 53. Binding molecule according to item 50, in which said VH region comprises SEQ ID NO:313 and said VL region comprises SEQ ID NO:428.

[0730] 54. Binding molecule according to any preceding item, wherein 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, said first KD value being lower than said second KD value, and said first and second KD values being determined under conditions that are identical except for the pH value, using bio-layer interferometry.

[0731] 55. Binding molecule according to item 54, wherein 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.

[0732] 56. Binding molecule according to any one of items 54-55, wherein said first KD value is no more than 1 x 10-6M, such as no more than 1 x 10-7M, such as no more than l x IO’8M.

[0733] 57. Binding molecule according to any preceding item, in which said VH / VL pair forms part of an antibody construct. 58. Binding molecule according to item 57, wherein said antibody construct has more than one binding specificity.

[0734] 59. Binding molecule according to item 58, wherein said antibody construct is bispecific.

[0735] 60. Binding molecule according to item 59, wherein the VH / VL pair as defined in any preceding item 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.

[0736] 61. Binding molecule according to item 60, wherein said antibody fragment is an scFv.

[0737] 62. Binding molecule according to any one of items 58-61, said antibody construct further comprising an antibody or antigen binding fragment thereof capable of selective binding to a target present in the brain of a mammal.

[0738] 63. Binding molecule according to item 62, wherein said target is selected from the group consisting of amyloid-p peptide or derivatives or fragments thereof, alpha- synuclein or derivatives or fragments thereof, TAR DNA-binding protein 43 (TDP-43) or derivatives or fragments thereof, triggering receptor expressed on myeloid cells 2 (TREM2), beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, Tau, phosphorylated Tau or fragments thereof, apolipoprotein E4, CD20, prion protein, leucine rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, amyloid-p precursor protein, p75 neurotrophin receptor, neuregulin and caspase 6.

[0739] 64. Binding molecule according to item 63, wherein said target is selected from the group consisting of amyloid-p peptide or derivatives or fragments thereof, alpha- synuclein or derivatives or fragments thereof, TAR DNA-binding protein 43 (TDP-43) or derivatives or fragments thereof, triggering receptor expressed on myeloid cells 2 (TREM2), Tau, phosphorylated Tau or fragments thereof and apolipoprotein E4.

[0740] 65. Binding molecule according to item 64, wherein said target is selected from the group consisting of amyloid-p peptide or derivatives or fragments thereof, alpha- synuclein or derivatives or fragments thereof and TAR DNA-binding protein 43 (TDP- 43) or derivatives or fragments thereof.

[0741] 66. Binding molecule according to any one of items 62-65, wherein said antibody or antigen binding fragment thereof capable of selective binding to a target present in the brain of a mammal is an a nti-AP antibody, for example an antibody selected from the group consisting of lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06 and KHK6640.

[0742] 67. Binding molecule according to any one of items 62-65, wherein said antibody or antigen binding fragment thereof capable of selective binding to a target present in the brain of a mammal is an anti-alpha-synuclein antibody, for example an antibody selected from the group consisting of prasinezumab, UCB7853, Lu AF82422, TAK-341 and exidavnemab.

[0743] 68. Pharmaceutical composition, comprising a binding molecule according to any preceding item and a pharmaceutically acceptable carrier or excipient.

[0744] 69. A binding molecule according to any one of items 1-67 or a pharmaceutical composition according to item 68 for use in treatment, such as for use in therapeutic treatment and / or for use in prophylactic treatment.

[0745] 70. A binding molecule according to any one of items 1-67 or a pharmaceutical composition according to item 68 for use in diagnosis in vivo and / or prognosis in vivo.

[0746] 71. Binding molecule or pharmaceutical composition for use according to any one of items 69-70, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a neurodegenerative disorder, for example a disorder selected from Alzheimer's disease and other disorders associated with A|3 protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), the Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy.

[0747] 72. Binding molecule or pharmaceutical composition for use according to item 71, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a disorder selected from Alzheimer's disease and other disorders associated with A|3 protein aggregation, Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, Parkinson's disease (PD), Parkinson's disease dementia (PDD) and the Lewy body variant of Alzheimer's disease.

[0748] 73. Binding molecule or pharmaceutical composition for use according to item 72, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a disorder selected from Alzheimer's disease and other disorders associated with A|3 protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD).

[0749] 74. Binding molecule or pharmaceutical composition for use according to item 73, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to Alzheimer's disease.

[0750] 75. Binding molecule or pharmaceutical composition for use according to any one of items 69-70, wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a disorder selected from brain cancer, multiple sclerosis and lysosomal storage diseases. 76. A method of therapeutic and / or prophylactic treatment of a mammal having, or being at risk of developing, a disorder, said method comprising administering to said mammal a therapeutically effective amount of a binding molecule according to any one of items 1-67 or a pharmaceutical composition according to item 68.

[0751] 77. A method according to item 76, wherein said disorder is a neurodegenerative disorder, for example a neurodegenerative disorder as defined in any one of items 71-73. 78. A method according to item 76, wherein said disorder is as defined in item 74.

Claims

IllCLAIMS1. A human transferrin receptor 1 (hTfRl) binding molecule, which- is 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,- comprises 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, which antigen-binding surface provides the binding molecule with the capacity to bind selectively to said epitope; and- comprises from two to four histidine residues in said VH / VL pair, and wherein the affinity of the binding molecule for hTfRl is higher at pH 7.4 than at pH 5.5.

2. Binding molecule according to claim 1, wherein said VH region comprises two histidine residues and said VL region comprises one histidine residue.

3. Binding molecule according to any preceding claim, in which said antigen-binding surface is composed of three complementarity-determining regions (CDRs) from said VH region and three CDRs from said VL region, and wherein 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; andX14 is selected from Y and A;VLCDR2: X15ASTRES (SEQ ID NO:5) whereinX15 is selected from W and A; andVLCDR3: QQX16X17X18X19PX20T (SEQ ID NO:6) whereinX16 is selected fromX17 is selected fromX18 is selected fromX19 is selected fromX20 is selected fromoptionally further comprisingVHCDR3: GGX21SGSSX22X23HPMX24X25 (SEQ ID NO:3) whereinX21 is selected from Y and A;X22 is selected from Y and A;X23 is selected from Y and A;X24 is selected from D and A; and X25 is selected from Y and A.

4. Binding molecule according to any preceding claim, wherein said VH region 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.

5. Binding molecule according to any preceding claim, wherein said VL region 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 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(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.

6. Binding molecule according to any one of claims 4-5, wherein said VH region is as defined in claim 4 and said VL region is as defined in claim 5.

7. Binding molecule according to claim 6, wherein the VH region and the VL region 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.

8. Binding molecule according to any one of claims 1-3, which comprises one first cysteine residue in said VH region and one second cysteine residue in said VL region, said first and second cysteine residues being arranged such that they form a disulfide bridge connecting the VH and VL regions, optionally wherein said first cysteine residue is located at VH position 44 and said second cysteine residue is located at VL position 100, as determined by reference to the Kabat numbering scheme.

9. Binding molecule according to claim 8, wherein said VH region comprises or consists of an amino acid sequence selected from(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.

10. Binding molecule according to any one of claims 8-9, wherein said VL region comprises or consists of an amino acid sequence selected from(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, 410-418and 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(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.

11. Binding molecule according to any one of claims 9-10, wherein said VH region is as defined in claim 9 and said VL region is as defined in claim 10.

12. Binding molecule according to claim 11, wherein the VH region and the VL region 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.

13. Binding molecule according to any preceding claim, wherein 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, said first KD value being lower than said second KD value, and said first and second KD values being determined under conditions that are identical except for the pH value, using bio-layer interferometry;optionally wherein 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; optionally wherein said first KD value is no more than 1 x 10-6M, such as no more than 1 x 10-7M, such as no more than 1 x IO-8M.

14. Binding molecule according to any preceding claim, in which said VH / VL pair forms part of an antibody construct, for example a bispecific antibody construct.

15. Binding molecule according to claim 14, wherein the VH / VL pair as defined in any preceding claim 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, for example wherein the VH / VL pair is present in an scFv.

16. Binding molecule according to any one of claims 14-15, said antibody construct further comprising an antibody or antigen binding fragment thereof capable of selective binding to a target present in the brain of a mammal, for example wherein said target is selected from the group consisting of amyloid-p peptide or derivatives or fragments thereof, alpha-synuclein or derivatives or fragments thereof, TAR DNA- binding protein 43 (TDP-43) or derivatives or fragments thereof, triggering receptor expressed on myeloid cells 2 (TREM2), beta-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, Tau, phosphorylated Tau or fragments thereof, apolipoprotein E4, CD20, prion protein, leucine rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, amyloid-p precursor protein, p75 neurotrophin receptor, neuregulin and caspase 6.

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

18. A binding molecule according to any one of claims 1-16 or a composition according to claim 17 for use in treatment, such as for use in therapeutic treatmentand / or for use in prophylactic treatment, or for use in diagnosis in vivo and / or prognosis in vivo, for example wherein the therapy, prophylaxis, in vivo diagnosis and / or in vivo prognosis is with respect to a neurodegenerative disorder, for example a disorder selected from Alzheimer's disease and other disorders associated with A|3 protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), the Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, and familial amyloid neuropathy.

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