Bispecific binding molecule

Abstract

Provided is a bispecific binding molecule comprising one single chain component, which is a polypeptide chain comprising two identical antibody light chains and two identical antibody heavy chains, linked to a single chain binding module having affinity for a target that mediates transport of the bispecific binding molecule across the blood-brain barrier (BBB). Also provided are therapeutic, prophylactic, prognostic and diagnostic uses of the bispecific binding molecule.

Description

[Technical field]

[0001] The present disclosure relates to a bispecific binding molecule comprising two identical antibody heavy chains and one single chain component, characterized in that the single chain component is a polypeptide chain comprising two identical antibody light chains linked to a single chain binding module having affinity for a target that mediates transport of the bispecific binding molecule across the blood-brain barrier (BBB), wherein the antibody heavy chain and the antibody light chain are derived from a monoclonal antibody having affinity for a target present in the mammalian brain.

[0002] The present disclosure also relates to therapeutic, prophylactic, prognostic and diagnostic uses of the bispecific binding molecules. [Background technology]

[0003] Treatment modalities for brain and neurological diseases are severely limited due to the impermeability of cerebral blood vessels to most substances carried in the bloodstream (Freskgard and Urich (2017), Neuropharmacology, 120:38-55; Stanimirovic et al. (2018), BioDrugs, 32:547-559). The brain's microvasculature (capillaries), collectively referred to as the blood-brain barrier (BBB), is unique compared to the blood vessels present around the body. The tight apposition of endothelial cells (ECs) of the BBB to neural cells such as astrocytes, pericytes, and neurons gives rise to phenotypic characteristics that contribute to the observed impermeability. Tight junctions between ECs in the BBB limit paracellular transport, while the lack of passive pinocytic vesicles and round window pits limits nonspecific intercellular transport. These factors work together to limit molecular flux from blood to brain to molecules that are generally less than 500 Da in size and lipophilic. Thus, unless a drug with the desired pharmacological properties happens to have a size and lipophilic properties that allow it to cross the BBB, it is best to rely on the large mass transport surface area of ​​the bloodstream (600 km of capillaries to 20 m in the human brain) as a transport medium. 2This limitation makes the promising possibility of using nanoparticles with a surface area of ​​1000 nm or greater almost impossible to realize. It is estimated that more than 98% of all small molecule drugs, and nearly 100% of the emerging classes of protein and gene therapeutics, do not cross the BBB due to these limitations.

[0004] International Publication WO91 / 03259 proposes a principle for transporting neurological drugs through the BBB, which includes conjugating the drug to an antibody that is reactive with the transferrin receptor.According to this disclosure, the binding of the conjugate to the transferrin receptor results in the active transport of the conjugate through the BBB.Subsequent studies have further developed this basic concept, proposing, for example, other receptors that may be useful for BBB transport as an alternative to the transferrin receptor.

[0005] International Publication WO2012 / 075037 discloses bispecific antibodies with one Fab portion specific for a BBB receptor and mediating transport and one Fab portion specific for a therapeutic target in the brain. Figure 3A of WO2012 / 075037 shows a representative embodiment of this teaching.

[0006] International Publication WO2014 / 033074 discloses a bispecific binding molecule comprising a standard monospecific and bivalent antibody directed to a therapeutic target in the brain and a binding domain specific for a BBB receptor. The binding domain is attached to the C-terminus of one of the two heavy chains of the antibody. Figure 1B of WO2014 / 033074 shows a representative embodiment of this teaching. It is further disclosed that monovalent binding to the BBB receptor results in more efficient BBB transport than bivalent binding. The biopharmaceutical candidate described in Romanian Patent RO7126209 from Roche appears to use the design described in International Publication WO2014 / 033074. Romanian Patent RO7126209 is in clinical trials registered on clinicaltrials.gov. (the joint NIH and FDA clinical trial registry) with trial identification numbers NCT04023994 and NCT04639050.

[0007] International Publication WO2018 / 011353 discloses another alternative construct in which a standard monospecific and bivalent antibody directed against a therapeutic target in the brain is equipped with multiple BBB receptor binding elements, which are nevertheless arranged in such a way as to allow the desired monovalent binding to the BBB receptor. Figure 1A of WO2018 / 011353 shows a representative embodiment of this teaching.

[0008] Despite the existence of a variety of experimental formats for providing antibodies and antibody-derived biopharmaceuticals with the ability to cross the BBB via active transport mediated by the transferrin receptor and other receptors, all of these have one or more drawbacks and there remains a need for novel biopharmaceuticals capable of crossing the BBB for further development of highly functional therapeutic, preventive, diagnostic and prognostic tools for detecting and treating diseases of the brain and central nervous system. Summary of the Invention

[0009] Description of the invention It is an object of the present invention to provide a designed format that allows antibody-based therapeutics to reach the brain.

[0010] Another object of the present invention is to provide a format for bispecific binding molecules that have affinity for targets in the brain and for BBB transport mediators.

[0011] Another object of the present invention is to provide a symmetric format that is easy to express and assemble while at the same time providing monovalent binding to BBB transport mediators.

[0012] Another object of the present invention is to provide a symmetric format that prevents the formation of any products that may lead to bivalent interactions with BBB transport mediators.

[0013] Another object of the present invention is to generate ready-to-use symmetric bispecific binding molecules from only two different contiguous polypeptide chains.

[0014] The various disclosed embodiments fulfill one or more of these objectives, as well as others that will be apparent to those of skill in the art from reading this disclosure in its entirety.

[0015] Thus, in a first aspect, the present disclosure provides a method for the preparation of a polypeptide comprising the following three polypeptide chains: (A) Two identical antibody heavy chains (HC) derived from a monoclonal antibody having affinity for a first target present in the mammalian brain; (B) The following five elements in a continuous polypeptide chain: i) two identical antibody light chains (LC) derived from said monoclonal antibody having affinity for said first target; ii) one single-chain binding module (scBM) having affinity for a second target that mediates transport of the bispecific binding molecule across the blood-brain barrier; and iii) two amino acid linkers L1 and L2 and one single-stranded component comprising A bispecific binding molecule comprising: The light chain (LC) and the single chain binding module (scBM) are separated by the linkers L1 and L2, which provide from N-terminus to C-terminus: [LC-L1-scBM-L2-LC], [LC-L1-LC-L2-scBM] and [scMB-L1-LC-L2-LC] The present invention provides a bispecific binding molecule comprising a sequence selected from the group consisting of:

[0016] A bispecific binding molecule as defined above will be formed through the association of each light chain LC element with each heavy chain HC in said single chain components such that said bispecific binding molecule adopts a standard antibody structure substantially reproducing a monoclonal antibody having affinity for a first target, where a single chain binding module scBM is coupled to said bispecific binding molecule via said linkers L1 and L2, and said heavy chain (HC) and light chain (LC) are derived from said monoclonal antibody (see Figures 1 and 2 for schematics of two different embodiments of bispecific binding molecules). Effect of the Invention

[0017] Without wishing to be bound by theory, by way of non-limiting example, the bispecific binding molecules of the present disclosure offer the following advantages over existing formats for transporting therapeutic antibodies across the BBB:

[0018] The constructs disclosed in International Publications WO2012 / 075037 and WO2014 / 033074 are both asymmetric constructs, requiring the expression of different heavy chain elements that need to be assembled, for example, using knob-into-hole technology. This can lead to inefficient production, potential chain imbalance issues, and the concomitant generation of unwanted by-products (e.g., the generation of homodimeric knob-knob and hole-hole by-products; see Kuglstatter et al. (2017), Protein. Eng. Des. Sel. 30:649-656). For any antibody heterodimerization technology, a low content or even the absence of homodimeric by-product impurities is desirable. This is the case when the unwanted homodimers have similar biophysical properties as the desired product, making them difficult to separate using purification steps at production scale. The situation is also applicable when the undesired homodimers retain biological activity, such as affinity for BBB transport mediators. This is particularly important when homodimer by-products have the potential to induce undesired biological activity, such as activity leading to dimerization or multimerization of BBB transport mediators, thus reducing the function of the molecule. On the other hand, the bispecific binding molecules of the present disclosure are composed of only one type of heavy chain, and in this sense are symmetrical constructs.

[0019] The constructs disclosed in International Publication WO2018 / 011353 do not have the drawbacks associated with heavy chain asymmetry, but instead contain two separate BBB binding components and may not be able to provide the necessary monovalent binding to BBB transport mediators. In WO2018 / 011353, the inventors disclose that bivalent constructs still confer monovalent binding in certain circumstances, but there remains uncertainty regarding the general applicability of the principles in WO2018 / 011353. The bispecific binding molecules of the present disclosure remove this uncertainty and achieve true monovalent binding to mediators of BBB transport. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Overall architecture of the single-chain components According to the present disclosure, the single-chain component comprises five elements in one continuous polypeptide chain. One of the five elements is a single-chain binding module (scBM) that has affinity for a target that mediates transport across the BBB. Two of the five elements are two identical antibody light chains (LC) that bind to two identical heavy chains (HC) to form the antibody portion of the bispecific binding molecule. As will be understood by those skilled in the art, the scBM and LC elements must be separated from each other in the single-chain component so that each of them can serve the purpose of either associating with a heavy chain (HC) (in the case of light chain elements) or providing a bispecific binding molecule with binding affinity for a BBB target (in the case of scBM). For this purpose, these three elements are separated by the remaining two elements in the single-chain component, namely two amino acid linkers L1 and L2. In this regard, it is contemplated that any order of each element is possible within the single-chain component, as long as the linker separates the three binding elements. In other words, the three possible sequences in the single chain component of the bispecific binding molecule are, from N-terminus to C-terminus, the following: [LC-L1-scBM-L2-LC], [LC-L1-LC-L2-scBM], and [scBM-L1-LC-L2-LC].

[0021] In one embodiment, the sequence of the element is selected from [LC-L1-scBM-L2-LC] and [LC-L1-LC-L2-scBM]. In a particular embodiment, the sequence of the element is [LC-L1-scBM-L2-LC]. In another particular embodiment, the sequence of the element is [LC-L1-LC-L2-scBM].

[0022] Antibody heavy and light chains In the bispecific binding molecules of the present disclosure, the antibody heavy and light chain elements, i.e., the HC and LC, respectively, are derived from a monoclonal antibody that has affinity for a first target present in the mammalian brain. As used in this particular context, "derived from" means that the amino acid sequence of each of the HC and LC is essentially unchanged compared to the sequence of the "parent" monoclonal antibody from which the HC and LC are derived. That is, the bispecific binding molecules of the present disclosure incorporate the heavy and light chains of a monoclonal antibody as the HC and LC, respectively.

[0023] In the bispecific binding molecules of the present disclosure, two antibody heavy chain (HC) elements and two light chain (LC) elements in a single chain component are combined to form a classical antibody structure, to which the scBM is attached via the L1 and L2 linkers. Thus, the bispecific binding molecules contain two VH-VL pairs from each pair of HC and LC, and have the ability to bind to a first target via the complementarity determining regions (CDRs) in the same or essentially the same way as the (parent) monoclonal antibody from which they are derived.

[0024] Through its linked HC and LC elements, the bispecific binding molecule of the present disclosure has affinity for a first target present in the mammalian brain. This target is typically associated with a disease in such a manner that it binds, blocks, activates or otherwise interacts with the target in the context of treating or preventing such disease. However, the particular advantages of the bispecific binding molecule of the present disclosure are primarily derived from the improved ability of the target binding activity to reach a targeted destination in the brain, and not from the fact that it binds to any particular first target. Thus, the particular nature of the first target is not limited and can be any target in the brain that may be important for interacting with that target.

[0025] Nevertheless, by way of example, the first target may in certain embodiments be selected from the group consisting of amyloid beta peptide or a derivative or fragment thereof, alpha-synuclein or a derivative or fragment thereof, TAR DNA-binding protein 43 (TDP-43) or a derivative or fragment 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 a fragment thereof, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, amyloid beta precursor protein, p75 neurotrophin receptor, neuregulin and caspase 6.

[0026] In more specific embodiments, the first target is selected from the group consisting of amyloid beta peptide or a derivative or fragment thereof, alpha-synuclein or a derivative or fragment thereof, TAR DNA binding protein 43 (TDP-43) or a derivative or fragment thereof, triggering receptor on myeloid cells 2 (TREM2), tau, phosphorylated tau or a fragment thereof, and apolipoprotein E4.

[0027] In particularly particular embodiments, the first target is selected from the group consisting of amyloid beta peptide or a derivative or fragment thereof, alpha-synuclein or a derivative or fragment thereof, and TAR DNA binding protein 43 (TDP-43) or a derivative or fragment thereof.

[0028] Any monoclonal antibody that binds to any one or more of the above-listed targets present in the mammalian brain is believed to be useful as a source of HC and LC elements in the bispecific binding molecules of the present disclosure. Those skilled in the art of biopharmaceutical research are familiar with the many monoclonal antibodies that have affinity for such targets.

[0029] In one embodiment, the (parent) monoclonal antibody from which the HC and LC elements are derived is an antibody of the IgG class. In a more specific embodiment, the antibody or antigen-binding fragment thereof is of a subclass selected from IgG1, IgG2 and IgG4, e.g. selected from IgG1 and IgG4. The desired subclass used depends, for example, on the required function of the monoclonal antibody. In a more specific embodiment, the antibody is of the subclass IgG1. IgG1 antibodies are particularly preferred, for example when effective antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) are desired.

[0030] In one embodiment, the (parent) monoclonal antibody from which the HC and LC elements are derived is selected from the group consisting of human antibodies, humanized antibodies, and antibodies that have been mutated to reduce effector functions, to increase plasma half-life, or to reduce their antigenicity in humans.

[0031] Non-limiting examples of specific monoclonal antibodies from which the HC and LC elements are derived are known antibodies directed against various forms of amyloid beta, such as those selected from the group consisting of lecanemab, gentenerumab, aducanumab, donanemab, PBD-C06 and KHK6640. Another example of a monoclonal antibody from which the HC and LC elements are derived is an antibody directed against α-synuclein, such as ABBV0805.

[0032] In the following examples, the bispecific binding molecule concepts of the present disclosure were tested and found to function in the intended manner. Specifically, Examples 10, 14 and 15 show various bispecific binding molecules as defined herein in in vivo mouse studies. To function as suitable proof-of-concept constructs in mice, the bispecific binding molecules tested in the examples have antibody heavy and light chain variable domains derived from a mouse monoclonal antibody (mAb158) with affinity for amyloid-β protofibrils, rather than from any of the human, humanized or chimeric antibodies contemplated for human use as described above. This mouse antibody mAb158 is the mouse precursor of the humanized monoclonal antibody lecanemab (also known as BAN2401; see International Publication WO 2007 / 108756).

[0033] Single-stranded binding module In one embodiment, the single-chain binding module (scBM) element of the single-chain component is derived from an antibody. The scBM can be selected, for example, from the group consisting of known single-chain formats, such as scFv, scFab, VHH and VNAR. In one embodiment, the scBM is selected from the group consisting of scFv and scFab. In a more specific embodiment, the scBM is scFv. In another specific embodiment, the scBM is scFab.

[0034] In another embodiment, the scBM element of the single chain component is not derived from an antibody. In this embodiment, the scBM is, for example, selected from the group consisting of known non-antibody scaffolds, such as monobodies (Adnectin® molecules), Protein Z variants (Affibody® molecules), lipocalins (Anticalin® proteins), bicyclic peptides, ankyrin repeat proteins (DARPin® molecules), finomers, and Kunitz domains.

[0035] The single-chain binding module (scBM) provided in the single-chain component of the bispecific binding molecule of the present disclosure has affinity for a second target, i.e., a second target that mediates transport of the bispecific binding molecule across the BBB when administered to a subject. This second target can be, for example, a receptor or other ligand found on the surface of endothelial cells of the BBB. One of skill in the art will appreciate the many different targets that have been tested for the purposes of BBB transport, so-called "brain shuttling," and will be able to select an appropriate single-chain binding module based on its affinity for such a target.

[0036] In one embodiment, the second target is selected from the group consisting of transferrin receptor 1 (TfR1), insulin receptor (InsR), insulin-like growth factor 1 receptor (IGF-1R), low density lipoprotein receptor-related protein 8 (Lrp8), low density lipoprotein receptor-related protein 1 (Lrp1), CD98, transmembrane protein 50A (TMEM50A), glucose transporter 1 (Glutl), basigin (BSG), and heparin-binding epidermal growth factor-like growth factor.

[0037] In a more specific embodiment, said second target is selected from the group consisting of transferrin receptor 1 (TfR1), insulin receptor (InsR), insulin-like growth factor 1 receptor (IGF-1R) and low-density lipoprotein receptor-related protein 8 (Lrp8).

[0038] In an even more specific embodiment, said second target is transferrin receptor 1 (TfR1).

[0039] In the proof-of-concept studies reported in the Examples below, the bispecific binding molecule tested uses as the scBM an scFv domain with affinity for mouse transferrin receptor 1 (mTfR1). The scFv domain is constructed from the VH and VL domains of the monoclonal mouse anti-mTfR1 antibody 8D3 (Kissel et al. (1998) Histochem. Cell. Biol. 110:63-72).

[0040] Linkers L1 and L2 With regard to the design of linkers L1 and L2, those skilled in the art understand that the construction of fusion proteins, such as the single-chain components of bispecific binding molecules, often involves the use of linkers between the functional components to be fused, and that there can be various types of linkers with different properties, such as flexible amino acid linkers, rigid amino acid linkers, cleavable amino acid linkers, etc. As described above, the single-chain components in the bispecific binding molecules according to the present disclosure include two linkers L1 and L2. In one embodiment, one or both of L1 and L2 are appropriately selected from flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid linkers. In one embodiment, at least one of L1 and L2 is a flexible amino acid linker. In another embodiment, both L1 and L2 are flexible amino acid linkers. As known by those skilled in the art, flexible linkers are frequently used when the domains or elements to be linked require some degree of movement or interaction, and can be particularly useful in some embodiments of the bispecific binding molecules of the present disclosure. Flexible linkers are usually composed of small amino acids, either non-polar (e.g., G or A) or polar (e.g., S or T). Some flexible linkers consist of stretches of primarily G and S residues, e.g., (GGGGS) pBy adjusting the copy number "p", it is possible to optimize the linker to achieve the appropriate separation between the functional components or to maintain the necessary interactions between the components. Apart from G and S linkers, other flexible linkers are known in the art, such as G and S linkers that contain additional amino acid residues such as T and A to maintain flexibility and / or polar amino acid residues to increase solubility.

[0041] In one embodiment of the bispecific binding molecule of the present disclosure, at least one of the linkers L1 and L2 is a flexible linker comprising glycine (G), serine (S), alanine (A) and / or threonine (T) residues. In another embodiment, both linkers L1 and L2 are such flexible linkers.

[0042] In one embodiment of the bispecific binding molecule of the present disclosure, at least one of the linkers L1 and L2 is n S m ) p and (S n G m ) p wherein, independently, n=1-7, m=0-7, n+m≦8 and p=1-10. In one embodiment, n=1-5. In one embodiment, m=0-5. In one embodiment, p=3-10. In a more particular embodiment, n=4, m=1 and p=1-4. In one embodiment, at least one of the linkers L1 and L2 is selected from (G4S)3 (SEQ ID NO: 1), (G4S)5 (SEQ ID NO: 2), (G4S)6 (SEQ ID NO: 3) and (G4S) 10 (SEQ ID NO: 4). In one particular embodiment, at least one of L1 and L2 is (G4S)3. In another embodiment, at least one of L1 and L2 is (G4S)5.

[0043] In one embodiment of the bispecific binding molecule of the present disclosure, at least one of the linkers L1 and L2 is a flexible linker comprising G, S, T and A residues. In one such embodiment, at least one of L1 and L2 has the amino acid sequence of SEQ ID NO: 5. In another embodiment, at least one of L1 and L2 has the amino acid sequence of SEQ ID NO: 6.

[0044] In one embodiment of the bispecific binding molecule of the present disclosure, L1 and L2 are the same. In another embodiment, L1 and L2 are different.

[0045] In one embodiment, L1 and L2 have the same length, i.e., the same number of amino acid residues. In another embodiment, L1 and L2 have different lengths. In such an embodiment, L1 can be longer than L2, or vice versa.

[0046] In one embodiment, L1 and / or L2 is 10 to 50 amino acid residues in length, such as 10 to 30 amino acid residues in length, such as 15 to 25 amino acid residues in length, or 10 to 20 amino acid residues in length.

[0047] Affinity for target As used herein, the terms "specific binding to X", "selective binding to X", "affinity for X" where X is a target (e.g., an antigen or epitope) refer to a property of a binding molecule, such as an antibody or antigen-binding fragment thereof, which can be tested, for example, by ELISA, by surface plasmon resonance (SPR), by Kinetic Exclusion Assay (KinExA®) or by Biolayer Interferometry (BLI). Those skilled in the art are familiar with these and other methods.

[0048] For example, the binding affinity to a target, antigen or epitope X can be tested in an experiment in which the binding molecule to be tested is captured on an ELISA plate coated with X or coated with a molecule containing epitope X, and then a biotinylated detection antibody is added, followed by the addition of streptavidin-conjugated horseradish peroxidase (HRP). Alternatively, the detection antibody can be directly conjugated to HRP. Tetramethylbenzidine (TMB) substrate is added, and the absorbance at 450 nm is measured using an ELISA multi-well plate reader. The skilled person can then interpret the results obtained by such an experiment to establish at least a qualitative measure of the binding affinity of the binding molecule to X. If a quantitative measurement is desired, ELISA can be used, for example, to determine the EC50 value (50% maximum effective concentration) for the interaction. The response of the binding molecule to a dilution series of X can be measured using the ELISA described above. The skilled person can then interpret the results obtained by such an experiment and calculate the EC50 value therefrom, for example, using GraphPad Prism v.9 and nonlinear regression.

[0049] As used herein, the term "EC50" refers to the 50% maximally effective concentration of a binding molecule that induces a response halfway between the baseline and maximum after a particular exposure time.

[0050] Furthermore, inhibition ELISA can be used to obtain a quantitative measure of the interaction by determining the "IC50" (50% maximal inhibitory concentration). In inhibition ELISA, the concentration of target X in a fluid sample is measured by detecting interference in the expected signal output. In theory, a known target or epitope-bearing substance is used to coat a multi-well plate. In parallel, a binding molecule with the expected affinity for the target is added and incubated with a solution containing the target at varying concentrations. Following standard blocking and washing steps, a sample containing a mixture of the binding molecule and the target is added to the well. A labeled detection antibody with affinity for the binding molecule is then applied for detection with a relevant substrate (e.g. TMB). In principle, if a high concentration of target is present in the fluid sample, a significant decrease in signal output is observed. In contrast, if only a small amount of target is present in the fluid sample, the decrease in the expected signal output will be very small. Those skilled in the art are aware that the signal output also depends on the affinity of the binding molecule for the target.

[0051] As used herein, the term "IC50" refers to the 50% maximum inhibitory concentration of a binding molecule that induces a response between baseline and maximum inhibition after a specific exposure time.As used herein, a lower IC50 value indicates that a lower concentration of target is required to inhibit the binding of detection antibody to the known target coated on the plate, compared to a higher IC50 value.Therefore, a lower IC50 value typically corresponds to a higher affinity.

[0052] The binding affinity of a binding molecule can also be tested by surface plasmon resonance (SPR). For example, affinity can be tested in an experiment in which a target or epitope X is immobilized on a sensor chip of an apparatus, and a sample containing the binding molecule to be tested is passed over the chip. Alternatively, the binding molecule to be tested can be immobilized on a sensor chip of an apparatus, and a sample containing X is passed over the chip. The skilled person can then interpret the results obtained by such an experiment to establish at least a qualitative measure of the binding affinity of the binding molecule to X. For example, the K D If a quantitative measure is desired, SPR may be used to determine the value. Binding titers can be determined, for example, using Biacore. TM (trademark) (Cytiva) or ProteOn TM The target or epitope is suitably immobilized on the sensor chip of the instrument, and a sample of the binding molecule for which affinity is to be determined is prepared by serial dilution and injected into the instrument. The results are then used to determine the affinity of the binding molecule, typically using a Biacore™ assay provided by the instrument manufacturer. TM The results are analyzed using a 1:1 Langmuir binding model in Insight™ Analysis Software 2.0 or other suitable software. D The value can be calculated.

[0053] Binding affinity can also be measured by Biolayer Interferometry (BLI), a label-free technique for measuring biomolecular interactions in the interactome field. It is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of proteins immobilized on a biosensor chip; and an internal standard layer. Binding between a ligand (target or epitope X) immobilized on the biosensor chip surface and an analyte in solution (e.g., a binding molecule with a putative affinity for X) leads to an increase in optical thickness at the biosensor chip, resulting in a wavelength shift Δλ. The wavelength shift Δλ is a direct measure of the change in thickness of the biological layer. The interaction is measured in real time, providing the ability to monitor binding specificity, association and dissociation rates, or concentration with accuracy and precision.

[0054] Those of skill in the art are familiar with these and other methods for measuring the affinity of a binding molecule for a target or epitope X, either qualitatively or quantitatively, or both.

[0055] Pharmaceutical Compositions In a second aspect, the disclosure provides a pharmaceutical composition comprising a bispecific binding molecule as described herein and at least one pharma- ceutically acceptable excipient or carrier.

[0056] 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.

[0057] Pharmaceutically acceptable excipients that can 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, phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, buffer substances such as salts or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0058] In certain embodiments, the pharmaceutical composition is formulated for administration to a subject via any suitable route of administration, including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, intranasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal administration. In preferred embodiments, the composition is formulated for intravenous or subcutaneous administration.

[0059] Methods of Prevention, Treatment, Diagnostics, Prognosis and Detection Bispecific binding molecules according to the present disclosure may be useful as therapeutic, prophylactic, diagnostic and / or prognostic agents.

[0060] Thus, in a further aspect of the disclosure there is provided a bispecific binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a medicament.

[0061] In yet another aspect of the disclosure there is provided a bispecific binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a diagnostic agent.

[0062] In yet another aspect of the disclosure there is provided a bispecific binding molecule according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a prognostic agent.

[0063] Also provided are methods for the prevention, treatment or diagnosis of a disease or for assessing the prognosis of a disease, comprising administering a bispecific binding molecule as disclosed herein to a subject, typically a human subject, in need thereof.

[0064] Also provided is the use of a bispecific binding molecule of the disclosure for the manufacture of a composition (e.g., a medicament) for use in the prevention, treatment, diagnosis and / or prognosis of any one of the listed diseases.

[0065] Thus, in one embodiment, the bispecific binding molecule, or a pharmaceutical composition comprising same, is useful in the treatment, prevention, diagnosis and / or prognosis of a neurodegenerative disease, e.g. a disease selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, traumatic brain injury (TBI), dementia with Lewy bodies (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's dementia (PDD), Lewy body degeneration of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creuferts-Jakob disease, Huntington's disease, and familial amyloidotic neuropathies.

[0066] In more specific embodiments, said disease is selected from Alzheimer's disease and Aβ protein aggregation, Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontal dementia, tauopathy, Parkinson's disease (PD), Parkinson's dementia (PDD) and other diseases associated with Lewy body degeneration of Alzheimer's disease.

[0067] In more specific embodiments, said disease is selected from Alzheimer's disease and Aβ protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), in particular other diseases related to Alzheimer's disease.

[0068] In alternative embodiments, the bispecific binding molecules, or pharmaceutical compositions comprising same, are useful in the treatment, prevention, diagnosis and / or prognosis of other diseases, e.g., diseases selected from brain tumors, multiple sclerosis and lysosomal storage diseases.

[0069] In another aspect, there is provided a method for the treatment, prevention, diagnosis and / or prognosis of a disease as listed above, comprising administering to said mammal an amount, such as a therapeutically effective amount, of the bispecific binding molecule or a pharmaceutical composition comprising same.

[0070] Incorporation by Reference Various publications are cited throughout this application, each of which is incorporated herein by reference in its entirety. [Brief description of the drawings]

[0071] [Figure 1] 1 is a schematic diagram of one alternative embodiment of a bispecific binding molecule of the present disclosure, in which each element of the single chain component is in the sequence [LC-L1-scBM-L2-LC]. Each heavy chain HC is composed of a VH, CH1, CH2 and CH3 immunoglobulin domain. The single chain component is composed of a first light chain LC (wherein said LC is composed of a VL and CL immunoglobulin domain); a first linker L1; a single chain binding module (scBM) in the form of an scFv; a second linker L2; and a second LC (identical to the first LC). [Diagram 2] 2 is a schematic diagram of another alternative embodiment of a bispecific binding molecule of the present disclosure, in which each element of the single chain component is in the sequence [LC-L1-LC-L2-scBM]. Each heavy chain HC is composed of a VH, CH1, CH2 and CH3 immunoglobulin domain. The single chain component is composed of a first light chain LC (wherein said LC is composed of a VL and CL immunoglobulin domain); a first linker L1; a second LC (identical to the first LC); a second linker L2; and a single chain binding module (scBM) in the form of an scFv. [Diagram 3]FIG. 3 shows the results of SDS-PAGE analysis after purification of the indicated expressed molecules. [Figure 4] FIG. 4 shows the binding of the indicated molecules to mTfR1 analyzed by indirect ELISA as described in Example 5. [Figure 5A] Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 5B] Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 5C] Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 5D]Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 5E] Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 5F] Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 5G] Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 5H]Figure 5 shows mTfR1 binding of the indicated molecules measured using Octet® Biolayer Interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.1; B) mAb158-scLc-8D3-Ig.2; C) mAb158-scLc-8D3-Ig.3; D) mAb158-scLc-8D3-Ig.4; E) mAb158-scLc-8D3-Ig.5; F) mAb158-scLc-Ig.6; G) 8D3Fab; and H) mAb158 IgG. [Figure 6] Figure 6 is a box plot showing binding of the indicated molecules to cEND cells analyzed using flow cytometry as described in Example 7. MFI (mean fluorescence intensity) represents the amount of test molecule bound to the cells. [Figure 7] FIG. 7 is a box plot showing internalization of bispecific binding molecules by cells, presented as the percentage of positive cells (cEND) in a cell population captured with a fluorescently labeled antibody as described in Example 7. [Figure 8] FIG. 8 shows binding of the indicated molecules to Aβ1-42 analyzed by indirect ELISA as described in Example 8. [Figure 9] FIG. 9 shows the in vitro plasma stability analyzed by Western blot as described in Example 9. [Figure 10] FIG. 10 is a series of box plots showing concentrations of 10 nmoles / kg mAb158 IgG, mAb158-scLc-8D3-Ig.1, or mAb158-scLc-8D3-Ig.3 in C57BL / 6J female mice 24 hours after intravenous (iv) administration. (A) Plasma, (B) TBS-Triton extracts of left hemisphere, both analyzed using MSD, and (C) brain-to-plasma concentration ratios for each construct are shown. Circles represent individual data points and bars represent mean ± SD. One-way ANOVA with Tukey's post hoc test was performed. ****P<0.0001. [Figure 11]FIG. 11 is a series of confocal images from immunohistochemical (IHC) analysis of the cerebral cortex 24 h after intravenous (iv) administration of 10 nmol / kg in C57BL / 6J wild-type (WT) female mice using mAb158 control IgG (left panel), mAb158-scLc-8D3-Ig.1 (middle panel), and mAb158-scLc-8D3-Ig.3 (right panel). mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3 were detected in brain capillaries (arrows), parenchyma, and surrounding brain cells (arrowheads). Scale bar: 50 μm. [Figure 12A] Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12B] Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12C]Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12D] Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12E] Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12F]Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12G] Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12H] Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 12I]Figure 12 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAb158-scLc-8D3-Ig.7; B) mAb158-scLc-8D3-Ig.8; C) mAb158-scLc-8D3-Ig.9; D) mAb158-scLc-8D3-Ig.10; E) mAb158-scLc-8D3-Ig.11; F) mAb158-scLc-Ig.12; G) mAb158-scLc-Ig.14; H) mAb158-scLc-Ig.15; and I) mAb158. [Figure 13A] 13 is a series of diagrams showing binding of the indicated molecules to Aβ1-42 analyzed by indirect ELISA as described in Example 8. A) mAb158-scLc-8D3-Ig.7, mAb158-scLc-8D3-Ig.8, mAb158-scLc-8D3-Ig.9, mAb158-scLc-8D3-Ig.11, mAb158-scLc8D3-Ig.12 and mAb158-scLc-Ig.15; B) mAb158-scLc-8D3-Ig.10, mAb158-scLc-Ig.12, mAb158-scLc-Ig.14 and mAb158. [Figure 13B] 13 is a series of diagrams showing binding of the indicated molecules to Aβ1-42 analyzed by indirect ELISA as described in Example 8. A) mAb158-scLc-8D3-Ig.7, mAb158-scLc-8D3-Ig.8, mAb158-scLc-8D3-Ig.9, mAb158-scLc-8D3-Ig.11, mAb158-scLc8D3-Ig.12 and mAb158-scLc-Ig.15; B) mAb158-scLc-8D3-Ig.10, mAb158-scLc-Ig.12, mAb158-scLc-Ig.14 and mAb158. [Figure 14] FIG. 14 shows binding to mTfR1 by the indicated molecules measured using surface plasmon resonance as described in Example 12. [Figure 15A]Figure 15 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAbB-scLc-8D3-Ig.2; B) mAbB-scLc-8D3-Ig.3; C) mAbB. [Figure 15B] Figure 15 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAbB-scLc-8D3-Ig.2; B) mAbB-scLc-8D3-Ig.3; C) mAbB. [Figure 15C] Figure 15 shows mTfR1 binding by the indicated molecules measured using Octet biolayer interferometry as described in Example 6. A) mAbB-scLc-8D3-Ig.2; B) mAbB-scLc-8D3-Ig.3; C) mAbB. [Figure 16] FIG. 16 shows mTfR1 binding by mAb B-scLc-8D3-Ig.1, measured using surface plasmon resonance as described in Example 13. [Figure 17A] Figure 17 shows Aβ binding by the indicated molecules as measured using surface plasmon resonance as described in Example 13. A) mAbB-scLc-8D3-Ig.1; B) mAbB-scLc-8D3-Ig.2; C) mAbB-scLc-8D3-Ig.3; D) mAbB. [Figure 17B] Figure 17 shows Aβ binding by the indicated molecules as measured using surface plasmon resonance as described in Example 13. A) mAbB-scLc-8D3-Ig.1; B) mAbB-scLc-8D3-Ig.2; C) mAbB-scLc-8D3-Ig.3; D) mAbB. [Figure 17C] Figure 17 shows Aβ binding by the indicated molecules as measured using surface plasmon resonance as described in Example 13. A) mAbB-scLc-8D3-Ig.1; B) mAbB-scLc-8D3-Ig.2; C) mAbB-scLc-8D3-Ig.3; D) mAbB. [Figure 17D]Figure 17 shows Aβ binding by the indicated molecules as measured using surface plasmon resonance as described in Example 13. A) mAbB-scLc-8D3-Ig.1; B) mAbB-scLc-8D3-Ig.2; C) mAbB-scLc-8D3-Ig.3; D) mAbB. [Figure 18] Figure 18 shows box plots of plasma and brain binding molecule concentrations 24 hours after intravenous (iv) administration of 40 nmoles / kg of mAbB and mAbB-scLc-8D3-Ig.1 in plasma (left) and in TBS-Triton extracts of left hemispheres (middle) in 5xFAD mice and wild-type (WT) littermates (both analyzed using MSD as described in Example 14), as well as box plots of brain-to-plasma concentration ratios (right) for each construct and transgene. Circles represent individual data points and bars represent the mean ± SD. [Figure 19] Figure 19 shows wide-field fluorescence microscopy images of intracerebral distribution of iv-administered antibody detected by anti-hIgG1 secondary antibody in the sagittal plane of 5xFAD mouse brains described in Example 15. The bottom panels show high magnification images from the cerebral cortex (CTX), hippocampus (HC), and thalamus (TH) co-stained for Aβ plaques (detected by the combination of 6E10+4G8). [Figure 20] FIG. 20 shows the results of image analysis of the number of amyloid-β plaques decorated with mAbB and mAbB-scLc-8D3-Ig.1 per field and their colocalization in the thalamus (% of 6E10 / 4G8 plaques labeled with hIgG), measured as described in Example 15. [Figure 21] FIG. 21 shows hTfR1 binding by mAb158-scLc-15G11-1-Ig.1 measured using surface plasmon resonance as described in Example 16. [Figure 22] FIG. 22 shows binding of the indicated molecules to Aβ1-42 as analyzed by indirect ELISA as described in Example 16. EXAMPLES

[0072] Although the present invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will recognize that various changes can be made and equivalents can be substituted for the elements without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or molecule to the teachings of the invention without departing from its essential scope. Therefore, the present invention is not limited to any particular embodiment, but rather is intended to include all embodiments falling within the scope of the appended claims.

[0073] The present invention will be further illustrated by the following non-limiting examples. These are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that 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, temperature, etc.), but some experimental error and deviations may exist. Unless otherwise indicated, the practice of the present invention employs conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. Such techniques are fully described in the existing literature. In addition, it will be apparent to those skilled in the art that the protein engineering methods applied to the specific linker designs herein can be applied to other constructs described herein and contemplated by the inventors, so as to fall within the scope of the present disclosure.

[0074] Example 1 Plasmid construction and protein expression Proteins were cultured in Chinese hamster ovary cells (ExpiCHO TM (Trademark) or human embryonic kidney cells (Expi293F TM (trademark) (Thermo Fisher Scientific) transient transfection.

[0075] For protein expression, the pcDNA3.4 plasmid (Thermo Fisher Scientific) was used and designed to contain the following functional elements: - promoter elements derived from cytomegalovirus (CMV), Mouse kappa light chain signal sequence (SS), the gene to be expressed, and Complete stop using double stop codons, e.g. TGA, TAA.

[0076] Example 2 Overall design and amino acid sequences of exemplary bispecific binding molecules of the present disclosure All binding molecules described in this example were designed with the same antibody heavy chain (HC). More specifically, the heavy chain comprises a mouse VH domain from Aβ protofibril-binding antibody mAb158 coupled with a mouse CH1 domain of IgG2 subclass followed by the CH2-CH3 portion of human IgG1, all encoded on the pcDNA3.4 plasmid. In other words, the heavy chain is a chimeric construct comprising a mouse VH-CH1 portion (Fab) and a human CH2-CH3 portion (Fc). The complete heavy chain amino acid sequence is given in SEQ ID NO:7, which is referred to herein as "mAb158 heavy chain".

[0077] Variation between the different constructs was achieved through the use of different linker lengths to connect each element of the continuous polypeptide chain to construct the single chain components.

[0078] In each tested single-chain component, two identical antibody light chains LC were derived from the light chain of the Aβ protofibril-binding antibody mAb158, and each LC consisted of a VL-CL portion of the κ class having the amino acid sequence of SEQ ID NO:8.

[0079] Also in each single chain component tested, the single chain binding module scBM was a single chain variable fragment (scFv) derived from the transferrin receptor binding antibody 8D3 (Kissel et al. (1998) Histochem. Cell. Biol. 110:63-72) and having the amino acid sequence of SEQ ID NO: 9. This scFv derived from 8D3 and used in the constructs is often referred to as "8D3" for simplicity.

[0080] The various test constructs were designed according to either the format given in Table 1 and depicted in FIG. 1 (mAb158-scLc-8D3-Ig.1, mAb158-scLc-8D3-Ig.2 and mAb158-scLc-8D3-Ig.3), the format depicted in FIG. 2 (mAb158-scLc-8D3-Ig.4 and mAb158-scLc-8D3-Ig.5) or as a negative control lacking any scBM elements (mAb158-scLc-Ig.6).

[0081] [Table 1]

[0082] Example 3 Production and purification of designed constructs Bispecific binding molecules of the present disclosure and controls were expressed by transient transfection of Chinese Hamster Ovary cells according to the manufacturer's instructions (ExpiCHO™ Expression System; Thermo Fisher Scientific). Equimolar ratios of heavy and light chain plasmids were added to the cells during transfection. Cell culture supernatants containing the expression constructs were harvested 8-10 days after transfection by centrifugation at 3200×g for 10 min. Supernatants were stored frozen until purification.

[0083] During purification, the frozen supernatant containing the expression construct was thawed and filtered. The filtered supernatant was applied to a MabSelectSuRe® column (Cytiva), which was subsequently washed with DPBS pH 7.4. The 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. The purified sample was further refined by size exclusion chromatography (SEC; HiLoad 26 / 600 Superdex® 200; Cytiva) in DPBS pH 7.4. The SEC-purified monomeric construct was concentrated to 10 μM using centrifugal concentrators AmiconUltra® (30 MWCO (molecular weight cut-off), Millipore) and stored at −80° C. for further analysis. Each purified expression construct was characterized using SDS-PAGE, size exclusion chromatography (Superdex® 200, Increase3.2 / 300; Cytiva), endotoxin assay and UV protein assay. An example of a representative SDS-PAGE analysis of the purified constructs is shown in FIG. 3.

[0084] Example 4 Antigen production and purification Recombinant mouse transferrin receptor 1 (mTfR1) was produced by transient transfection of human embryonic kidney cells according to the manufacturer's instructions (Expi293F™ Expression System; Thermo Fisher Scientific). The expression plasmid contained the extracellular domain of mTfR1 (amino acids 89-763; SEQ ID NO: 16) fused to a His-tag for purification.

[0085] mTfR1 protein was purified from the filtered cell culture supernatant. The supernatant was applied to a His Trap Excel column (Cytiva) and washed with 20 mM Tris, 200 mM NaCl and 5 mM imidazole. The protein was eluted with 20 mM Tris, 200 mM NaCl and 500 mM imidazole, followed by buffer exchange into DPBS (pH 7.4) using a HiPrep® 26 / 10 desalting column (Cytiva). The protein was concentrated using an Amicon Ultra® centrifugal concentrator (30 MWCO; Millipore). The protein was stored at -80°C immediately after purification to prevent aggregation. Analytical characterization of the protein was performed by UV protein measurement and SDS-PAGE, concluding that the purification was successful.

[0086] Example 5 ELISA analysis of binding to the mouse transferrin receptor Binding of specific expression constructs to mTfR1 was assessed by indirect ELISA (Figure 4). Briefly, half-areas of 96-well plates (Corning, #3690) were coated with 1 μg / mL recombinant mTfR1 produced as described in Example 4 in PBS overnight at 4°C. The coated plates were blocked with Pierce Protein-Free Blocking Solution (Thermo Fisher Scientific, #37572) for 1 h at room temperature with shaking, followed by 4 washes in PBS with 0.1% TWEEN® 20. Serial dilutions (1:3) of the expression constructs in incubation buffer (1% BSA, 0.1% TWEEN® 20 in PBS) were incubated for 1 h at room temperature. Following four washing steps, bound test constructs were detected by adding anti-mouse IgG F(ab′)2-HRP antibody (Jackson Immuno Research, #115-035-006) at a 1:1250 dilution in incubation buffer (1 h, room temperature). Following four washing steps, K-Blue® aqueous TMB substrate (Neogen, #331177) was added to the wells for 15 min at room temperature, after which the reaction was stopped with a 1:1 dilution of 0.5 M H2SO4. The optical density at 450 nm was recorded (Spark®, Tecan) and background signals were subtracted before analysis. Figure 4 shows mTfR1 binding for mAb158-scLc-8D3-Ig.1, mAb158-scLc-8D3-Ig.2 and mAb158-scLc-8D3-Ig.3 as measured by indirect ELISA. The affinity for mTfR1 for all mAb158-scLc-8D3 variants is comparable to the monovalent 8D3 Fab control (having a light chain of SEQ ID NO: 17 and a heavy chain of SEQ ID NO: 18).

[0087] Example 6 Biolayer Interferometry Analysis of Binding to Mouse Transferrin Receptor Binding of the expression constructs to mTfR1 was assessed by biolayer interferometry (Octet RED384, ForteBio). High-precision streptavidin sensors were loaded in two consecutive steps: first with 20 μg / mL biotinylated human holotransferrin (Sigma) for 180 s, then with 20 μg / mL recombinant mTfR1 produced as described in Example 4 for 180 s. Association of the sample to the loaded sensor was then measured for 120 s, followed by dissociation for 300 s. Binding was analyzed using samples diluted to 17.5 μg / mL for mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.2, 18 μg / mL for mAb158-scLc-8D3-Ig.3, and 4.9 μg / mL for 8D3Fab (corresponding to 100 nM of each binding molecule). Responses from buffer samples were subtracted. All samples were diluted in 1× kinetics buffer (ForteBio). The same buffer was used for baseline and dissociation steps. The resulting binding curves are shown in Figure 5. This figure shows that mAb158-scLc-8D3-Ig.1, mAb158-scLc-8D3-Ig.2, mAb158-scLc-8D3-lg.3, mAb158-scLc-8D3-Ig.4 and mAb158-scLc-8D3-Ig.5 all bind to the complex of holo-transferrin and mTfR1. mAb158-scLc-Ig.6, which lacks the TfR1 binding module, does not bind to the complex of holo-transferrin and mTfR1. As controls, 8D3 Fab and antibody mAb158 IgG were included in the experiment.

[0088] Example 7 Binding to and uptake by cells TfR1-mediated binding and uptake was measured in immortalized mouse brain capillary endothelial cells (cEND) (ABM, #T0290).

[0089] The pellet was resuspended in PBS and 200,000 cells per well were seeded into a U-bottom 96-well Corning plate for flow cytometry staining. The cells were then washed with PBS and incubated with 100 nM of one of the mAb158-8D3 constructs mAb158-scLc-8D3-Ig.1, mAb158-scLc-8D3-Ig.2 and mAb158-scLc-8D3-Ig.3, control 8D3 IgG (light chain SEQ ID NO: 17, heavy chain SEQ ID NO: 19) or mouse IgG control for 45 minutes at 4°C. After the primary incubation, the cells were washed twice with ice-cold PBS and the mAb158-8D3 constructs were captured using a fluorescently labeled secondary anti-mouse IgG-PE antibody (BD Biosciences, 550589). After incubation, cells were washed twice with PBS and resuspended in 200 μL of PBS. Cells were acquired on a BD FACSLyric™ flow cytometer system (BD Biosciences) and samples were analyzed using FCS Express™ software (DeNovo Software). The measured mean fluorescence intensity is displayed in FIG. 6, which shows that the bispecific binding molecule construct binds to mTfR1 expressed on the cell surface.

[0090] For measurements of internalization / uptake of constructs, cEND cells were cultured in T75 cell culture flasks for 2–3 days to >80% confluence. Cells were harvested using TrypIE reagent and washed with fresh cell medium by centrifugation at 1500 rpm for 5 min. Cells were resuspended in DPBS (Gibco) and seeded at a density of 300,000 cells / well in 96-well U-bottom plates. Cells were treated with 100 nM mAb158-scLc-8D3-Ig.1, mAb158-scLc-8D3-Ig.2, mAb158-scLc-8D3-Ig.3, or positive control (bivalent 8D3 IgG) for 1 h at 37°C, 5% CO2 or left untreated. The cells were then washed twice with ice-cold PBS and permeabilized using BD Cytofix / Cytoperm™ reagent (BD Biosciences) to access the internalized constructs. The cells were stained with anti-mouse IgG-PE in 1% BD Perm / Wash™ buffer for 45 minutes at room temperature. After staining, the cells were washed twice with 1% BD Perm / Wash™ buffer. Finally, the cells were resuspended in PBS and acquired using a BD FACSLyric™ flow cytometer. The samples were analyzed for the PE-positive population using FCS Express™ software. The percentage of positive cells, an indicator of mTfR1-mediated uptake, is displayed in Figure 7.

[0091] Taken together, these results demonstrated that the bispecific binding molecules mAb158-scLc-8D3-Ig.1, mAb158-scLc-8D3-Ig.2 and mAb158-scLc-8D3-Ig.3 bound to mTfR1 on cEND cells and were internalized at a higher rate than bivalent 8D3 IgG.

[0092] Example 8 ELISA measurement of binding to amyloid β (Aβ1-42) Binding of mAb158IgG and mAb158-scLc-8D3 constructs to Aβ1-42 was assessed by indirect ELISA. Half-areas of 96-well plates (Corning, #3690) were coated with 0.5 μg / mL recombinant Aβ1-42 (SEQ ID NO: 20) and analyzed as described for mTfR1 in Example 5. The results are shown in Figure 8. Binding to Aβ1-42 by all tested constructs was comparable to that of the unmodified mAb158IgG control, demonstrating that binding to Aβ1-42 is maintained in a bispecific format with a single-chain component containing a single-chain binding module (scBM).

[0093] Example 9 Stability of expression constructs in plasma The plasma stability of the bispecific binding molecules mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3 was measured by immunoblot (Western blot). The bispecific binding molecules were incubated in C57BL / 6 mouse plasma or PBS for 0, 1, 24 and 168 hours at 37°C (1.5 μg binding molecules diluted 1:10 in plasma or PBS). Samples were freeze-thawed twice and subjected to SDS-PAGE and Western blot. Briefly, 0.38 μg (2.5 μL) plasma / PBS samples were diluted with RIPA buffer and LDS sample buffer (Thermo Fisher Scientific, NP0007) and subjected to NuPAGE. TMThe mAbs were loaded onto a 4-12% Bis-Tris gel (Thermo Fisher Scientific, AP0329). Following SDS-PAGE and transfer onto a nitrocellulose membrane (BioRad, #1704158), they were detected with a goat anti-human IgG-IRDye® 800CW secondary antibody (LI-COR Biosciences, #926-32232). The results are shown in Figure 9. The bispecific binding molecules were detected at the expected molecular weight of approximately 175 kDa with a slight mobility shift between plasma and PBS. Western blot analysis showed that mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3 were stable in vitro in mouse plasma over a period of at least 7 days.

[0094] Example 10 In vivo brain uptake of mAb158-scLc-8D3 construct To assess mTfR1-mediated brain uptake in vivo, bispecific binding molecules mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3, as well as mAb158 IgG (chimeric IgG1, human Fc), were injected intravenously (iv) into C57BL / 6J female mice (n=3-5 mice per construct) at equimolar doses of 10 nmol / kg (corresponding to 1.82 mg / kg, 1.78 mg / kg, and 1.50 mg / kg, respectively). Plasma and brain exposure was assessed 24 hours after dosing. Animals were anesthetized with isoflurane and peripheral blood samples were collected from the orbital plexus into BD Microtainer® K2EDTA blood collection tubes. Samples were inverted and centrifuged at 2400 x g for 10 min at 4 °C. Plasma was extracted, transferred to Eppendorf tubes, and frozen at -80°C. Immediately after blood sampling, the animal's abdomen was opened and a cannula (21 gauge) was inserted into the left ventricle of the heart. A small incision was made in the right atrium and transcardial perfusion with a minimum of 50 mL of cold PBS was performed. After perfusion, the brain was extracted and the olfactory bulbs were removed. The cerebrum was separated into left and right hemispheres and the cerebellum was removed from the left hemisphere, after which the left hemisphere was weighed, frozen on dry ice, and stored at -80°C until further preparation of the injected constructs and further concentration analysis using Meso Scale Discovery (MSD)-based assays. The right hemispheres with the associated intact cerebellum were placed in 4% formaldehyde and stored at 4°C for 24 hours, after which they were rinsed in cold PBS, transferred to cold 30% sucrose / PBS solution, and stored at 4°C for further immunohistochemistry (IHC) processing.

[0095] For the measurement of intracerebral concentration, the frozen left hemisphere was thawed on ice and homogenized in TBS by automated bead homogenization. Triton® was added to the homogenate to a final concentration of 0.5%, followed by centrifugation at 16000×g, after which the supernatant was collected.

[0096] Brain and plasma concentrations of mAb158-scLc-8D3-Ig.1, mAb158-scLc-8D3-Ig.3 and mAb158 IgG were determined using the MSD platform. Standard 96-well MSD plates (MSD, #L15XA-3) were coated with 0.5 μg / mL goat anti-human IgGFc gamma fragment specific antibody (Jackson Immunо Research Europe Ltd, #109-005-098) diluted in 1×PBS-TWEEN® (Fisher Scientific, #09-9400-100) in a volume of 50 μL / well. After overnight incubation at 4° C., plates were washed 4 times with 1×PBS-TWEEN® (Fisher Scientific, #09-9410-100) and blocked with Blocker A (MSD, #R93BA-4). Samples and corresponding standards were diluted in BlockerA ranging from 83 pM to 0.02 pM in 1:4 dilution steps and added in duplicate in a volume of 50 μL / well. After 2 h incubation at room temperature and 900 rpm, wells were washed four times. 50 μL of sulfo-TAG conjugated anti-mouse antibody (MSD, R32AC-1) diluted to 0.5 μg / mL in BlockerA was added to each well and the plate was incubated for an additional hour at room temperature with agitation at 900 rpm. After four washes, 150 μL of 2×MSD read buffer (MSD, R92TC) was added to each well and the plate was read on MSD's SECTOR® imager. Analyte concentrations in samples were evaluated using the MSD Workbench software using a 4PL (four parameter logistic) curve fitting algorithm and 1 / Y2 curve weighting for the standard curve. Statistical analysis was performed in GraphPad Prism (version 9.0.0) using one-way ANOVA with Tukey's post hoc test.

[0097] The results are shown in Figure 10. As shown in Figure 10A, the plasma concentrations of the two mAb158-scLc-8D3-Ig formats were well below that of mAb158 IgG at 24 hours post-dose. The difference between the plasma data is expected since the mAb158-scLc-8D3-Ig format is likely cleared more rapidly from plasma compared to normal (wild-type) IgG due to the contribution of mTfR1-related clearance. Despite the lower plasma concentrations, significantly higher brain concentrations (Figure 10B, p<0.0001) were observed for both mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3 compared to mAb158 IgG at 24 hours post-dose. Brain-to-plasma concentration ratios of mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3 after 24 hours also showed significantly enhanced brain exposure relative to plasma in comparison to mAb158 IgG (FIG. 10C, mAb158-scLc-8D3-Ig.1: p<0.0001; mAb158-scLc-8D3-Ig.3: p<0.0001). Collectively, this data results in high brain concentrations of the tested bispecific binding molecules and supports mTfR1-mediated BBB transport.

[0098] The BBB transcytosis and brain uptake of mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3 were further supported by qualitative IHC analysis. Briefly, 20 μm thick coronal brain sections were obtained from the cerebral hemispheres after PBS perfusion using a cryostat (Microm HM 500 OM). Each section was collected on a Superfrost® plus slide (Menzel-Glaser, #J1800AMNZ) and air-dried before IHC. 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 the intravenously (iv) administered constructs, brain sections were incubated with secondary goat anti-human IgG (heavy and light chain specific) conjugated to Alexa Fluor® 488 (Invitrogen, #A11013) for 90 min at room temperature, followed by 3×15 min washes in PBS. To visualize brain capillaries, rabbit anti-collagen IV antibody (Bio-Rad, #2150-1470) was applied and detected with secondary affinity-purified goat anti-rabbit IgG (heavy and light chain specific) conjugated to Alexa Fluor® 568 (Invitrogen, #A-11011) (data not shown). Slides were loaded with Fluoromount®-G (Invitrogen, #00-4958-02) for imaging analysis. Confocal images from the cerebral cortex were captured using a Leica Stellaris® 5 confocal imaging system equipped with a HC PL APO 40x / 1.25GLYC (glycerol immersion) mоtCORR (with correction collar) CS2 objective lens (Leica, #11506423). Single confocal plane images with a resolution of 2048 x 2048 pixels were acquired at 600 Hz with a pinhole setting of 1 AU (Airy Unit).

[0099] Minimal IHC signal was detected in brain sections from animals injected with mAb158 IgG (Figure 11, left panel). In contrast, in animals administered mAb158-scLc-8D3-Ig.1 and mAb158-scLc-8D3-Ig.3, clear IHC signal was observed in brain capillaries, along with enhanced parenchymal signal and immunostained brain cells (Figure 11, center and right panels). This result indicates mTfR1-mediated engagement and transcytosis across the BBB. Taken together, MSD and IHC analysis demonstrate that mAb158-scLc-8D3-Ig.1 and Ig.3 bispecific binding molecules confer enhanced brain exposure relative to control mAb158 IgG.

[0100] Example 11 Design and characterization of additional mAb158-scLc-8D3 constructs All binding molecules described in this example were generated using the same antibody IgG1 heavy chain (HC) as in Example 2, ie, SEQ ID NO:7.

[0101] Single chain components were created by linking elements of a contiguous polypeptide chain using different linker lengths, and variation was built into each single chain component between different constructs. All binding molecules described in this example were designed using the same LC as in Example 2, i.e., SEQ ID NO:8.

[0102] The different constructs provided in this example contained either the same single-chain binding module scBM as described in Example 2, i.e. SEQ ID NO: 9 (present in mAb158-scLc-8D3-Ig.7, mAb158-scLc-8D3-Ig.8, mAb158-scLc-8D3-Ig.9, mAb158-scLc-8D3-Ig.10, mAb158-scLc-8D3-Ig.11 and mAb158-scLc-8D3-Ig.12) or the single-chain binding module scBM also derived from 8D3 but with the order of heavy and light chains reversed, SEQ ID NO: 21 (present in mAb158-scLc-8D3-Ig.13, mAb158-scLc-8D3-Ig.14 and mAb158-scLc-8D3-Ig.15).

[0103] The different test constructs are presented in Table 2, but mAb158-scLc-8D3-Ig.13, mAb158-scLc-8D3-Ig.14 and mAb158-scLc-8D3-Ig.15 were designed according to the format depicted in Figure 1, except that they had a scBM in which the order of the heavy and light chain variable regions was reversed.

[0104] [Table 2]

[0105] The bispecific binding molecules were expressed and purified as described in Example 3.

[0106] Binding of the expression constructs to mTfR1 was evaluated similarly to that described in Example 6, except that the concentration during the loading step was 10 μg / mL, the duration of the loading step was 200 and 300 seconds, and the concentration of the analyte was 50 nM. The resulting binding curves are shown in FIG. 12. All tested constructs bound to mTfR1 (FIG. 12A-12H). The antibody mAb158 without scBM was included in the experiment as a negative control (FIG. 12I).

[0107] Binding of the specific expression constructs to Aβ1-42 was assessed by indirect ELISA as described in Example 8. The results are shown in Figure 13 and demonstrate that binding to Aβ1-42 by all tested constructs was comparable to that by the unmodified mAb158 control lacking 8D3 scBM. The experiment demonstrated that binding to Aβ1-42 is maintained in the bispecific format by the Fab arm of the standard antibody structure.

[0108] Example 12 Validation of monovalent binding to TfR1 for selected constructs The monovalent binding interaction of mAb158-scLc-8D3-Ig.8 to mouse TfR1 was measured using surface plasmon resonance (Biacore8K, Cytiva). 3 μg / mL mTfR1 was immobilized on a Cm5 sensor chip (Cytiva, #BR100399) using the Amine Coupling Kit Type 2 (Cytiva, #BR100633) according to the manufacturer's instructions. HBS-EP starting with 50 nM analyte was used. + A series of two-fold dilutions in 100 mM Tris-Acetate (Cytiva, #BR100669) were injected in duplicate over the immobilized ligand and interactions were measured using a single-cycle kinetic method with a 160 s contact time followed by a 1000 s dissociation time at a flow rate of 70 μL / mL.

[0109] The resulting sensorgram is shown in Figure 14 and confirms that mAb158-scLc-8D3-Ig.8, a binding molecule according to the present disclosure, binds monovalently to mTfR1. As a control, monovalent 8D3 Fab was compared to standard bivalent 8D3 hIgG. The figure clearly shows the bivalent binding profile of 8D3 hIgG compared to monovalent 8D3 Fab.

[0110] Example 13 Design and characterization of further bispecific binding molecules according to the present disclosure The binding molecules described in this example were designed starting from a different Aβ-binding antibody derived from mAb158 used in the previous examples.

[0111] The heavy chains (HC) of the bispecific binding molecules in this study comprised a mouse VH domain derived from an Aβ-binding antibody, referred to herein as mAbB, linked to a human CH1-CH3 portion (mAbB-scLc-8D3-Ig.1 and mAbB-scLc-8D3-Ig.2) or to a mouse CH1 and a human CH2-CH3 portion (mAbB-scLc-8D3-Ig.3). In other words, the heavy chains were chimeric constructs comprising a mouse VH portion and a human CH1-CH3 portion, or a mouse VH-CH1 portion and a human CH2-CH3 portion.

[0112] In each tested single-chain component, two identical antibody light chains LC were derived from the mouse VL domain of the light chain of the Aβ-binding antibody mAbB linked to a human CL portion of the κ class (mAbB-scLc-8D3-Ig.1 and mAbB-scLc-8D3-Ig.2) or a mouse CL portion (mAbB-scLc-8D3-Ig.3).

[0113] All the different constructs presented in this example contained the same single chain binding module scBM as in Example 2, ie SEQ ID NO: 9.

[0114] The various test constructs are given in Table 3, which were designed according to the format shown in FIG.

[0115] [Table 3]

[0116] The bispecific binding molecules mAbB-scLc-8D3-Ig.1, mAbB-scLc-8D3-Ig.2 and mAbB-scLc-8D3-Ig.3 were expressed and purified as described in Example 3.

[0117] Binding of the expression constructs to mTfR1 was assessed essentially as described in Example 6 for Biolayer Interferometry (Octet) and as described in Example 12 for Surface Plasmon Resonance (Biacore).

[0118] Representative results from Octet measurements of binding to mTfR1 are shown in FIG. 15 for the bispecific binding molecules mAbB-scLc-8D3-Ig.2 and mAbB-scLc-8D3-Ig.3, as well as for the negative control mAbB, i.e., mAbB as a standard antibody format that does not contain any TfR1 binding module.

[0119] Representative results from Biacore SCK measurements of binding to mTfR1 are shown in FIG. 16 for the bispecific binding molecule mAbB-scLc-8D3-Ig.1.

[0120] Binding of expression constructs to Aβ was assessed using a Biacore 8K instrument (Cytiva) following standard procedures. Binding to the target was measured using single cycle kinetics (SCK) with binding molecules immobilized on a CM5 chip. For measurements, 5 μg / mL of analyte binding molecules were immobilized on the chip. Aβ target was then injected over the chip using five two-fold dilutions starting at 250 nM. Regeneration of the surface between cycles was performed by injecting 30 μL of 10 mM glycine-HCl (pH 1.7). Binding data were fitted to a 1:1 interaction model. 1× HBS-EP + (Cytiva, Cat. No. BR100669) was used to dilute the binding molecules and target antigens. Experiments were performed at 25°C.

[0121] Representative results from Biacore SCK binding assays for Aβ are shown in Figure 17. Binding of mAbB-scLc-8D3-Ig.1, mAbB-scLc-8D3-Ig.2 and mAbB-scLc-8D3-Ig.3 to their Aβ target was assessed by Biacore and found to be comparable to that of mAbB in the standard IgG format.

[0122] Example 14 In vivo target binding by bispecific binding molecules of the present disclosure To further evaluate target engagement in vivo, mAbB and mAbB-scLc-8D3-Ig.1 produced as described in Example 13 were examined in B6SJL-Tg 5xFAD mice (Northwestern University). The 5xFAD mouse model is an Alzheimer's disease (AD) model using mice expressing human APP and PSEN1 transgenes, which harbor a total of five AD-associated mutations, including the Swedish (K670N / M671L), Florida (I716V) and London (V717I) mutations in APP, and the M146L and L286V mutations in PSEN1. 7-8 month old 5xFAD mice and wild type (WT) littermates were intravenously (iv) injected with equimolar amounts of 40 nmol / kg mAbB and mAbB-scLc-8D3-Ig.1 (corresponding to 5.8 mg / kg and 7.0 mg / kg, respectively). Plasma and brain exposure to each binding molecule was assessed 24 hours post-dose in both 5xFAD and WT mice, as well as binding to brain parenchymal Aβ plaques in 5xFAD mice. Peripheral blood samples were collected from all animals by cardiac puncture into MiniCollect® K2EDTA tubes. Samples were inverted and centrifuged at 2400×g for 10 min at 4° C. Plasma was extracted, transferred to LoBind™ Eppendorf tubes, and frozen at −80° C. Immediately after blood sampling, animals were perfused with 0.9% saline and 4% paraformaldehyde (PFA; pH 7.4) or 0.9% saline alone for brain immunohistochemistry (IHC) or antibody exposure analysis, respectively. After perfusion, the cerebrum was extracted and the olfactory bulb was removed. The cerebrum was separated into left and right hemispheres, and the cerebellum was removed from the left hemisphere in the case of animals perfused with saline alone. The left hemisphere was then weighed, immediately frozen on dry ice, and stored at -80°C before further concentration measurements were performed using the Meso Scale Discovery (MSD) platform.The right cerebral hemisphere, and in the case of animals perfused with both saline and PFA, the left hemisphere with intact cerebellum, were post-fixed by immersion in freshly prepared 4% PFA (pH 7.4) for 2 h at room temperature, after which they were rinsed in cold PBS, transferred to 15% sucrose / PBS, and stored at 4°C until they sank to the bottom of the tube. Each hemisphere was transferred to a cryomold (plastic embedding dish), embedded in OCT medium, frozen in isopentane cooled on dry ice, and stored at -80°C.

[0123] For brain concentration measurements, frozen left hemispheres were thawed on ice and homogenized in TBS by automated bead homogenization. Beads were added to the homogenate to a final Triton concentration of 0.5% and then centrifuged at 16,000 × g, after which the supernatant was collected.

[0124] Brain and plasma concentrations of mAbB and mAbB-scLc-8D3-Ig.1 were determined using the MSD platform. A sandwich setup was used in standard 96-well MSD plates (MSD, #L15XA-3) coated with goat anti-human IgG (0.5 μg / mL, Fcγ-specific antibody, Jackson Immunо Research Europe Ltd, #109-005-098) diluted in 1× PBS (Fisher Scientific, #09-9400-100). Plates were blocked for 1 h in Blocker A (MSD, #R93BA-4) and then incubated with samples and standards for 2 h. A 1-hour incubation step of mouse anti-human IgG1 (0.5 μg / mL, Mabtech, #3850-1-1000) was included followed by a 1-hour incubation step of SULFO-TAG-conjugated anti-mouse antibody (0.5 μg / mL, MSD, #R32AC-1). After adding MSD read buffer (MSD, #R92TC) to the wells, the plate was read on an MSD SECTOR® imager. Between each incubation step, 4 washes in 1×PBS-Tween® (Fisher Scientific, #09-9410-100) were performed. All binding molecules, except for the coating antibodies, were diluted in Blocker A.

[0125] The results are shown in Figure 18. The bispecific binding molecule mAbB-scLc-8D3-Ig.1, a mAb with 8D3 scBM, shows lower plasma exposure but higher brain exposure compared to mAbB in conventional antibody format. Furthermore, mAbB-scLc-8D3-Ig.1 provides a higher brain to plasma ratio (B:P ratio).

[0126] Example 15 Immunohistochemical analysis of in vivo binding by bispecific binding molecules of the present disclosure Transcytosis across the blood-brain barrier and binding to Aβ targets by the binding molecule of the present disclosure, mAbB-scLc-8D3-Ig.1, was further evaluated by IHC in the transgenic mouse model 5xFAD.

[0127] Briefly, 10 μm thick sagittal brain sections were obtained from 5xFAD mice using a cryostat (Leica CM1950 or CryoStar NX70). Section collection began approximately 0.50 mm lateral to the midline and spanned the entire hemisphere. Sections were stored at -20°C.

[0128] Non-specific binding was blocked with MOM blocking reagent (MKB-2213, Vector Laboratories) in 0.1% Triton®-100 / PBS for 60 min, followed by rinsing in PBS. Amyloid plaques were labeled with primary antibodies 6E10 (Biozym Scientific, B803001, 1:1000) and 4G8 (Biozym Scientific, B800701, 1:500) in MOM diluent overnight at 4°C. Intravenously administered constructs containing human IgG chains were labeled with anti-human IgG (H+L) AlexaFluor 1000 (1:1000) and 4G8 (1:500) in MOM diluent overnight at 4°C. TM The two primary mIgG antibodies were detected by DyLight™ 647 (Jackson Immuno Research, 709-605-149) and the donkey anti-mouse IgG (H+L) secondary antibody was TM The slides were visualized using Mowiol™ 550 labeling (Thermo Scientific, SA5-10167). Cell nuclei were labeled with DAPI. TM (trademark) (mounting agent) and mounted on a Zeiss Axiocam TM (Trademark) 506mоnо and Hitachi 3CCD HV-F202SCL camera and Zeiss TM Sections were imaged with a Zeiss automated microscope AxioScan Z1 scanner equipped with a high numerical aperture lens and ZEN 3.3 software.

[0129] Automated IHC analysis of plaque number and colocalization in the thalamus (N=5 mice / treatment) was performed using the Colocalization Threshold & Analyze Particle plugin in Fujifilm ImageJ (v1.53c; Schindelin et al. (2012), Nat. Meth. 9(7):676-682). Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test.

[0130] The results are shown in Figures 19 and 20. The bispecific binding molecule of mAbB with 8D3 scBM, mAbB-scLc-8D3-Ig.1, shows much stronger target engagement compared to mAbB in a conventional antibody format. The arrows indicate that mAbB-scLc-8D3-Ig.1 and mAbB each reached targets in the brain based on hIgG staining (based on 6E10 / 4G8 staining). hIgG staining was much more extensive and intense for mAbB-scLc-8D3-Ig.1 compared to that seen for mAbB.

[0131] Example 16 Design and characterization of further bispecific binding molecules of the present disclosure The binding molecules described in this example were designed similarly to previously studied binding molecules, but using a different single-chain binding module, scBM.

[0132] The heavy chain (HC) of this experimental bispecific binding molecule contained a mouse VH domain from mAb158 linked to a human CH1-CH3 portion. The complete heavy chain amino acid sequence is given in SEQ ID NO:33.

[0133] Two identical antibody light chains LC were derived from the VL domain of the light chain of mAb158 linked to a human CL portion of the kappa class. The amino acid sequence of each LC is shown by SEQ ID NO:34.

[0134] The single-chain binding module scBM used in this example was a single-chain variable fragment (scFv) derived from the transferrin receptor binding antibody 15G11-1 (Yu et al. (2014) Sci. Transl. Med. 6(261):261ra154) and having the amino acid sequence of SEQ ID NO:35.

[0135] The test constructs were designed according to the format depicted in FIG. 1 and are presented in Table 4, including their linker sequences.

[0136] [Table 4]

[0137] The bispecific binding molecule mAb158-scLc-15G11-1-Ig.1 was expressed and purified as described in Example 3.

[0138] Binding to nTfR1 was assessed as described in Example 12, except that hTfR1 was used for immobilization and 3M MgCl2 was used for regeneration. The results are shown in Figure 21 and demonstrate that the affinity of the expressed variants for hTfR1 is comparable to that of the 15G11-1 Fab control.

[0139] Binding of the expression constructs to Aβ1-42 was assessed by indirect ELISA as described in Example 8. The results are shown in Figure 22 and demonstrate that binding to Aβ1-42 by mAb158-scLc-15G11-1-Ig.1 was comparable to that by the unmodified mAb158 control.

[0140] Itemized List of Embodiments [1] A bispecific binding molecule comprising three polypeptide chains: (A) two identical antibody heavy chains (HC) derived from a monoclonal antibody having affinity for a first target present in the mammalian brain; (B) The following five elements in a continuous polypeptide chain: i) two identical antibody light chains (LC) derived from said monoclonal antibody having affinity for said first target; ii) one single-chain binding module (scBM) with affinity for a second target that mediates transport of the bispecific binding molecule across the blood-brain barrier; and iii) two amino acid linkers L1 and L2 and one single-stranded component comprising Consisting of wherein the light chain (LC) and the single chain binding module (scBM) are separated by the linkers L1 and L2, thereby comprising from N-terminus to C-terminus: [LC-L1-scBM-L2-LC], [LC-L1-LC-L2-scBM], and [scBM-L1-LC-L2-LC] The bispecific binding molecule comprising a sequence selected from the group consisting of: [2] The sequence of the element from the N-terminus to the C-terminus in the single-chain component is [LC-L1-scBM-L2-LC] and [LC-L1-LC-L2-scBM] 2. The bispecific binding molecule according to item 1, selected from the group consisting of: [3] The bispecific binding molecule according to item 2, wherein the sequence of the elements from the N-terminus to the C-terminus in the single-chain component is [LC-L1-scBM-L2-LC]. [4] The bispecific binding molecule according to item 2, wherein the sequence of the elements in the single-chain component from the N-terminus to the C-terminus is [LC-L1-LC-L2-scBM]. [5] The bispecific binding molecule of any of the preceding claims, wherein the first target is selected from the group consisting of amyloid beta peptide or a derivative or fragment thereof, alpha-synuclein or a derivative or fragment thereof, TAR DNA-binding protein 43 (TDP-43) or a derivative or fragment 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 a fragment thereof, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, gamma secretase, death receptor 6, amyloid beta precursor protein, p75 neurotrophin receptor, neuregulin and caspase 6. [6] The bispecific binding molecule of item 5, wherein the first target is selected from the group consisting of amyloid beta peptide or a derivative or fragment thereof, alpha-synuclein or a derivative or fragment thereof, TAR DNA-binding protein 43 (TDP-43) or a derivative or fragment thereof, triggering receptor expressed on myeloid cells 2 (TREM2), tau, phosphorylated tau or a fragment thereof, and apolipoprotein E4. [7] The bispecific binding molecule of item 6, wherein the first target is selected from the group consisting of amyloid beta peptide or a derivative or fragment thereof, alpha-synuclein or a derivative or fragment thereof, and TAR DNA binding protein 43 (TDP-43) or a derivative or fragment thereof. [8] The bispecific binding molecule of any of the preceding claims, wherein the monoclonal antibody having affinity for the first target is an anti-Aβ antibody selected from the group consisting of lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06 and KHK6640. [9] The bispecific binding molecule of any one of items 1 to 7, wherein the monoclonal antibody having affinity for the first target is the anti-α-synuclein antibody ABBV0805.

[10] The bispecific binding molecule of any of the preceding claims, wherein the scBM is of a type selected from the group consisting of scFv, scFab, VHH and VNAR.

[11] The bispecific binding molecule of item 10, wherein the scBM is selected from the group consisting of scFv and scFab.

[12] The bispecific binding molecule of item 11, wherein the scBM is an scFv.

[13] The bispecific binding molecule of item 11, wherein the scBM is an scFab.

[14] The bispecific binding molecule of any of the preceding claims, wherein the second target is selected from the group consisting of transferrin receptor 1 (TfR1), insulin receptor (InsR), insulin-like growth factor 1 receptor (IGF-1R), low-density lipoprotein receptor-related protein 8 (Lrp8), low-density lipoprotein receptor-related protein 1 (Lrp1), CD98, transmembrane protein 50A (TMEM50A), glucose transporter 1 (Glutl), basigin (BSG), and heparin-binding epidermal growth factor-like growth factor.

[15] The bispecific binding molecule of item 14, wherein the second target is selected from the group consisting of transferrin receptor 1 (TfR1), insulin receptor (InsR), insulin-like growth factor 1 receptor (IGF-1R) and low-density lipoprotein receptor-related protein 8 (Lrp8).

[16] The bispecific binding molecule of item 15, wherein the second target is transferrin receptor 1.

[17] The bispecific binding molecule of any of the preceding claims, wherein at least one of the amino acid linkers L1 and L2 is a flexible linker.

[18] The bispecific binding molecule of item 17, wherein both of the amino acid linkers L1 and L2 are flexible linkers.

[19] The bispecific binding molecule of any one of items 17 to 18, wherein the flexible linker comprises glycine, serine, alanine and / or threonine residues.

[20] The linker is n S m ) p and (S n G m ) p 20. The bispecific binding molecule according to item 19, having a general formula selected from:

[21] The bispecific binding molecule of any of the preceding items, wherein at least one of the amino acid linkers L1 and L2 is 10 to 50 amino acid residues in length, such as 10 to 30 amino acid residues in length, 15 to 25 amino acid residues in length, or 10 to 20 amino acids in length.

[22] The bispecific binding molecule according to item 21, wherein both of the amino acid linkers L1 and L2 are 10 to 50 amino acid residues in length, such as 10 to 30 amino acid residues in length, 15 to 25 amino acid residues in length, or 10 to 20 amino acids in length.

[23] The bispecific binding molecule of any of the preceding claims, wherein the amino acid linkers L1 and L2 are the same length.

[24] The bispecific binding molecule of any one of items 1 to 22, wherein the amino acid linkers L1 and L2 are of different lengths.

[25] The bispecific binding molecule of item 24, wherein the amino acid linker L1 is longer than the amino acid linker L2.

[26] The bispecific binding molecule of item 24, wherein the amino acid linker L2 is longer than the amino acid linker L1.

[27] A pharmaceutical composition comprising the bispecific binding molecule of any of the preceding items and a pharma- ceutically acceptable carrier or excipient.

[28] The bispecific binding molecule of any one of items 1 to 26 or the composition of item 27 for use in treatment, such as use in therapeutic or prophylactic treatment.

[29] The bispecific binding molecule of any one of items 1 to 26 or the composition of item 27 for use in in vivo diagnosis or in vivo prognosis.

[30] The bispecific binding molecule or composition for use according to any one of items 28 to 29, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is for a neurodegenerative disease, such as for a disease selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease, familial amyloidotic neuropathies.

[31] The bispecific binding molecule or composition for use according to item 30, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is for a disease selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, Lewy body dementia (LBD), Down's syndrome (DS), amyotrophic lateral sclerosis (ALS), frontal dementia, tauopathy, Parkinson's disease (PD), Parkinson's disease dementia (PDD) and Lewy body degeneration of Alzheimer's disease.

[32] The bispecific binding molecule or composition for use according to item 31, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is for a disease selected from Alzheimer's disease and other diseases associated with Aβ protein aggregation, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD).

[33] The bispecific binding molecule or composition for use according to item 32, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is for Alzheimer's disease.

[34] The bispecific binding molecule or composition according to any one of items 28 to 29, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is for a disease selected from brain tumors, multiple sclerosis and lysosomal storage diseases.

[35] A method for the therapeutic or prophylactic treatment of a mammal having or at risk of developing a disease, comprising administering to said mammal a therapeutically effective amount of a bispecific binding molecule according to any one of items 1 to 26 or a composition according to item 27.

[36] The method according to item 35, wherein the disease is a neurodegenerative disease, for example a neurodegenerative disease as defined in any one of items 30 to 33.

[37] The method of claim 35, wherein the disease is a disease as defined in claim 34.

Claims

1. The following three polypeptide chains: (A) Two identical antibody heavy chains (HC) derived from a monoclonal antibody having affinity for a first target present in the mammalian brain, (B) Five elements in a continuous polypeptide chain: i) Two identical antibody light chains (LC) derived from the monoclonal antibody having affinity for the first target; ii) One single-chain binding module (scBM) having affinity for a second target that mediates the transport of the bispecific binding molecule across the blood-brain barrier; and iii) Two amino acid linkers L1 and L2 A single-chain component containing A bispecific binding molecule comprising The light chain (LC) and the single-chain binding module (scBM) are separated by the linkers L1 and L2, thereby forming the following group from the N-terminus to the C-terminus: [LC-L1-scBM-L2-LC], [LC-L1-LC-L2-scBM], and [scMB-l1-LC-L2-LC] A bispecific binding molecule that forms a sequence selected from the group consisting of

2. The sequence of the elements from the N-terminus to the C-terminus in the single-chain component is [LC-L1-scBM-L2-LC] and [LC-L1-LC-L2-scBM] The bispecific binding molecule according to claim 1, which is selected from the group consisting of

3. The first target is selected from the group consisting of amyloid-β peptide or its derivative or fragment, α-synuclein or its derivative or fragment, TAR DNA-binding protein 43 (TDP-43) or its derivative or fragment, triggering receptor expressed on myeloid cells 2 (TREM2), β-secretase 1 (BACE1), superoxide dismutase (SOD), huntingtin, transthyretin, P-secretase 1, epidermal growth factor, epidermal growth factor receptor 2, tau, phosphorylated tau or its fragment, apolipoprotein E4, CD20, prion protein, leucine-rich repeat kinase 2, parkin, presenilin 2, γ-secretase, death receptor 6, amyloid β precursor protein, p75 neurotrophin receptor, neuregulin, and caspase 6. The bispecific binding molecule according to claim 1

4. The bispecific binding molecule according to claim 1, wherein the monoclonal antibody having affinity for the first target is selected from the group consisting of lecanemab, gantenerumab, aducanumab, donanemab, PBD-C06, and KHK6640.

5. The bispecific binding molecule according to claim 1, wherein the monoclonal antibody having affinity for the first target is the anti-α-synuclein antibody ABBV0805.

6. The bispecific binding molecule according to claim 1, wherein the scBM is of a type selected from the group consisting of scFv, scFab, VHH, and VNAR.

7. The bispecific binding molecule according to claim 6, wherein the scBM is scFv.

8. The bispecific binding molecule according to claim 1, wherein the second target is selected from the group consisting of transferrin receptor 1 (TfR1), insulin receptor (InsR), insulin-like growth factor 1 receptor (IGF-1R), low-density lipoprotein receptor-related protein 8 (Lrp8), low-density lipoprotein receptor-related protein 1 (Lrp1), CD98, transmembrane protein 50A (TMEM50A), glucose transporter 1 (Glut1), basigin (BSG), and heparin-binding epidermal growth factor-like growth factor.

9. The bispecific binding molecule according to claim 1, wherein both of the amino acid linkers L1 and L2 are flexible linkers.

10. The bispecific binding molecule according to claim 9, wherein the flexible linker contains glycine, serine, alanine, and / or threonine residues.

11. The bispecific binding molecule according to claim 1, wherein at least one of the amino acid linkers L1 and L2 is 10 to 50 amino acid residues in length, such as 10 to 30 amino acid residues in length, such as 15 to 25 amino acid residues in length, or 10 to 20 amino acids in length.

12. The bispecific binding molecule according to claim 1, wherein the amino acid linkers L1 and L2 are of the same length or the amino acid linkers L1 and L2 are of different lengths.

13. A pharmaceutical composition comprising the bispecific binding molecule according to claim 1 and a pharmaceutically acceptable carrier or excipient.

14. The bispecific binding molecule according to claim 1 or the pharmaceutical composition according to claim 13 for use in a treatment, for example for use in a therapeutic or prophylactic treatment, or for use in an in vivo diagnosis or an in vivo prognostic diagnosis.

15. wherein the treatment, prophylaxis, in vivo diagnosis or in vivo prognostic diagnosis is - a disease selected from neurodegenerative diseases such as Alzheimer's disease and other diseases associated with Aβ protein aggregation, traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia, Creutzfeldt-Jakob disease, Huntington's disease and familial amyloid neuropathy; or - a disease selected from brain tumors, multiple sclerosis and lysosomal storage diseases The specific binding molecule or composition for use according to claim 14, which is related to.

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