Blood-brain barrier crossing antibodies

Abstract

The present invention relates to antibodies or antibody fragments that bind to human and non-human primate transferrin receptors. The antibodies described herein can be used as agents to deliver pharmaceutical compounds into or through cells during receptor-mediated endocytosis and / or transendocytosis processes. As transferrin receptors are also present in the blood brain barrier endothelial cells, one aspect of the invention provides means and methods to increase delivery of pharmaceutical compounds to the central nervous system.

Description

TECHNICAL FIELD

[0001] The present application relates to antibodies or antibody fragments that bind to the transferrin receptor (TfR) of humans and non-human primates. The antibodies and methods described herein can be used to increase the delivery of a pharmaceutical compound to the central nervous system in a receptor-mediated endocytosis and / or transcytosis process. BACKGROUND

[0002] The central nervous system (CNS) is separated from the rest of the organism by a very special organ, the blood brain barrier (BBB), which protects the central nervous system from harmful circulating substances in the peripheral blood stream, while still allowing selective influx of desired elements such as nutrients. The BBB thus represents a bottleneck for the treatment of neurological diseases, as most biologies cannot reach their brain targets (Freskgard & Ulrich 2017 Neuropharmacology 120, 28-55). At best, they reach their brain targets in very small amounts, thus requiring the administration of high doses of biologies, leading to potential side effects and high treatment costs (Freskgard & Ulrich 2017 Neuropharmacology 120, 28-55; St-Amour et al. 2013 J Cereb Blood Flow Metab 33, 1983-1992; Poduslo et al. 1994 PNAS 91, 5705-5709). The blood brain barrier is composed of an endothelial layer surrounded by pericytes and astrocytic end-feet. In contrast to other endothelium in the organism, the endothelium in the BBB expresses tight junctions that limit the paracellular diffusion of substances. In contrast, most desired substances from the periphery in the brain follow an active route of entry through specific channels and transporters. Receptor-mediated transcytosis (RMT) is one such physiological mechanism, where nutrients are recognized by specific receptors expressed on the surface of endothelial cells, internalized in intracellular vesicles, and finally released in the brain parenchyma. It has been demonstrated that targeting such RMT receptors with antibodies is an effective strategy to increase the brain penetration of biologies (Pardridge 1986 Endocrine Reviews 7, 314-330), and the transferrin receptor (TfR) is one of the most exploited RMT mechanisms for brain drug delivery (Sehlin et al. 2020 FASEB J 34, 13272-13283; Su et al. 2022 PLoS One 17; Sonoda et al. 2018 Molecular Therapy 26, 1366-1374). Recently, a anti-TfR-idursulfase conjugate drug (Izcargo®) was approved in Japan for the treatment of Hunter syndrome (Giugliani et al. 2021 Molecular Therapy 29, 2378-2386).

[0003] We previously identified TfR nanobodies that successfully deliver biologies across the BBB. We obtained a set of mouse TfR binders as well as a set of human TfR binders (Wouters et al. 2020 Fluids Barriers CNS 17, 62; Wouters et al. 2020 Fluids Barriers CNS 19, 79). Unfortunately, our human TfR nanobodies do not bind non-human primate (NHP) TfR, despite having high sequence homology between the two proteins. Lack of binding to NHP TfR represents an obstacle to determine preclinical efficacy and safety of potential therapeutic conjugates. In the present application, the identification of two human / macaque TfR binding nanobodies, and the in vivo validation of their potential to shuttle therapeutic agents into the brain is disclosed. More particularly, single domain antibodies, more particularly VHHs, that bind to human and NHP transferrin receptor (TfR) are disclosed. The antibodies described herein can deliver compounds including therapeutic and / or diagnostic antibodies and small molecules across the BBB after a single systemic administration in mice. The VHH sequences described herein are compared to VHH sequences that bind to TfR (disclosed in patent application WO2020144233 (Vect-Horus)). From a therapeutic biology development perspective, the Vect-Horus VHHs show many serious liabilities, and the VHHs disclosed herein have improved chemical stability performance, have higher thermal stability, and have lower oligomerization and lower aggregation propensity. BRIEF DESCRIPTION OF DRAWINGS

[0004] Figure 1 The identification of human / macaque TfR binders is summarized. Figure 1 A shows the immunization, selection and screening strategy followed to select human and macaque TfR nanobody binders. Figure 1 B-E show the binding of VHHs to CHO cells overexpressing hTfR (B), macaque TfR (C), mouse TfR (D) and GFP (E). Figure 1 F summarizes the results of the kinetic analysis of VHHs binding to recombinant material of macaque and human TfR assessed with SPR.

[0005] Figure 2 The shuttle of anti-TfR / anti-BACE1 compositions across the BBB is shown. Figure 2 A is an illustration of the design of bispecific antibodies. Figure 2 B-C show the binding of bispecific antibodies to human TfR (B) or GFP overexpressing cells (C). Figure 1 D-F summarize the BLI kinetic analysis of antibody binding to BACE1.

[0006] Figure 3 Aβ40 levels in plasma (A) and brain (B) of human TfR knock-in mice as readout for BACE1 inhibition when peripherally administering anti-BACE1 conjugated VHHs of the application.

[0007] Figure 4 A SEC profile of exemplary humanized variants disclosed in WO2020144233 (Vect-Horus) compared to BBB00515 and BBB00533.

[0008] Figure 5 SYPRO Orange fluorescence spectra of exemplary humanized variants disclosed in WO2020144233 (Vect-Horus) compared to BBB00515 and BBB00533.

[0009] Figure 6 Sequence alignment of BBB00515 and humanized variants thereof is described. CDR1, 2 and 3 regions are shown in grey shading.

[0010] Figure 7 Sequence alignment of BBB00533 and humanized variants thereof is described. CDR1, 2 and 3 regions are shown in grey shading.

[0011] Figure 8 Binding properties and biophysical parameters of humanized variants of BBB00533 are described.

[0012] Figure 9 Binding properties and biophysical parameters of humanized variants of BBB00515 are described.

[0013] Figure 10 pH-dependent binding of histidine mutants of BBB00515 to human TfR is shown.

[0014] Figure 11 pH-dependent binding of histidine mutants of BBB00533 to human TfR is shown.

[0015] Figure 12 Details and alignment of histidine mutants of BBB00515 and BBB00533 are described. DETAILED DESCRIPTION

[0016] Definitions

[0017] To make this specification easier to understand, certain terms are first defined. Further definitions are set forth throughout the detailed description. The invention is described with reference to specific embodiments and certain accompanying drawings, but is not limited thereto; rather, it is defined solely by the claims. Any reference numerals in the claims should not be construed as limiting the scope. The described drawings are illustrative only and not restrictive. In the drawings, for illustrative purposes, the dimensions of some elements may be enlarged and not drawn to scale. It should be noted that the term “a” (or “an”) refers to one or more of such entities; for example, “a nucleotide sequence” should be understood to mean one or more nucleotide sequences. Therefore, the terms “a” (or “an”), “one or more”, and “at least one” are used interchangeably herein. Furthermore, “and / or” as used herein should be considered as each of two specified features or components being, or not, a specific disclosure of the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” is intended herein to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). When referring to a singular noun, the use of indefinite or definite articles, such as "a" or "the," includes the plural form of that noun unless otherwise specified. Furthermore, the terms first, second, third, etc., in the specification and claims are used to distinguish similar elements and are not necessarily used to describe sequence or chronological order. It should be understood that such terms are interchangeable where appropriate, and embodiments of the invention described herein can operate in a different order than that described or illustrated herein.

[0018] It should be understood that wherever the term "comprising" is used to describe an aspect or embodiment, similar aspects or embodiments described in the terms "consisting of" and / or "substantially consisting of" are also provided. Where the term "comprising" is used in this specification and claims, it does not exclude other elements or steps. Unless specifically defined herein, all terms used herein have the same meaning as would be apparent to those skilled in the art.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. For additional relevant definitions and terms, practitioners can refer to, inter alia, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012). The definitions provided herein are not to be construed as having a scope less than understood by a person of ordinary skill in the art.

[0020] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxyl orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined herein have meanings ascribed to them by reference to the specification as a whole and, as such, are not limited to a specific listed meaning, but encompass myriad

[0021] The term“about” is used herein to refer to approximately, roughly, in the region of, or in the region of. When the term“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries of the range by the stated value above and below the given range. Generally, the term“about” can allow for a certain amount of variation as well as a certain degree of error as understood by one of ordinary skill in the art. For example, a dissociation constant, koff, of about 1.50 x 10 -2 / s means that koffis in the range of 1.45 x 10-2 / s to 1.55 x 10 -2 / s to 1.55 x 10

[0022] The present application relates to antibodies that bind to human and NHP transferrin receptors.

[0023] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain that specifically binds with an antigen. An "antibody" can be an intact immunoglobulin derived from natural sources or from recombinant sources, and can be an immunoreactive portion of an intact immunoglobulin. Antibodies are generally tetramers of immunoglobulin molecules. The term "immunoglobulin (Ig) domain" as used herein refers to the globular region of an antibody chain, or to a polypeptide that essentially constitutes such a globular region. Immunoglobulin domains are characterized by their retention of the characteristic immunoglobulin fold of antibody molecules (the Ig fold as named herein), which consists of two layers of sandwich of about seven to nine strands of anti-parallel beta-chains arranged in two beta-sheets, optionally stabilized by conserved disulfide bonds. The term "immunoglobulin (Ig) domain" includes "immunoglobulin constant domains" and "immunoglobulin variable domains" (abbreviated "IVD"), wherein the latter refers to an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1", respectively; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively; wherein the framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1", respectively; "complementarity determining region 2" or "CDR2"; and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain (IVD) that confers specificity to antibodies for antigens by carrying the antigen binding site.

[0024] The "immunoglobulin domain" of the present application also includes an "immunoglobulin single variable domain" (abbreviated as "ISVD"), which is equivalent to the term "single variable domain" and defines a molecule in which the antigen binding site is present on and formed by a single immunoglobulin domain. This distinguishes the immunoglobulin single variable domain from a "conventional" immunoglobulin or fragment thereof, in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen binding site. Typically, in a conventional immunoglobulin, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form the antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will participate in the antigen binding site formation. In view of the above definition, the antigen binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or a Fab fragment, a F(ab')2 fragment, a Fv fragment such as a disulfide linked Fv or a scFv fragment, or a diabody (known in the art) derived from such a conventional 4-chain antibody is typically not considered to be an immunoglobulin single variable domain, because in these cases the binding to the respective epitope of an antigen typically does not occur by one (single) immunoglobulin domain, but by a pair (associated) of immunoglobulin domains such as a light and a heavy chain variable domain, i.e. by a VH-VL pair of immunoglobulin domains, which jointly bind to the epitope of the respective antigen. In contrast, an immunoglobulin single variable domain is capable of specifically binding to an epitope of an antigen without pairing with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Thus, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. Thus, the single variable domain can be a light chain variable domain sequence (e.g. a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a VH-sequence or a VHH-sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e. a functional antigen binding unit consisting essentially of a single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the present application, the immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g. a VH-sequence); more specifically, the immunoglobulin single variable domain can be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. For example, the immunoglobulin single variable domain can be a (single) domain antibody (or an amino acid sequence suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) or a nanobody (as defined herein and including but not limited to a VHH); other single variable domains, or any suitable fragment of any of the same.In particular, the immunoglobulin single variable domain can be a Nanobody® (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. For a general description of Nanobodies®, reference is made to the further description below, as well as to the prior art cited herein, e.g. as described in WO2008 / 020079.

[0025] The immunoglobulin domains herein also include "VHH domains", also known as VHH, VHH domain, VHH antibody fragment, and VHH antibody, which were originally described as the antigen-binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al. (1993) Nature 363: 446-448). The term "VHH domain" is chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as "VH domains") and the light chain variable domains present in conventional 4-chain antibodies (referred to herein as "VL domains").For further description of VHHs and Nanobodies, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001) and the following patent applications mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 to Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 to Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 to Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 to Algonomics N.V. and Ablynx N.V.; WO 01 / 90190 to National Research Council of Canada; WO 03 / 025020 (= EP 1433793) to Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 to Ablynx N.V., and further published patent applications by Ablynx N.V. as described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) are characterized, inter alia, by the presence of one or more “Hallmark residues” in one or more framework sequences. For further description of Nanobodies, including humanization and / or camelization of Nanobodies, as well as other modifications, portions or fragments, derivatives or “Nanobody fusions”, multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of Nanobodies and their preparation, see for example WO 08 / 101985 and WO 08 / 142164.

[0026] “Domain antibodies” also known as “Dabs”, “Domain Antibodies” and “dAbs” (the terms “Domain Antibodies” and “dAbs” are trademarks of the GlaxoSmithKline group of companies) have been described in e.g. EP 0368684, Ward et al. (Nature 341 : 544-546, 1989), Holt et al. (Tends in Biotechnology 21 : 484-490, 2003) and WO 03 / 002609 and e.g. WO 04 / 068820, WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammals, in particular of humans, 4-chain antibodies. For binding an epitope in a single antigen binding domain, i.e. not paired with a VL or VH domain, respectively, specific selection for such antigen binding properties is required, e.g. by using libraries of human single VH or VL domain sequences. Domain antibodies have a molecular weight of about 13 to about 16 kDa like VHHs and, if derived from fully human sequences, do not require humanization for therapeutic use in e.g. humans. It is also noted that single variable domains can be from certain shark species (e.g. so-called “IgNAR domains”, see e.g. WO 05 / 18629).

[0027] Immunoglobulin single variable domains such as domain antibodies and nanobodies (including VHH domains and humanized VHH domains) represent macromolecules that mature in vivo after their production, but can be further subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs that result in an improved affinity of the resulting immunoglobulin single variable domain to its corresponding antigen as compared to the corresponding parent molecule. Affinity matured immunoglobulin single variable domain molecules of the present invention can be prepared by methods known in the art, for example as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas et al. (Proc. Nat. Acad. Sci, USA 91 :3809-3813, 1994), Shier et al. (Gene 169: 147-155, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154:3310-9, 1995), Hawkins et al. (J. MoI. Biol. 226:889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996). Starting from an immunoglobulin single variable domain such as a domain antibody or nanobody, the process of designing / selecting and / or preparing a polypeptide from the immunoglobulin single variable domain is also referred to herein as "formatting" the immunoglobulin single variable domain; and an immunoglobulin single variable domain that forms part of a polypeptide is referred to as "formatted" or "in the format of the polypeptide". Based on the disclosure herein, the skilled person will be clear on examples of ways in which an immunoglobulin single variable domain can be formatted and examples of such formats that for example avoid glycosylation.

[0028] Immunoglobulin single variable domains, such as domain antibodies and Nanobodies® (including VHH domains) can be humanized, i.e. increased in the degree of sequence identity to the closest human germline sequence. In particular, a humanized immunoglobulin single variable domain, such as a Nanobody® (including VHH domain) can be one in which at least one amino acid residue (and in particular, at least one framework residue) is present and / or corresponds to a humanization substitution (as further defined herein). By comparing the framework region sequences of a naturally occurring VHH sequence to the corresponding framework sequences of one or more closely related human VH sequences, potential useful humanization substitutions can be determined, after which one or more of the thus determined potential useful humanization substitutions (or combinations thereof) can be introduced into the VHH sequence (in any manner known per se, as further described herein), and the resulting humanized VHH sequence can be tested for affinity for the target, stability, ease and level of expression, and / or other desired properties. In this manner, the skilled person can determine other suitable humanization substitutions (or suitable combinations thereof) by limited degrees of trial and error. Furthermore, based on the foregoing, the framework region of an immunoglobulin single variable domain, such as a Nanobody® (including VHH domain) can be partially humanized or fully humanized.

[0029] Humanized immunoglobulin single variable domains, in particular Nanobodies® can have several advantages, for example reduced immunogenicity compared to the corresponding naturally occurring VHH domains. Humanization refers to mutations that render the immunogenicity less or non-existent upon administration in a human patient. The humanization substitutions should be chosen such that the resulting humanized amino acid sequence and / or VHH still retains the advantageous properties of a VHH, for example the antigen binding capacity. Based on the description provided herein, the skilled person will be able to select humanization substitutions or suitable combinations of humanization substitutions that optimize or achieve a desired or suitable balance between the advantageous properties provided by the humanization substitutions on the one hand and the advantageous properties of the naturally occurring VHH domain on the other hand. These methods are known to the person skilled in the art. The human consensus sequence can be used as a target sequence for humanization, but other methods are also known in the art. One alternative includes a method wherein the skilled person aligns a plurality of human germline alleles, for example but not limited to an alignment of IGHV3 alleles, to use said alignment to identify residues suitable for humanization in the target sequence. Furthermore, a subset of the human germline alleles that are most homologous to the target sequence can be aligned as a starting point to identify suitable humanization residues. Alternatively, the VHH is analyzed to identify its closest homologues in human alleles and used for humanization construct design. Humanization techniques applied to camelid VHHs can also be performed by methods comprising replacing specific amino acids individually or in combination. Said replacements can be selected based on what is known from the literature, from known humanization efforts, and from the human consensus sequence compared to the native VHH sequence or the human alleles that are most similar to the VHH sequence of interest. From the data on VHH entropy and VHH variability given in Tables A-5 to A-8 of WO 08 / 020079 it can be seen that some amino acid residues in the framework regions are more conserved between humans and camelids than in other species. In general, although the application is not limited thereto in its broadest aspect, it is preferred that any substitutions, deletions or insertions are made at positions which are less conserved. Also, in general, amino acid substitutions are preferred over amino acid deletions or insertions. For example, camelid single domain antibodies of the human-like class contain hydrophobic FR2 residues that are typically found in conventional antibodies of human origin or from other species, but this loss of hydrophobicity is compensated by other substitutions at position 103 which replaces a conserved tryptophan residue present in the VH of a conventional antibody. Thus, peptides belonging to these two classes display a high degree of amino acid sequence homology to human VH framework regions and said peptides can be directly administered to humans without expecting an unwanted immune response thereby and without the burden of further humanization. Indeed, some camelid VHH sequences display a high sequence homology to human VH framework regions, thus said VHHs can be directly administered to a patient without expecting an immune response thereby and without the additional burden of humanization.

[0030] Suitable mutations, particularly substitutions, can be introduced during humanization to produce peptides with reduced binding to pre-existing antibodies (see, for example, WO 2012 / 175741 and WO2015 / 173325), for example at at least one position: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108. The amino acid sequence and / or VHH of the present invention can be appropriately humanized at any frame residue, for example at one or more marker residues (as defined below) or at one or more other frame residues (i.e., non-marker residues) or any suitable combination thereof. Depending on the host organism used to express the amino acid sequence, VHH, or peptide of the present invention, such deletions and / or substitutions can also be designed by removing one or more post-translational modification sites (e.g., one or more glycosylation sites), which is within the capabilities of those skilled in the art. Alternatively, substitutions or insertions can be designed to introduce one or more sites for attaching functional groups (as described herein), for example, to allow site-specific polyethylene glycol esterification.

[0031] In some cases, substitutions are made for at least one of the typical Camelidae hallmark residues with hydrophilic properties at positions 37, 44, 45, and / or 47 (see Table A-03 of WO2008 / 020079). Another example of humanization includes substitutions of residues in FR1, such as positions 1, 5, 11, 14, 16, and / or 28; substitutions of residues in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93, and / or 94; and substitutions of residues in FR4, such as positions 103, 104, 108, and / or 111 (see Tables A-05-A08 of WO2008 / 020079; all numbered according to Kabat).

[0032] As used herein, an "epitope" refers to an antigenic determinant of a polypeptide that constitutes a binding site or binding pocket on a target molecule (e.g. a protein to which an immunoglobulin or a portion thereof, an antibody, a VHH or an ISVD binds). "Binding" refers to any direct or indirect interaction. Direct interaction refers to contact (e.g. physical or chemical) between two binding partners. Indirect interaction refers to any interaction by which the interaction partners interact in a complex of more than two molecules. The interaction can be entirely indirect (e.g. two molecules are part of the same complex with the help of one or more bridging molecules, but do not bind in the absence of the bridging molecule(s)). The interaction can be partly direct or partly indirect: there is still direct contact between the two interaction partners, but this contact is e.g. unstable and stabilized by interaction with one or more additional molecules. The term "binding pocket" or "binding site" refers to a region of a molecule or a complex of molecules that, because of its shape and charge, associates with another chemical entity, compound, protein, peptide, antibody, single domain antibody or ISVD or VHH.

[0033] An epitope can comprise 1, 2, or 3 amino acids in a spatial conformation that is unique to that epitope. Typically, an epitope consists of at least 4, 5, 6, 7, more typically at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, including, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a "conformational epitope" refers to an epitope comprising amino acids in a spatial conformation that is unique to the folded 3-dimensional conformation of a polypeptide. Typically, a conformational epitope consists of amino acids that are non-contiguous in the linear sequence but which come together in the folded structure of the protein. However, a conformational epitope can also consist of a linear sequence of amino acids that adopt a conformation unique to the folded 3-dimensional conformation of a polypeptide (and which does not exist in a denatured state). In a protein complex, a conformational epitope consists of amino acids that are non-contiguous in the linear sequence of one or more polypeptides that come together when the different folded polypeptides fold and associate to form their unique quaternary structure. Similarly, a conformational epitope can also consist here of a linear sequence of amino acids of one or more polypeptides that come together and adopt a conformation unique to the quaternary structure. The term "conformation" or "conformational state" of a protein generally refers to the range of structures that a protein can adopt at any instant. Those skilled in the art will recognize that determinants of conformation or conformational state include, for example, the primary structure of the protein as reflected in the amino acid sequence of the protein (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as, for example, the secondary structure of the protein (e.g., alpha-helices, beta- sheets, etc.), the tertiary structure (e.g., 3-dimensional folding of the polypeptide chain), and the quaternary structure (e.g., interactions of the polypeptide chain with other protein subunits). Post-translational and other modifications to the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, etc., can influence the conformation of the protein. In addition, environmental factors, such as the pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interactions with other proteins and cofactors, etc., can influence the conformation of the protein. The conformational state of a protein can be determined by functional activity assays or binding to another molecule assays or by physical methods such as X-ray crystallography, NMR, or spin labeling, etc.For a general discussion of protein conformation and conformational states, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

[0034] As used herein, "paratope" refers to the antigen binding site and is the part of an antibody that recognizes and binds to an antigen. Thus the paratope of a TfR binding agent is composed of the amino acid residues of the binding agent that bind the epitope of the TfR protein.

[0035] As used herein, the term "affinity" generally refers to the extent to which an antibody or other binding protein (as further defined herein) binds to a target protein so as to shift the equilibrium of the target protein and the binding protein toward the presence of a complex formed by their binding. Thus, for example, when an antibody and an antigen are combined in relatively equal concentrations, a high affinity antibody will bind the antigen so as to shift the equilibrium toward a high concentration of the resulting complex. The equilibrium dissociation constant KD (or K D ) is commonly used to describe the affinity between a ligand and a target protein, or between an antibody and its antigen. KD is the calculated ratio of k off / k on off between an antibody and its antigen, thus measuring the tendency of the complex to break apart into its component molecules. The association constant (K on or Kon) is used to characterize the speed at which an antibody binds its target. The dissociation constant (k off or koff, also known as kdis, Kdis, Kd, or kd) is used to measure the speed at which an antibody dissociates from its target, and is expressed as the number of units that dissociate from the target per second. Thus, the lower the koff, the higher the affinity for the target. koff, as well as KD, are inversely related to affinity. High affinity interactions are characterized by low KD, fast recognition (high kon), and strong stability of the complex formed (low koff).

[0036] It will be appreciated that within the scope of the present application, the term "affinity" is used in the context of an antibody or antibody fragment that binds to the transferrin receptor TfR epitope, and more particularly, that the antibody or antibody fragment is "functional" in binding its target through the CDR regions of its immunoglobulin (Ig) domains.

[0037] As used herein, "amino acid" refers to the structural units (monomers) that make up proteins. They are linked together to form short polymer chains called peptides or longer chains called polypeptides or proteins. These chains are linear and unbranched, with each amino acid residue within the chain attached to two adjacent amino acids. The twenty amino acids encoded by the universal genetic code are naturally incorporated into polypeptides, referred to as proteinogenic amino acids or natural amino acids. The natural amino acids or naturally occurring amino acids are glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (lie or I), methionine (Met or M), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W), serine (Ser or S), threonine (Thr or T), asparagine (Asn or N), glutamine (Gin or Q), tyrosine (Tyr or Y), cysteine (Cys or C), lysine (Lys or K), arginine (Arg or R), histidine (His or H), aspartic acid (Asp or D), and glutamic acid (Glu or E).

[0038] As used herein, the terms "nucleic acid," "nucleic acid sequence," or "nucleic acid molecule" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs. Nucleic acids can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of nucleic acids include a gene, a gene fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule can be linear or circular. A nucleic acid can include promoters, introns, enhancer regions, polyadenylation sites, translation initiation

[0039] As used herein, "nucleotide sequence," "DNA sequence," or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double-stranded and single-stranded DNA, (reverse) complementary DNA and RNA. It also includes known types of modifications, for example, methylation, one or more of the naturally occurring nucleotides are substituted for by analogs "caps" or "hats." A "nucleic acid construct" refers to a nucleic acid sequence which has been constructed or arranged to contain one or more functional elements which are not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing the COS sequences from lambda phage), viral genomes containing non-native nucleic acid sequences, and the like. A "coding sequence" is a nucleotide sequence which is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' terminus and a translation stop codon at the 3' terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and in some instances can also include introns.

[0040] As used herein, an "expression cassette" comprises any nucleic acid construct capable of directing expression of a gene / coding sequence of interest operably linked to a promoter of the expression cassette. An expression cassette is typically a DNA construct, preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homolog, variant or fragment thereof operably linked to a transcriptional initiation region, and a termination sequence including an RNA polymerase termination signal and a polyadenylation signal. It will be appreciated that all of these regions should be operable in the biological cell, such as a prokaryotic or eukaryotic cell, to be transformed. The promoter region including the transcriptional initiation region, which preferably includes an RNA polymerase binding site, and the polyadenylation signal can be native to the biological cell to be transformed, or can be derived from other sources, where the regions are functional in the biological cell. Such cassettes can be constructed into "vectors". The term "vector" or alternatively "vector construct", "expression vector" or "gene transfer vector" means a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked, and includes any of a number of known vehicles, including any of a number of types, including but not limited to, for example, plasmid vectors, cosmid vectors, phage vectors (e.g., lambda phage), viral vectors (e.g., adenoviral, AAV or baculoviral vectors) or artificial chromosome vectors, such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) or P1 artificial chromosomes (PACs). Expression vectors include plasmids as well as viral vectors, and typically contain the necessary coding sequences and appropriate DNA sequences necessary for the expression of operably linked coding sequences in a particular host organism (e.g., bacteria, yeast, plants, insects or mammals) or in an in vitro expression system. Cloning vectors are typically used for engineering and amplification of a certain desired DNA fragment, but can lack functional sequences required for expression of the desired DNA fragment. Construction of expression vectors for cell transfection is also well known in the art, and can therefore be accomplished by standard techniques (e.g., see Sambrook, Fritsch and Maniatis, in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, E. J. Murray, ed., The Humana Press Inc., Clif ton, N.J.); and see Ambion 1998 Catalog (Ambion, Austin, Tex.).

[0041] In the context of two or more nucleic acid or amino acid sequences, the term "identical" or percent "identity" means that two or more sequences are the same or have a specified percentage of nucleotides or amino acid residues, respectively, identical if aligned for maximum correspondence (introducing gaps if necessary) over the comparison window. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software known in the art are available for obtaining sequence alignments of nucleotide or amino acid sequences.

[0042] The term "percent sequence identity" or "sequence identity %" or "percent identity" or "identity %" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions in the sequence shared by the two sequences, taking into account additions or deletions (i.e., gaps) that must be introduced in order to achieve optimal alignment of the two sequences. A matching position is any position in the target sequence and the reference sequence that is occupied by the same nucleotide or amino acid. Gaps are not counted as nucleotides or amino acids, so gaps in the target sequence are not counted. Likewise, gaps in the reference sequence are not counted because it is the nucleotides or amino acids of the target sequence that are being counted, not the nucleotides or amino acids from the reference sequence.

[0043] One non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, modified as in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain aspects, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Nucleic Acids Res., 25:3389-3402) can also be used. et al. , 1996, Methods in Enzymology(266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are other publicly available software programs that can be used for sequence alignment. In some respects, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using the NWSgapDNA.CMP matrix with vacancy weights of 40, 50, 60, 70, or 90 and length weights of 1, 2, 3, 4, 5, or 6). In some alternatives, the GAP program in the GCG software package (which incorporates the algorithms of Needleman and Wunsch)... J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percentage of identity between two amino acid sequences (e.g., using a BLOSUM 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5). Alternatively, in some respects, the percentage of identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percentage of identity can be determined using the ALIGN program (version 2.0) and using PAM120 with residue tables, vacancy length penalties of 12, and vacancy penalties of 4. Those skilled in the art can determine appropriate parameters to achieve maximum alignment using specific alignment software. In some respects, the default parameters of the alignment software are used.

[0044] Those skilled in the art will appreciate that the generation of sequence alignments for the calculation of percent sequence identity is not limited to binary sequence-sequence comparisons driven exclusively by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com / muscle / . ClustalW2 and MUSCLE are alternatively available from, e.g., EBI (European Bioinformatics Institute). In certain aspects, the percent identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y / Z), where Y is the number of nucleotide residues scored as identical matches in an alignment (as by visual inspection or by alignment by a particular sequence alignment program) of the first and second sequences, and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. Different regions within a single polynucleotide target sequence aligned to a polynucleotide reference sequence can each have their own percent sequence identity. It is noted that percent sequence identity values are rounded to one decimal place. For example, 80.11, 80.12, 80.13, and 80.14 are rounded to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded to 80.2. It is also noted that length values will always be integers.

[0045] According to the present application, the degree of identity between a given reference nucleotide sequence and a nucleotide sequence which is a homologue of said given nucleotide sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Preferably, the degree of identity is given for a nucleic acid region which represents at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the full length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is preferably given for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180 or 200 nucleotides, preferably consecutive nucleotides. In one particular embodiment, the degree / percentage of similarity or identity is given for the full length of the reference nucleic acid sequence.

[0046] The term "amino acid identity" as used herein refers to the extent to which sequences are identical on an amino acid by amino acid basis in a comparison window. Thus, the "percent sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of position at which the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e. the window size), and multiplying the result by 100 to yield the percent sequence identity. According to the present application, the extent of identity between a given reference amino acid sequence and an amino acid sequence which is a homologue of said given amino acid sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Preferably, the extent of identity is given for an amino acid region which represents at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the extent of identity is preferably given for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180 or 200 amino acids, preferably consecutive amino acids. In a particular embodiment, the extent / percentage of similarity or identity is given for the full length of the reference amino acid sequence.

[0047] A "homologue" of a protein includes peptides, oligopeptides, polypeptides, proteins and enzymes which have amino acid substitutions, deletions and / or insertions relative to the unmodified protein in question and which have similar biological and functional activities to the unmodified protein from which they are derived.

[0048] The term "defined by SEQ ID No. X" or "as shown in SEQ ID No. X" as used herein refers to a biological sequence consisting of the amino acid or nucleotide sequence given in SEQ ID No. X. For example, a protein defined by / through SEQ ID No. X consists of the amino acid sequence given in SEQ ID No. X. Another example is an amino acid sequence comprising the amino acid sequence of SEQ ID No. X, which refers to an amino acid sequence that is longer than the amino acid sequence given in SEQ ID No. X but completely contains the amino acid sequence given in SEQ ID No. X (wherein the amino acid sequence given in SEQ ID No. X can be located at the N- or C-terminus of the longer amino acid sequence or can be embedded in the longer amino acid sequence), or which consists of the amino acid sequence given in SEQ ID No. X.

[0049] The term "in vivo medical imaging" refers to techniques and procedures used to visualize the interior of the body (or parts and / or functions thereof) of a living organism for clinical purposes (e.g. disease diagnosis, prognosis or therapy monitoring) or medical science (e.g. anatomical and physiological research). Examples of medical imaging methods include invasive techniques such as intravascular ultrasound (IVUS) and non-invasive techniques such as magnetic resonance imaging (MRI), ultrasound (US) and nuclear imaging. Examples of nuclear imaging include positron emission tomography (PET) and single photon emission computed tomography (SPECT). In a preferred embodiment, a nuclear imaging method is used for in vivo medical imaging. According to one specific embodiment, in vivo pinhole SPECT / micro-CT (computed tomography) imaging is used as in vivo imaging method.

[0050] As used herein, the term "radionuclide" relates to a radiolabel, which is a compound in which one or more atoms have been replaced by a radioisotope. Radionuclides vary based on their characteristics, which include half-life, energy emission characteristics, and type of decay. This allows one to select a radionuclide with a desired mixture of characteristics suitable for diagnostic and / or therapeutic use. For example, gamma emitters are typically used for diagnosis, while alpha and beta emitters are typically used for therapy. However, some radionuclides are both gamma emitters, as well as alpha and / or beta emitters, and thus, can be suitable for both uses. Radionuclides, as used herein, include, for example, but are not limited to, actinium-225, astatine-209, astatine-210, astatine-211, bismuth-212, bismuth-213, bromine-76, cesium-137, carbon-11, chromium-51, cobalt-60, copper-64, copper-67, dysprosium-165, erbium-169, fermium-255, fluorine-18, gallium-67, gallium-68, gold-198, holmium-166, indium-111, iodine-123, iodine-124, iodine-125, iodine-131, iridium-192, iron-59, krypton-81m, lead-212, lutetium-177, molybdenum-99, nitrogen-13, oxygen-15, palladium-103, phosphorus-32, potassium-42, radium-223, rhenium-186, rhenium-188, samarium-153, technetium-99m, radium-223, rubidium-82, ruthenium-106, sodium-24, strontium-89, terbium-149, thallium-201, thorium-227, xenon-133, ytterbium-169, ytterbium-177, yttrium-86, yttrium-90, zirconium-89. In certain embodiments, the radionuclide is selected from the radionuclides described above. In a particular embodiment, the radionuclide is selected from technetium-99m, gallium-68, fluorine-18, indium-111, zirconium-89, iodine-123, iodine-124, iodine-131, astatine-211, bismuth-213, lutetium-177, and yttrium-86.

[0051] For the purposes of the present application, a "patient" or "subject" relates to any organism, such as a vertebrate, particularly any mammal, including a human and another mammal, such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a llama, a pig, or a non-human primate (e.g. a monkey). In one embodiment, the patient is a human, a rat, or a non-human primate. Preferably, the patient is a human. In one embodiment, the patient is a subject suffering from or suspected of suffering from a disease or disorder or an injury. In the context of the present application, the disease is a cancer, more particularly a cancer characterized by tumor cells expressing TfR.

[0052] The term "treatment" and its various parts of speech are used interchangeably and are defined by a therapeutic intervention that slows, interrupts, prevents, controls, stops, reduces, or reverses the progression or severity of signs, symptoms, conditions, ailments, injuries, or diseases, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or ailments. The population requiring treatment includes those already diagnosed with a condition, those predisposed to or susceptible to exposure to the condition, or those requiring prevention of the disease. For example, in the treatment of tumors (e.g., cancer), a therapeutic agent can directly reduce the pathology of tumor cells or make tumor cells more sensitive to other therapeutic agents or treatment by the subject's own immune system.

[0053] As used in this article, the term "therapeutic effective amount" refers to the amount required to achieve the desired outcome when used in treatment.

[0054] As used herein, the terms “diagnosis,” “prognosis,” and / or “prediction” include the diagnosis, prognosis, and / or prediction of a disease and / or condition, thereby predicting the onset and / or presence of a disease and / or condition, and / or predicting the progression and / or duration of a disease and / or condition, and / or predicting the response of a patient with a disease and / or condition to treatment.

[0055] The term "statistically significant" difference is well-known to those skilled in the art. Statistical significance plays a crucial role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained. The null hypothesis is the default hypothesis that nothing has occurred or changed. For a null hypothesis to be rejected, the observed result must be statistically significant, i.e., the observed p-value is less than a pre-specified significance level α. Assuming the null hypothesis is true, the p-value of the result p is the probability of obtaining a result at least as extreme as the current result. In one implementation, α is 0.05. In a more particular implementation, α is 0.01. In even more particular implementations, α is 0.001.

[0056] transferrin receptor

[0057] Transferrin receptors are cell surface receptors essential for the uptake of cellular iron through receptor-mediated endocytosis. These receptors are necessary for erythropoiesis and neural development.

[0058] In this application, unless otherwise stated, "transferrin receptor" or "TfR" are used interchangeably and refer to the human transferrin receptor as described above and in SEQ ID No. 1. The amino acid sequence of the cynomolgus monkey transferrin receptor is shown in SEQ ID No. 10.

[0059] SEQ ID No. 1 (amino acid sequence of human TfR):

[0060] SEQ ID No. 10 (amino acid sequence of cynomolgus TfR):

[0061] The present application provides antibodies and antibody fragments that bind to human transferrin receptor. Developing antibodies against human TfR is part of a promising strategy for targeted therapy and immunotherapy.

[0062] TfR binding agents

[0063] In a first aspect, the present application discloses binding agents, more particularly antibodies, even more particularly single variable domain antibodies, most particularly VHHs, that recognize and bind to human and / or NHP transferrin receptor. These antibodies are thus TfR binding agents. In various embodiments, the TfR binding agents bind to, but do not modulate iron transport. In other embodiments, the TfR binding agents are also able to dissociate from TfR after binding to TfR. This is particularly useful in the process of transferrin receptor mediated transcytosis, in which the transferrin receptor binds to a cargo in the periphery, e.g. of a BBB endothelial cell, transports the cargo through the cell, and releases the cargo on the brain side of the BBB endothelial cell. The TfR binding agents of the present application are thus very useful in the brain delivery of drugs that are administered directly or indirectly in the peripheral blood. The TfR binding agents of the present application can likewise be used to deliver therapeutic and / or imaging compounds to cancer cells. The present application thus also provides compositions comprising a TfR binding agent (see below). The compositions can be pharmaceutical and / or imaging compositions, and the present application contemplates their use in the treatment and / or study of various CNS diseases and / or cancers expressing TfR.

[0064] In various embodiments, the TfR binding agents of the present application comprise a targeting moiety having an antigen recognition domain that recognizes an epitope present on TfR. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on TfR. As used herein, a linear epitope refers to any contiguous sequence of amino acids present on TfR. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on TfR. As used herein, a conformational epitope refers to one or more segments of amino acids (which can be non-contiguous) that form a three-dimensional surface having a signature and / or shape and / or tertiary structure that can be recognized by an antigen recognition domain.

[0065] In one embodiment, the TfR binding agent of the application comprises a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human TfR. In some embodiments, the human TfR comprises the amino acid sequence of SEQ ID No. 1. In more particular embodiments, the human TfR consists of the amino acid sequence of SEQ ID No. 1. In more particular embodiments, the TfR binding agent of the application does not compete with iron transport.

[0066] In one embodiment, the TfR binding agent of the application comprises a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g. VH) and at least three constant regions (e.g. CH1, CH2 and CH3), and each light chain comprises one variable region (VL) and one constant region (CL). As described in the Definitions section above, the variable region determines the specificity of the antibody and comprises three hypervariable regions, also known as complementarity determining regions (CDRs), which contribute to the antibody binding specificity.

[0067] In some embodiments, the TfR binding agent comprises a targeting moiety that is an antibody fragment. The term "antibody fragment" refers to any portion of an antibody or antibody-like structure that itself has high affinity for an antigenic determinant or epitope and contains one or more CDRs, and thus has this specificity. In some particular embodiments, the TfR binding agent of the present application comprises a targeting moiety that is a single-domain antibody, an immunoglobulin single variable domain, a heavy chain-only antibody (VHH), a single-chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a microbe protein (cysteine knot protein, knottin), a DARPin, a Tetranectin, an Affibody, an Affimer, a Transbody, an Anticalin, an AdNectin, an Affilin, a Microbody, a peptide aptamer, an alterase, a plastic antibody, a phylomer, a stradobody, a maxibody, an evibody, a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody, a pepbody, a vaccibody, a UniBody, a DuoBody, an Fv, a Fab, a Fab', a F(ab')2, a peptidomimetic molecule, or a synthetic molecule as described in U.S. Patent Nos. or Patent Publication Nos. US 7,417,130, US 2004 / 132094, US 5,831,012, US 2004 / 023334, US 7,250,297, US 6,818,418, US 2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004 / 146938, US 2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US 2010 / 119446, and / or US 7,166,697, the contents of which are incorporated by reference in their entirety. See also Storz 2011 MAbs 3: 310-317.

[0068] In a particular embodiment, the TfR binding agent of the application comprises a targeting moiety which is a single domain antibody, such as a VHH. The VHH can be derived from, for example, an organism producing VHH antibodies, such as a Camelid, a shark, or the VHH can be a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies (see definition section above). In some embodiments, the single domain antibody described herein is an immunoglobulin single variable domain or ISVD. In a most particular embodiment, the TfR binding agent comprises a targeting moiety which is a VHH.

[0069] In a particular embodiment, the TfR binding agent of the application, more particularly the ISVD or VHH of the application, comprises a CDR3 as set forth in SEQ ID No. 5 or SEQ ID No. 9, or an amino acid sequence having at most two amino acid differences to SEQ ID No. 5 or SEQ ID No. 9 or at most one amino acid difference to SEQ ID No. 5 or SEQ ID No. 9, or comprises a CDR3 comprising or consisting of the amino acid sequence as set forth in SEQ ID No. 5 or SEQ ID No. 9. By "at most two" is meant 0, 1 or 2.

[0070] The CDR3 sequence represents an essential feature of the ISVD family, more particularly the VHH, which binds TfR with specificity at the same binding site. An ISVD family is defined herein as a group of ISVD amino acid sequences having a high similarity or even identity in the CDR3 sequence. By default, ISVDs belong to the same family if they bind the same target epitope. If the expression / stability / affinity / crystallizability of a representative ISVD family is poor, variations in this family can be of interest as small differences, such as single amino acid mutations, occurring in one family can improve these properties.

[0071] In yet another specific embodiment, the TFR binding agent of the present application, more particularly the ISVD or VHH of the present application, comprises a CDR3 as shown in SEQ ID No. 5 or SEQ ID No. 9, or an amino acid sequence which differs from SEQ ID No. 5 or SEQ ID No. 9 by at most two amino acids or which differs from SEQ ID No. 5 or SEQ ID No. 9 by at most one amino acid, or comprises a CDR3 comprising or consisting of the amino acid sequence as shown in SEQ ID No. 5 or SEQ ID No. 9, and / or a CDR2 as shown in SEQ ID No. 4 or SEQ ID No. 8, or an amino acid sequence which differs from SEQ ID No. 4 or SEQ ID No. 8 by at most two amino acids or which differs from SEQ ID No. 4 or SEQ ID No. 8 by at most one amino acid, or comprises a CDR2 comprising or consisting of the amino acid sequence as shown in SEQ ID No. 4 or SEQ ID No. 8, and / or a CDR1 as shown in SEQ ID No. 3 or SEQ ID No. 7, or an amino acid sequence which differs from SEQ ID No. 3 or SEQ ID No. 7 by at most two amino acids or which differs from SEQ ID No. 3 or SEQ ID No. 7 by at most one amino acid, or comprises a CDR1 comprising or consisting of the amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No. 7.

[0072] One embodiment relates to an ISVD of the present application, said ISVD comprising SEQ ID No. 2 or SEQ ID No. 6, or a homologue thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% homology at the amino acid level, or a humanized variant thereof.

[0073] In another embodiment, the present application provides an ISVD comprising the sequence as shown in SEQ ID No. 11, 12, 13, 14,... to SEQ ID No. 31 or the sequence as shown in SEQ ID No. 37, 38, 39,... to SEQ ID No. 68.

[0074] In one specific embodiment, the present application provides a humanized variant of the ISVD as shown in SEQ ID No. 2 or SEQ ID No. 6, wherein said humanized variant is shown in sequences SEQ ID No. 11 to SEQ ID No. 31.

[0075] In another embodiment, the present application provides a variant of the ISVD as shown in SEQ ID No. 2 or SEQ ID No. 6, wherein said variant has an amino acid substitution in CDR1 and / or CDR2 and / or CDR3 to a histidine amino acid, and wherein said variant is shown in sequence SEQ ID No. 37 to SEQ ID No. 68.

[0076] In another embodiment, the present application provides a variant of the ISVD as shown in SEQ ID No. 2 or SEQ ID No. 6, wherein said variant has an amino acid substitution in CDR1 and / or CDR2 and / or CDR3 to a histidine amino acid, and wherein said variant is shown in sequence SEQ ID No. 37 to SEQ ID No. 68, and wherein said variant is a further humanized variant.

[0077] In another embodiment, the present application provides a variant of the ISVD as shown in SEQ ID No. 2 or SEQ ID No. 6, wherein said variant has an amino acid substitution in CDR1 and / or CDR2 and / or CDR3 to a histidine amino acid, and wherein said variant is obtained by one amino acid substitution in CDR1 and CDR2 or CDR1 and CDR2 and CDR3 or CDR2 and CDR3 to a histidine, wherein said variant is a combination of the variants shown in sequence SEQ ID No. 37 to SEQ ID No. 68.

[0078] Table 1 provides an overview of the full length and CDR sequences of the anti-TfR VHHs disclosed herein.

[0079] Table 1. Overview of the full length and CDR sequences of the obtained TfR binding VHHs

[0080] In some embodiments, the TfR binding agent of the present application comprises a targeting moiety that is a VHH comprising a single chain of amino acids having four "framework regions" and three "complementarity determining regions" or CDRs. As used herein, "framework region" refers to a region in the variable domain between CDRs. As used herein, "complementarity determining region" or "CDR" refers to a variable region in a VHH that contains an amino acid sequence capable of specifically binding an antigenic target. In various embodiments, the TfR binding agent comprises a VHH having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. In some embodiments, the CDR1 sequence is selected from SEQ ID No. 3 or 7. In some embodiments, the CDR2 sequence is selected from SEQ ID No. 4 or 8. In some embodiments, the CDR3 sequence is selected from SEQ ID No. 5 or 9.

[0081] In a specific embodiment, a TfR binding agent is provided having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to SEQ ID No. 2, said TfR binding agent comprising three complementarity determining regions (CDR1, CDR2 and CDR3), wherein CDR1 comprises or consists of SEQ ID No. 3, CDR2 comprises or consists of SEQ ID No. 4, and CDR3 comprises or consists of SEQ ID No. 5. In specific embodiments, the differences in the amino acid sequence between the homolog and SEQ ID No. 2 are present in the framework regions. The role of framework regions in specific binding to a target is rather limited, thus allowing for variation of the framework sequence to obtain similar efficacy as ISVDs (see, e.g., De Groeve et al. 2010 J Nuclear Medicine 51:782; Saerens et al. 2005 J Mol Biol 352:597-607). In specific embodiments, the differences in the amino acid sequence are introduced, e.g., for humanization purposes (see below). In more specific embodiments, the differences in the amino acid sequence are limited to substitution of conserved amino acids (see below). In the most specific embodiment, a TfR binding agent is provided, wherein said TfR binding agent is represented by SEQ ID NO. 2.

[0082] In a specific embodiment, a TfR binding agent is provided having an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to SEQ ID No. 6, said TfR binding agent comprising three complementarity determining regions (CDR1, CDR2 and CDR3), wherein CDR1 comprises or consists of SEQ ID No. 7, CDR2 comprises or consists of SEQ ID No. 8, and CDR3 comprises or consists of SEQ ID No. 9. In a further specific embodiment, the differences in the amino acid sequence between the homologue and SEQ ID No. 6 are present in the framework regions. In the most specific embodiment, a TfR binding agent is provided, wherein said TfR binding agent is represented by SEQ ID NO. 6.

[0083] In a specific embodiment, a TfR binding agent is provided having an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to SEQ ID No. 6, said TfR binding agent comprising three complementarity determining regions (CDR1, CDR2 and CDR3), wherein CDR1 comprises or consists of SEQ ID No. 7, CDR2 comprises or consists of SEQ ID No. 8, and CDR3 comprises or consists of SEQ ID No. 9. In a further specific embodiment, the differences in the amino acid sequence between the homologue and SEQ ID No. 6 are present in the framework regions. In the most specific embodiment, a TfR binding agent is provided, wherein said TfR binding agent is represented by SEQ ID NO. 6.

[0084] Humanization

[0085] In one embodiment, the TfR binding agent of the present application comprises an immunoglobulin single variable domain or a VHH that has been "humanized", i.e., one or more amino acid residues in the amino acid sequence of the VHH as obtained by immunization are replaced by one or more amino acid residues occurring at the corresponding position in a VH domain of a conventional 4-chain antibody from a human, to increase the degree of sequence identity to a recent human germline sequence. By comparing the sequences of the framework regions of a naturally occurring VHH sequence to the corresponding framework sequences of one or more closely related human VH sequences, potential useful humanization substitutions can be determined, after which one or more of the thus determined potential useful humanization substitutions (or combinations thereof) can be introduced into the VHH sequence (in any manner known per se, as further described herein), and the resulting humanized VHH sequence can be tested for affinity for the target, stability, ease and level of expression, and / or other desired properties. In this manner, other suitable humanization substitutions (or suitable combinations thereof) can be determined by the skilled person through a limited degree of trial and error. Likewise, based on the foregoing, the framework regions of the immunoglobulin single variable domain (such as a VHH domain) can be partially humanized or fully humanized.

[0086] Thus, in various embodiments, the TfR binding agent of the present application comprises a targeting moiety comprising an amino acid sequence having one or more amino acid mutations relative to SEQ ID No. 2. In various embodiments, the TfR binding agent comprises a targeting moiety comprising an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations relative to SEQ ID No. 2. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and can include conservative and / or non-conservative substitutions. In particular embodiments, the one or more amino acid mutations can be in a CDR (e.g., a CDR1, CDR2, or CDR3 region) of the targeting moiety. In other particular embodiments, the one or more amino acid mutations can be in a framework region (e.g., a FR1, FR2, FR3, or FR4 region) of the targeting moiety. In most particular embodiments, the one or more amino acid mutations are present only in the framework regions of the TfR binding agent.

[0087] For example, "conservative substitutions" can be made in accordance with the similarity of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into six standard groups based on their polar, charge, size, solubility, hydrophobic, hydrophilic, and / or amphipathic properties: (1) hydrophobic: Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, "conservative substitutions" are defined as replacements of an amino acid by another amino acid listed in the same group of the six standard groups described above. For example, replacement of Asp by Glu retains a negative charge in the so modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt alpha-helices.

[0088] As used herein, "non-conservative substitutions" are defined as replacements of an amino acid by another amino acid listed in a different group of the six standard groups described above.

[0089] In various embodiments, substitutions can also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine beta-alanine, GABA, and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminobutyric acid, 4-aminobutanoic acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropanoic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, C alpha-methyl amino acids, N alpha-methyl amino acids, and general amino acid analogs).

[0090] Humanization can be performed using humanization techniques known in the art. In some embodiments, possible humanization substitutions or combinations of humanization substitutions can be determined by methods known in the art, such as, but not limited to, by comparison between the sequence of a VHH and the sequence of a naturally occurring human VH domain. In some embodiments, the humanization substitutions are selected so that the resulting humanized VHH still retains advantageous functional properties. Typically, as a result of humanization, a VHH of the application can become more "human-like" while still retaining advantageous properties compared to the corresponding naturally occurring VHH domain, such as reduced immunogenicity. In various embodiments, a humanized VHH of the application can be obtained in any suitable manner known in the art, and thus is not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VHH domains as starting materials. Indeed, modifications of the amino acid sequence can be achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. These techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

[0091] In various embodiments, the mutations introduced, e.g., for humanization of a TfR binding agent, do not substantially reduce the ability of the TfR binding agent to specifically bind human TfR. In various embodiments, the mutations do not substantially reduce the ability of the TfR binding agent to specifically bind TfR without neutralizing TfR.

[0092] Association kinetics of TfR binding agents

[0093] In various embodiments, the TfR binding agents of the present application can have a binding affinity for the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other naturally occurring or synthetic analog, variant or mutant of human TfR (including monomeric and / or dimeric and / or tetrameric forms) that can be described by an equilibrium dissociation constant (KD), or by an off rate constant koff. In various embodiments, the TfR binding agents comprise a targeting moiety that binds the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other naturally occurring or synthetic analog, variant or mutant of human TfR (including monomeric and / or dimeric and / or tetrameric forms) with a KD of less than 10 µM, or more particularly less than 1 µM and / or greater than 1 nM. In other embodiments, the TfR binding agents of the present application comprise a targeting moiety that binds the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other naturally occurring or synthetic analog, variant or mutant of human TfR (including monomeric and / or dimeric and / or tetrameric forms) with a KD between 1 nM and 1 µM, or between 5 nM and 950 nM, or between 10 nM and 900 nM, or between 20 nM and 850 nM, or between 30 nM and 800 nM, or between 40 nM and 700 nM, or between 50 nM and 600 nM, or between 50 nM and 500 nM. In a more particular embodiment, the KD for human TfR is between 1 nM and 100 nM, or between 2 nM and 75 nM, or between 3 nM and 50 nM, or between 4 nM and 40 nM, or between 5 nM and 30 nM, or between 6 nM and 25 nM, or between 7 nM and 20 nM. In other embodiments, the TfR binding agents comprise a targeting moiety that binds the full-length and / or mature form and / or isoform and / or splice variant and / or fragment and / or any other naturally occurring or synthetic analog, variant or mutant of human TfR (including monomeric and / or dimeric and / or tetrameric forms) with a KD of about 300 nM, about 250 nM, about 275 nM, about 100 nM, about 75 nM, about 50 nM, about 25 nM, or about 15 nM.

[0094] According to another embodiment of the present application, the TfR binding agents of the present application have an affinity for human and NHP TfR ranging from about 1 nM to about 1 µM, or ranging from about 2 nM to about 700 nM, or ranging from about 2 nM to about 60 nM, or ranging from about 20 nM to 300 nM, for example as measured by Bio-Layer Interferometry (BLI) and / or ELISA.

[0095] In various embodiments, the ISVDs or VHHs of the application are not limited to a particular biological source or a particular method of preparation. The ISVD or VHH sequences can generally be produced or obtained by immunizing a Camelid species with a human and / or NHP TfR molecule as appropriate (i.e., so as to generate an immune response against TfR and / or heavy chain antibodies), by obtaining an appropriate biological sample (e.g., a blood sample or any B cell sample) from a Camelid animal, and by using any appropriate known techniques to generate a VHH sequence against TfR starting from the sample. VHHs can also be obtained by expressing a nucleotide sequence encoding a naturally occurring VHH domain, by "humanizing" a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain, by using synthetic or semi-synthetic techniques to make proteins, polypeptides or other amino acid sequences known in the art, by using nucleic acid synthesis techniques known in the art to make a nucleic acid encoding a VHH, followed by expressing the nucleic acid so obtained, and / or by any combination of one or more of the foregoing.

[0096] Production of transferrin receptor binding agents

[0097] The TfR binding agents, particularly the TfR antibodies, more particularly the ISVDs or VHHs, of the application are not limited to a particular biological source or a particular method of preparation. Methods of producing the TfR binding agents of the application are described herein. For example, DNA sequences encoding the TfR binding agents of the application can be readily prepared using techniques known in the art, such as cloning, hybridization screening, and polymerase chain reaction (PCR). Standard techniques of cloning, DNA isolation, amplification, and purification, as well as various separation techniques involving enzymatic reactions of DNA ligases, DNA polymerases, restriction enzymes, and the like, are known and commonly used by those skilled in the art. Many of the standard techniques are described by Sambrook et al. (1989), Maniatis et al. (1982), Wu (ed.) (1993), and Ausubel et al. (1992). Alternatively, DNA sequences encoding the TfR binding agents of the application can be chemically synthesized using methods known in the art. The synthesized DNA sequences can be ligated to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce a genetic expression construct encoding the desired TfR binding agent.

[0098] Accordingly, in various embodiments, the present application provides an isolated nucleic acid comprising a nucleotide sequence encoding a TfR binding agent described herein. One embodiment further discloses an expression cassette comprising the nucleic acid molecule. More particular embodiments disclose expression cassettes in which elements for cell-specific expression or tissue-specific expression are present. Further embodiments relate to vectors comprising the expression cassette or the nucleic acid molecule. More particularly, the vector can be a viral vector, and even more particularly can be a lentiviral or AAV vector.

[0099] To produce a TfR binding agent of the present application, an expression vector comprising a nucleic acid sequence encoding the TfR binding agent can be introduced into a host cell by transfection, transformation, or transduction techniques. Accordingly, in various embodiments, the present application provides a host cell comprising a nucleic acid encoding one of the TfR binding agents of the present application. For example, a nucleic acid encoding a TfR binding agent of the present application can be introduced into a host cell by retroviral transduction. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, yeast cells such as Pichia species, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cells can be grown under conditions that allow the host cells to express the gene encoding a TfR binding agent of the present application.

[0100] Following expression, the TfR binding agent can be harvested and purified using techniques well known in the art, for example, affinity tags such as glutathione-S-transferase (GST) and histidine (His) tags, or by chromatography. The specific expression and purification conditions will vary depending on the expression system used. For example, if the gene is to be expressed in E. coli, it is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter (e.g., Trp or Tac) and a prokaryotic signal sequence. In another example, if the engineered gene is to be expressed in a eukaryotic host cell, e.g., a CHO cell, it is first inserted into an expression vector comprising, for example, a suitable eukaryotic promoter, a secretion signal, enhancers, and various introns. In one embodiment, a TfR binding agent of the present application comprises a His tag, a FLAG tag, and / or a Myc tag. In one embodiment, a TfR binding agent of the present application comprises a His tag and a proteolytic site that allows cleavage of the His tag.

[0101] Therefore, this application also provides host cells comprising one of the TfR binding agents described herein. This application also provides host cells comprising one of the nucleic acid molecules, expression cassettes, or vectors of this application. The host cells can be prokaryotic or eukaryotic. Representative host cells that can be used in this invention include, but are not limited to, bacterial cells, yeast cells, plant cells, and animal cells. Bacterial host cells suitable for this invention include Escherichia coli, Bacillus, Streptomyces, Erwinia, Klebsiella, Serratia, Pseudomonas, and Salmonella species cells. Yeast host cells suitable for this invention include yeast species (…). Saccharomyces ), genus *Fissionyomyces* Schizosaccharomyces ), Kluyveromyces ( Kluyveromyces ), Pichia pastoris ( Pichia (For example, Pichia pastoris) Pichia pastoris )), Hansenula genus ( Hansenula (For example, Hansenula polymorpha) Hansenula polymorpha )), Yersinia genus ( Yarrowia ), Schuwania genus ( Schwaniomyces ), *Schizosaccharomyces*, *Zygosaccharomyces* ( Zygosaccharomyces Species within the range of ) and others. Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Carlsberg yeast ( S. carlsbergensis ) and Kluyveromyces lactis ( K. *Lactis* is the most commonly used yeast host and a convenient fungal host. Animal host cells suitable for this invention include insect cells and mammalian cells (most particularly derived from Chinese hamsters (e.g., CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells and baculovirus-insect cell systems (e.g., review Jarvis 2003 Virology 310:1-7). Non-limiting examples of plant cells include tobacco cells, Arabidopsis cells, tomato cells, corn cells, algal cells, etc. Host cells can be provided in suspension or flask cultures, tissue cultures, organ cultures, etc. Alternatively, the host cell can also be a transgenic animal.

[0102] Animal or mammalian host cells suitable for carrying, expressing, and producing one of the TfR binding agents of this application include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al. 1986 Som Cell Mol Genet 12:555-556; Kolkekar et al. 1997 Biochemistry 36:10901-10909), CHO-K1Tet-On cell line (Clontech), CHO cells with ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, Italy), CHO clone B (GEIMG, Genova, Italy), CHO-K1 / SF cells with ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), and RR-CHOK1 cells with ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub & Chasin 1980 PNAS 77:4216), and dp12.CHO cells (U.S. Patent No. 5,721,121); monkey kidney cells (CV1 ATCC CRL- 1577) transformed by SV40 (e.g., COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, 293T cells, or subclones thereof for growth in suspension culture, Graham et al. 1977 J Gen Virol 36:59, or GnTI KO HEK293S cells, Reeves et al. 2002 PNAS 99:13419); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CRL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse

[0103] The host cells described above can be transiently or stably transfected. Such transfection of DNA, such as a nucleic acid molecule, expression cassette or expression vector, into prokaryotic and eukaryotic cells can be achieved by any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE dextran-, polycation-, or virus-mediated transfection. For all standard techniques, see, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, N.Y.). The host cell can also be a recombinant host cell, which involves a cell that has been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the application. The DNA can be introduced by any method known in the art, suitable for the particular type of cell, including but not limited to transformation, lipofection, electroporation or virus-mediated transduction.

[0104] Furthermore, in an alternative embodiment, use of a nucleic acid molecule, expression cassette or vector as described herein encoding a TfR binding agent, in particular a TfR antibody, more particularly a TfR ISVD or VHH, is provided for the production of said TfR binding agent, antibody, ISVD or VHH. In a particular embodiment, said use is provided for the production of an intrabody. An intrabody or "intracellular antibody" is an antibody or antibody fragment that is heterologously expressed in a specified intracellular compartment, a process made possible by the in-frame incorporation of intracellular trafficking signals. Intrabodies can be expressed in any shape or form, e.g. as intact IgG molecules or Fab fragments, more particularly as genetically engineered antibody fragments, e.g. as single domain intrabodies or VHHs. For review see Zhu and Marasco, 2008 (Therapeutic Antibodies. Handbook of Experimental Pharmacology 181. Ed. Springer-Verlag Berlin Heidelberg).

[0105] Chimeras and fusions

[0106] In various embodiments, any of the TfR binding agents of the present application are provided as part of a chimera or fusion with one or more other active agents. In particular embodiments, the other active agent is a cytotoxic agent, a therapeutic agent, an imaging agent, a radionuclide, an antisense oligonucleotide, an interfering RNA, an antibody or an antibody fragment comprising another VHH. In other specific embodiments, the other active agent is a nanoparticle, a liponanoparticle or an exosome. Alternatively, a composition, more specifically a pharmaceutical composition, is provided comprising any of the TfR binding agents of the present application coupled to one or more other active agents. In one embodiment, the active agent is a chemical entity. As used herein, the term "chemical entity" refers to simple or complex organic and inorganic molecules. Non-limiting examples of chemical entities used in the present application are peptides, peptidomimetics, proteins, antibodies (including antibody fragments such as ISVDs and VHHs), carbohydrates, nucleic acids or derivatives thereof, ligands, substrates, phosphates, agonists, antagonists, neurotransmitters, inhibitors, drugs. In one embodiment, the chemical entity is a biologic, a small molecule, a therapeutic agent, an imaging agent or a test compound.

[0107] As used herein, "biologic" refers to a substance made from a living organism or a product thereof. Biologics can consist of combinations of sugars, proteins, or nucleic acids or complex combinations of these substances, or can be living entities such as cells and tissues. Biologics are isolated from a variety of natural sources - humans, animals, or microorganisms - and can be produced through biotechnological methods and other frontier technologies. A non-limiting example of a biologic is an antibody.

[0108] As used herein, "small molecule" (as in the fields of molecular biology and pharmacology) refers to a low molecular weight (<900 daltons) organic compound that can modulate a biological process. Most drugs are small molecules. Larger structures such as nucleic acids and proteins, as well as many polysaccharides, are not small molecules, although their constituent monomers (ribonucleotides or deoxyribonucleotides, amino acids, and monosaccharides, respectively) are considered to be small molecules. Small molecules can have a variety of biological functions or applications, serving as cellular signal transduction molecules, medical drugs, agricultural pesticides, and many other roles, such as by inhibiting a specific function of a protein or disrupting a protein-protein interaction. These compounds can be natural (such as secondary metabolites) or artificial (such as peptidomimetics).

[0109] As used herein, "therapeutic agent" refers to a substance capable of slowing, interrupting, preventing, controlling, stopping, reducing or reversing the progression or severity of a sign, symptom, disorder, condition, injury or disease, but not necessarily involving the complete elimination of all disease-associated signs, symptoms, conditions or disorders. Non-limiting examples of therapeutic agents are pharmaceutical agents, antibodies, antibody fragments, enzymes, antibiotics, anti-proliferative agents, hormones, neurotransmitters, small molecules.

[0110] An "imaging agent" is a compound that possesses one or more properties that allow for the direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins and small molecule compounds that incorporate a labeled moiety (e.g., fluorescent or radioactive) that allows for detection.

[0111] The term "test compound" is used herein in the context of "drug candidate compounds" or "lead optimization candidate compounds" in therapy, in conjunction with the methods of the present application. Thus, "test compounds" are not used as such in a commercial setting, but rather can be used in lead optimization. These compounds include organic or inorganic compounds that are synthetically derived or from natural sources. The compounds include polynucleotides, lipids, or hormone analogs characterized by low molecular weight. Other biopolymer organic test compounds include small peptides or peptidomimetic molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and larger polypeptides, such as antibodies or antibody conjugates, comprising from about 40 to about 500 amino acids.

[0112] The conjugation between the TfR binding agent of the present application and the compound described above can be achieved by chemical crosslinkers or by creating a fusion protein. The covalent conjugation can be direct or through a linker. In certain embodiments, the direct conjugation is by constructing a protein fusion (i.e., by genetic fusion of two or more genes - encoding one of the TfR binding agents of the present application and one or more other proteins - and expressed as a single protein). In certain embodiments, the direct conjugation is by forming a covalent bond between a reactive group on one or more portions of the TfR binding agent of the present application and a corresponding group or acceptor on the chemical entity (e.g., a neurotropic drug). In certain embodiments, the direct conjugation is by modifying (i.e., genetically modifying) one of the two molecules to be conjugated to include a reactive group (as non-limiting examples, a thiol or a carboxyl group) that forms a covalent attachment to the other molecule to be conjugated under appropriate conditions. As a non-limiting example, a molecule (i.e., an amino acid) having a desired reactive group (i.e., a cysteine residue) can be introduced into, for example, a TfR antibody and a disulfide bond formed with a chemical entity (e.g., a neurotropic drug). Methods of covalently conjugating nucleic acids to proteins are also known in the art (i.e., photocrosslinking, see, e.g., Zatsepin et al. 2005 Russ Chem Rev 74:77-95). Non-covalent conjugation can be through any non-covalent means of attachment, including hydrophobic bonds, ionic bonds, electrostatic interactions, and the like, as will be readily appreciated by one of ordinary skill in the art. Conjugation can also be performed using a variety of linkers. For example, a TfR antibody and a neurotropic drug can be conjugated using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidophenyl) hexanediamine), bis-diazonium bi-sulfite compounds (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). Peptide linkers consisting of one to twenty amino acids linked by peptide bonds can also be used. In certain such embodiments, the amino acids are selected from the twenty naturally occurring amino acids. In certain other such embodiments, one or more of the amino acids are selected from the group consisting of glycine, alanine, proline, asparagine, glutamine, and lysine. The linker can be, for example, a "cleavable linker" that facilitates release of the chemical entity, for example, when delivering a neurotropic drug to the brain or when delivering a therapeutic drug to a cancer cell.Non-limiting examples that can be used are acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0113] According to one specific embodiment, “coupling” can be achieved by generating a multispecific antibody (e.g., a bispecific antibody). Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In one embodiment, the multispecific antibody comprises a first and a second antigen binding site that bind to human and / or NHP TfR. In one embodiment, the second antigen binding site is an antigen, more particularly a brain antigen selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta protein, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau protein, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), TREM2, and caspase 6. In another embodiment, the second antigen binding site is a cancer antigen. A “cancer antigen” or “tumor antigen” refers to an antigenic substance produced in cancer or tumor cells, i.e., it triggers an immune response in the host. Tumor antigens are useful tumor markers for identifying tumor cells with diagnostic tests and are potential candidates for cancer therapy. Non-limiting examples of cancer antigens are MAGE-1, NY-ESO-1a, and BAGE (see Renkvist et al. 2001 Cancer Immunology).

[0114] Therapeutic and diagnostic applications of the TfR binding agents of the present application

[0115] Blood brain barrier shuttles: TfR binding agents as blood brain barrier transporters

[0116] Accordingly, in one aspect of the application, any of the TfR binding agents of the application are provided for use in transporting a chemical entity across the blood brain barrier or for use in transporting a chemical entity to the brain. Accordingly, use of a TfR binding agent of the application in transporting a chemical entity across the blood brain barrier or to the brain is provided. Use of a TfR binding agent of the application for facilitating, effecting, increasing or improving CNS uptake of a chemical entity across the blood brain barrier is also provided. Uptake is increased or enhanced when the amount of the chemical entity in the brain is statistically significantly more, or at least 10%, 15%, 20%, 25%, 50%, 75%, 100%, or at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold more, when the chemical entity is bound to one of the TfR binding agents of the application than when the chemical entity is not bound to one of the TfR binding agents of the application.

[0117] Use of a TfR binding agent of the application as a medicament, for in vivo medical imaging and for treating a neurological disease, neuropathic pain or cancer, in particular a TfR expressing cancer or for preventing brain damage after brain injury is also provided.

[0118] In another embodiment, the TfR binding agent is capable of cross-reacting with human and non-human primate TfR.

[0119] Also provided is a TfR binding agent when coupled to a chemical entity to facilitate uptake of the chemical entity into the central nervous system (CNS) across the blood brain barrier (BBB). The chemical entity can be a biological, a small molecule, a therapeutic agent, a radionuclide, an antisense oligonucleotide, an imaging agent or a test compound. In a particular embodiment, the chemical entity is a neurotensin or a neurotensin analogue. In a most particular embodiment, the TfR binding agent comprises or consists of an antibody or an antibody fragment, more particularly an immunoglobulin single variable domain or VHH.

[0120] Having demonstrated the in vivo BBB shuttling capacity of the antibodies disclosed herein, the present application provides novel human blood brain barrier shuttles. The shuttles are effective in delivering a chemical entity to the brain, more particularly to the CNS. Alternatively, the present application provides a TfR binding agent suitable for delivering a chemical entity to the brain, the binding agent being one of the TfR binding agents disclosed in the present application. Transport of the chemical entity to the brain is significantly increased when compared to transport of the chemical entity which is not part of the shuttle or which is not bound to a TfR binding agent of the present application. In a particular embodiment, the chemical entity is a neuropharmaceutical.

[0121] The blood brain barrier shuttle comprises a TfR binding agent comprising a CDR3 sequence that differs in at most two amino acids from SEQ ID No. 5 or 9, or differs in at most one amino acid from SEQ ID No. 5 or 9, or is as set forth in SEQ ID No. 5 or 9, wherein the dissociation constant koff of the shuttle for human TfR is less than 5x10 -2 / s, more particularly less than 4x10 -2 , 3.5x10 -2 , 3x10 -2 , 2.9x10 -2 , 2.8x10 -2 , 2.7x10 -2 , 2.6x10 -2 , 2.5x10 -2 , 2.4x10 -2 , 2.3x10 -2 , 2.2x10 -2 , 2.15x10 -2 , 2.1x10 -2 , 2x10 -2 , 1.9x10 - 2, 1.8x10 -2 , 1.7x10 -2 , 1.6x10 -2 , 1.5x10 -2 , 1.4x10 -2 , 1.3x10 -2 , 1.2x10 -2 , 1.1x10 -2 , 1x10 -2 , 9x10 -3 , or 8x10 -3 / s, as determined by BLI. In one embodiment, the koff of the shuttle for human TfR is between 3x10 -4 / s and 3x10 -2 / s, or between 5x10 -4 / s and 2x10 -2 / s, or between 8x10 -4 and 1x10 -2 / s, between 9x10 -4 and 9x10 -3 / s, or between 1x10 -3 and 8x10 -3between / s as determined by BLI. In addition to the TfR binding agent, the shuttle comprises a molecule or moiety to be transported to the CNS, more particularly across the BBB. In one embodiment, the TfR binding agent comprises a CDR2 sequence which differs in at most two amino acids from SEQ ID No. 4 or 8, or in at most one amino acid from SEQ ID No. 4 or 8, or a CDR1 sequence which differs in at most two amino acids from SEQ ID No. 3 or 7, or in at most one amino acid from SEQ ID No. 3 or 7, or as shown in SEQ ID Nos. 3 and 7, or more particularly the TfR binding agent comprises or consists of an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 2 or 6 over the full length of said sequence. In a particular embodiment, the differences in the amino acid sequence are outside the CDR regions. The molecule or moiety which is part of the blood brain barrier shuttle can be a neurological disorder drug, an imaging compound, a nanoparticle or an exosome.

[0122] The blood brain barrier shuttle described above can alternatively be referred to as a blood CNS barrier shuttle, a composition or a pharmaceutical composition, or more particularly a BBB shuttle.

[0123] In another aspect, there is provided a blood brain barrier shuttle, a blood CNS barrier shuttle, a BBB shuttle, said composition or said pharmaceutical composition for use as a medicament, more particularly for use in the treatment or diagnosis of a neurological disorder. In one embodiment, the shuttle or composition comprises, in addition to any of the above-mentioned TfR binding agents, a neurological disorder drug, a cancer drug, a nanoparticle or an imaging compound. In a particular embodiment, the neurological disorder drug of the shuttle or composition is a biologic, a small molecule, a therapeutic agent, a radionuclide, an antisense oligonucleotide or a test compound.

[0124] In another specific embodiment, the composition or shuttle is a multispecific antibody comprising a human TfR binding agent as described above and a second antigen binding site that binds a brain antigen. Non-limiting examples of brain antigens are beta-secretase 1 (BACE1), amyloid beta protein, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau protein, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. The multispecific antibody can also comprise a second or further antigen binding site that binds a tumor or cancer antigen. The multispecific antibody is particularly advantageous in the treatment and / or diagnosis of brain cancer.

[0125] In one specific embodiment, the molecule or moiety that is part of the above-described shuttle or composition is neurotensin or a neurotensin agonist. Neurotensin body temperature assay serves as a precise system to evaluate the activity of antibodies to cross the BBB. However, the VHH-neurotensin fusions described herein are also of clinical relevance. First, studies have shown that neurotensin (or neurotensin agonists)-induced hypothermia has potential therapeutic uses. Choi et al. (2012 FASEB J 26:2799-2810) showed that administration of the NT agonist ABS-201 immediately or up to 60 minutes after stroke onset significantly reduced infarct formation and brain cell death in a focal ischemia animal model and effectively promoted long-term functional recovery of animals after stroke. A similar study of NT agonist-induced regulated hypothermia reduced oxidative stress in the brain during reperfusion due to asphyxial cardiac arrest (Katz et al. 2004 Brain Res 1017:85-91). Furthermore, in a rat model of near-drowning, lowering body temperature with neurotensin or NT agonists provided better neurological outcomes than brief external cooling (Katz et al. 2004 Crit Care Med 32:806-810). Thus, a shuttle or composition as described above is also provided that has a Koff for human TfR of between 8x10 4 and 4x10 -2 / s, or between 9x10 -4 and 3x10 -2 / s, or between 1x10 -3 and 2.5x10 -2 / s, or between 1x103 and 2 x 10 -2 / s, or 3 x 10 -4 / s and 3 x 10 -2 / s, or 5 x 10 -4 / s and 2 x 10 -2 / s, or 8 x 10 -4 and 1 x 10 -2 / s, 9 x 10 -4 and 9 x 10 -3 / s, or 1 x 10 -3 and 8 x 10 -3 / s, for treating or preventing stroke, post-stroke brain cell death, or brain injury following brain injury. In a particular embodiment, the shuttle or composition comprises neurotensin or a neurotensin agonist.

[0126] In addition to its ability to induce hypothermia, Nemeroff et al. (1979 PNAS 76:5368-5371) demonstrated that neurotensin is an important modulator of nociceptive sensory transmission and is even more potent than morphine as an analgesic on a molar basis. Neurotensin provides strong analgesia when administered directly into the brain and reverses pain behaviors induced by the development of neuropathic and bone cancer pain in animal models (Demeule et al. 2014 JIC 124:1199-1213). Neurotensin as part of a brain-permeable neurotherapeutic agent (e.g., by conjugation to one of the TfR binders of the present application) is effective for the clinical management of persistent and chronic pain. Thus, also provided are shuttles or compositions described herein comprising neurotensin or a neurotensin agonist, which have a Koff for human TfR of 8 x 10 -4 and 4 x 10 -2 / s, or 9 x 10 -4 / s and 3 x 10 -2 / s, or 1 x 10 -3 and 2.5 x 10 -2 / s, or 1 x 10 -3 and 2 x 10 -2 / s, or 3 x 10 -4 / s and 3 x 10 -2 / s, or 5 x 10 -4 / s and 2 x 10 -2 / s, or 8 x 10 -4 and 1 x 10 -2 / s, 9 x 10 -4 and 9 x 10 -3 / s, or 1 x 10 -3 and 8 x 10-3 Between / s, used to treat neuropathic pain.

[0127] As used in this article, “neuropathy” refers to a disease or condition that affects the central nervous system (CNS) and / or has a cause in the CNS. The “central nervous system” or “CNS” refers to a complex of neural tissues that control bodily functions, including the brain and spinal cord. Exemplary CNS diseases or conditions include, but are not limited to, neurodegenerative diseases (including but not limited to Lewy body disease, Parkinson's disease, taupathies (including but not limited to Alzheimer's disease and supranuclear palsy)), post-poliomyelitis syndrome, Shy-Draeger syndrome, oligopontocerebellar atrophy, multiple system atrophy, striatal substantia nigra degeneration, prions (including but not limited to bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting diseases, and fatal familial insomnia), bulbar palsy, dystonia (including but not limited to DYT1 dystonia), motor neuron diseases (including but not limited to multiple sclerosis, Charcot-Marie-Tooth (CMT) disease), amyotrophic lateral sclerosis (ALS), and heterogeneous neurodegenerative diseases (including but not limited to Canavan disease). Diseases including: Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, Laforadisease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome; dementia (including but not limited to Pick's disease and spinocerebellar ataxia); cancer (e.g., cancers of the CNS and / or brain, including brain metastases from cancers in other parts of the body); neuropathy; amyloidosis; eye diseases or conditions; viral or microbial infections; inflammation; ischemia; epilepsy; behavioral disorders; and lysosomal storage diseases.

[0128] A "neurological disorder drug" is a drug or therapeutic agent that treats one or more neurological disorders. Neurological disorder drugs contemplated in the present application include, but are not limited to, an antibody, a peptide, a protein, a natural ligand of one or more CNS targets, a modified form of a natural ligand of one or more CNS targets, an aptamer, an inhibitory nucleic acid or antisense oligonucleotide (i.e., a small inhibitory RNA (siRNA), a short hairpin RNA (shRNA), or a gapmer), a ribozyme, and a small molecule, or an active fragment of any of the foregoing, which is itself or specifically recognizes and / or acts upon (i.e., inhibits, activates, or detects) a central nervous system antigen or target molecule. A "CNS antigen" or "brain antigen" is an antigen expressed in the CNS (including the brain) that can be targeted by an antibody or small molecule. Non-limiting examples of the CNS antigens or target molecules are amyloid precursor protein or a portion thereof, amyloid beta protein, beta-secretase BACE1, gamma-secretase, Tau protein, alpha-synuclein protein, parkin protein, huntingtin protein, DR6, presenilin 1, presenilin 2, ApoE, glioma or other CNS cancer marker, and neurotrophin. Non-limiting examples of neurological disorder drugs and disorders that can be treated are anti-BACE1 antibodies (e.g., WO2009121948, WO2010146058, WO2012064836) and anti-HER2 antibodies (e.g., trastuzumab) (e.g., WO2003087131).

[0129] In another aspect of the application, there is provided a method of treating a subject, the method comprising the step of administering to the patient a shuttle or (pharmaceutical) composition described above comprising a neurological disorder drug, the shuttle or (pharmaceutical) composition having a koff for human TfR of less than 5x10 -2 / s, or less than 4x10 -2 / s, 3.5x10 -2 / s, 3x10 -2 / s, 2.9x10 -2 / s, 2.8x10 2 / s, 2.7x10 -2 / s, 2.6x10 -2 / s, 2.5x10 -2 / s, 2.4x10 -2 / s, 2.3x10 -2 / s, 2.2x10 -2 / s, 2.15x10 -2 / s, 2.1x10 -2 / s, 2x10 -2 / s, 1.9x10 -2 / s, 1.8x10 -2 / s, 1.7x10 -2 / s, 1.6x10-2 , 1.5 x 10 -2 / s, 1.4 x 10 -2 / s, 1.3 x 10 -2 / s, 1.2 x 10 -2 / s, 1.1 x 10 -2 / s, 1 x 10 -2 / s, 9 x 10 -3 / s, or 8 x 10 -3 / s, as determined by BLI, or a koff for human TfR between 3 x 10 -4 / s and 3 x 10 -2 / s, or between 5 x 10 -4 / s and 2 x 10 -2 / s, or between 8 x 10 -4 / s and 1 x 10 -2 / s, or between 9 x 10 -4 / s and 9 x 10 -3 / s, or between 1 x 10 -3 / s and 8 x 10 -3 / s, as determined by BLI, wherein the subject has a neurological disorder. Also provided are methods of in vivo medical imaging of a body region or tissue, more particularly a brain region, of a subject, the method comprising administering to the subject an effective amount of any of the blood-brain barrier shuttles comprising an imaging compound disclosed herein, and detecting the imaging compound in the body region of the subject. The method further comprises collecting one or more images of the subject and displaying one or more images of the subject. The images can be taken over a period of time, including multiple images over a period of time. The collection and display of the images can be accomplished by commercially available scanners and accompanying computer hardware and software. For example, PET and SPECT scanners can be used. The imaging compound can be any compound that allows for effective in vivo medical imaging. Non-limiting examples are radionuclides, such as technetium (99mTC) or lutetium-177. Also provided are methods of transporting a composition or shuttle described herein from the peripheral bloodstream of a subject to the CSF, more particularly from the basal side of the CPE cells to the apical side, the method comprising the step of administering to the subject any of the shuttles or (pharmaceutical) compositions described herein.

[0130] In one embodiment of the above method, the composition or shuttle is administered to the patient using a route selected from the group consisting of oral administration, nasal administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intradermal administration, topical administration, and enteral administration. In one embodiment, the composition is not administered intracerebrally or intracerebroventricularly or epidurally or is not administered to the brain by any other direct administration.

[0131] Anti-cancer methods: anti-cancer therapies targeting TfR

[0132] Surgery, chemotherapy and radiotherapy have long been considered the best options for cancer treatment. However, these treatments are a form of indiscriminate warfare, accompanied by harmful side effects, and do not prevent the recurrence of cancer cells (Lecocq, 2019). The identification of molecular accelerators of cancer cells, such as HER2, led to the development of molecularly targeted therapies, designed to bind and overcome defective molecules in cancer cells (Lecocq, 2019). Modern cancer treatments and diagnostics focus on targeting and thus specifically delivering high doses of chemotherapeutic drugs or diagnostic agents to tumor sites, while not harming normal tissues and thus overcoming the high systemic toxicity of drugs. Targeted therapies in the clinic require high-affinity, tumor-specific agents and efficient targeting vehicles to deliver therapeutic agents to tumor sites (Xing, 2018).

[0133] As described by Ying Shen et al. (2018) Am. J. Cancer Res. 8(6): 916-931, TfR is an attractive anticancer target.

[0134] Based on the molecular “Trojan horse” principle, anti-tumor prodrugs linked to anti-TfR binding agents as described in the present application can be taken up inside tumor cells expressing TfR. Because TfR quantitatively recycles between the cell surface and intracellular compartments, the TfR-mediated endocytic machinery can be used as a portal for delivering large payloads of anticancer therapeutics (Kalim et al. 2017 Drug Des Devel Ther 11).

[0135] In another aspect, any TfR binding agent of the present application is provided for use in in vivo medical imaging or for use in the treatment of cancer, in particular a TfR-expressing cancer, even more particularly a TfR-expressing cancer selected from the group consisting of ovarian cancer, breast cancer, pleural cancer, lung cancer, cervical cancer, endometrial cancer, colon cancer, kidney cancer, bladder cancer and brain cancer. The present application teaches that for transport across the BBB into the CNS, the TfR binding agents of the present application should have a dissociation constant koff in a specific range. However, for binding to or at the surface of TfR-expressing cancer cells, the specific dissociation constant is not an essential feature. Thus, any TfR binding agent disclosed herein is provided for use in cancer diagnosis and treatment methods, for example by coupling to an anticancer agent or an imaging compound.

[0136] In one embodiment, there is provided a TfR binding agent having a KD for human TfR of 50 nM to 500 nM, which improves uptake of a chemical entity into a cancer cell expressing TfR or improves binding of a chemical entity to the surface of a cancer cell expressing TfR when coupled to the chemical entity. In a specific embodiment, the TfR binding agent is one of the TfR binding agents of the application. In a most specific embodiment, the TfR binding agent is one of the VHHs from the application.

[0137] VHHs have been extensively studied in the context of targeted cancer therapies and immunotherapies. VHHs have been incorporated into various anti-cancer strategies: (1) inhibition of oncogenic signals, (2) delivery of a lethal punch to cancer cells, (3) design of cancer vaccines, (4) engagement of cytolytic, and (5) prevention of immunosuppressive events (Lecocq, 2019).

[0138] VHHs that lack antagonistic properties but target cancer cells have been coupled to other technological platforms to deliver targeted lethal punches to cancer cells (Lecocq, 2019). VHHs have been coupled to death-inducing ligands (e.g., TRAIL), truncated forms of Pseudomonas exotoxin A, multiple drugs and drug-loaded nanoparticles, photosensitizers (i.e., irradiation of photosensitizers with a specific wavelength of light in an oxygen-containing environment leads to the formation of ROS), therapeutic radionuclides (i.e., radiolabels such as lutetium-177, iodine-131, astatine-211, actinium-225, and bismuth-213 can be used to release their energy in the vicinity of cancer cells, thereby causing irreparable DNA damage), and enzymes for prodrug activation (e.g., beta-lactamase can convert the prodrug 7-(4-carboxybutylamido)cephalosporin mustard to phenylenediamine mustard) (Lecocq, 2019). Similar to photosensitizers, branched gold nanoparticles kill cancer cells when excited by NIR light, but do so by generating heat rather than ROS (Lecocq, 2019). VHHs can also bring these toxic moieties in close proximity to cancer cells while minimizing toxic effects on healthy tissues, thus reducing potential side effects (Lecocq, 2019).

[0139] Various bifunctional molecules have been designed (e.g., anti-EGFR VHHs coupled to TRAIL) (Lecocq, 2019). Drugs that are often used to treat various cancer types are cisplatin and its analogs, carboplatin and oxaliplatin, and doxorubicin, RTK inhibitors, and death effectors. Because these drugs lack selectivity, VHHs have been used to target them to cancer cells (Lecocq, 2019).

[0140] In various embodiments, pharmaceutical compositions are provided comprising any TfR binding agent of the present application conjugated to a chemotherapeutic agent for use as a medicament, more particularly for use in the treatment of cancer, even more particularly for use in the treatment of a TfR expressing cancer.

[0141] Non-limiting examples of the chemotherapeutic agents are alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (such as bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; cally statin; CC-1065 (including its synthetic analogs, adozelesin, carzelesin and bizelesin); a cryptophycin (such as cryptophycin 1® and cryptophycin 8); dolastatin; a duocarmycin (including the synthetic analogs, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromophores, e.g., chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin; anti-metabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogues, e.g., denopterin, methotrexate; pteroic acid; trimetrexate;purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; def of amine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE Cremophor-free; albumin-engineered nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, 111.); and doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumz analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including irinotecan in combination with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); lapatinib (Tykerb);inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A (these drugs reduce cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the method of treatment can further comprise the use of photodynamic therapy.

[0142] In some embodiments, the TfR binding agent or pharmaceutical composition described herein acts synergistically when co-administered with another therapeutic agent. In such embodiments, the dose of the TfR binding agent and the other therapeutic agent can be lower than the dose used when the therapeutic agent is used in monotherapy.

[0143] TfR targeting for tumor imaging

[0144] When a cancer is diagnosed, one wishes to know as much as possible about the tumor, for example the presence of tumor antigens that can be targeted and the immune context, to plan and monitor the most effective treatment (Lecocq, 2019). TfR targeting for non-invasive imaging of TfR-positive primary and metastatic tumors allows reliable selection of patients for personalized anticancer treatment with TfR-targeting therapeutic agents and allows systemic monitoring of the TfR expression status of the tumor throughout the treatment period (Cheung et al. 2016 Oncotarget 7). Imaging techniques based on TfR-specific active agents also help surgeons to perform better resections in patients with tumors expressing TfR (Scalrani, 2020).

[0145] One approach is to use a labeled TfR binding agent.

[0146] VHH-based imaging has been extensively studied in preclinical studies to detect cancer cells (e.g. antigens CEA, EGFR, HER2, PSMA, CD20, CD38) (Lecocq, 2019). For clinical purposes, the most advanced VHH-based imaging agent is the68Ga-coupled anti-HER2 nanobody 2Rs15d for PET imaging of BC patients (Lecocq, 2019). The first clinical trial in 2016 showed that HER2 in primary tumors and local or distant metastases can be detected and imaged 60 minutes after injection without side effects such as kidney toxicity and tracer-induced antibodies and is highly specific (Lecocq, 2019). Moreover, the background uptake was very low except for the signals observed in the kidneys, intestines and liver (Lecocq, 2019). Recently, a phase II clinical trial to evaluate the potential of68Ga-NOTA-2Rs15d to detect brain metastases has been initiated (NCT03924466) (Lecocq, 2019). The implementation of VHH-based imaging of cancer markers can guide therapy selection, especially when targeted therapies have been developed for many of these cancer markers, some of which are based on the use of VHHs (e.g. anti-HER2 VHHs for targeted therapy) (Lecocq, 2019). Moreover, VHH-based probes have been developed to image the expression of immune checkpoints (Lecocq, 2019).

[0147] For non-invasive imaging, VHHs need to be labeled with imaging probes that can consist of (1) radioisotopes, (2) fluorescent dyes, (3) microbubbles or (4) chemicals such as gadolinium that allow imaging by techniques such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), optical imaging (OI), ultrasound (US) and MRI (Lecocq, 2019). Most VHH-mediated imaging studies use SPECT and PET as these radioisotope-based techniques have high sensitivity, resolution and provide quantitative information (Lecocq, 2019). In preclinical studies, VHHs usually contain a C-terminal hexahistidine tag inserted for purification purposes that can complex with99mTc (CO3), a gamma-emitting radionuclide that can be easily detected by using SPECT (Lecocq, 2019). For PET, which is more clinically relevant, VHHs are labeled with positron-emitting radionuclides (18F (half-life 68 min),64Cu,68Ga (half-life 110 min) and89Zr) (Lecocq, 2019). The half-lives match the biological half-life of VHHs when injected intravenously (Lecocq, 2019). Site-specific labeling is required to obtain homogeneous and consistent tracers (e.g., transpeptidase sortase A-mediated ligation, catalyzing the formation of a peptide bond between the LPXTG peptide motif expressed at the C-terminus of the VHH and the N-terminal oligoglycine motif on the label) (Lecocq, 2019). An alternative to radiolabeling of VHHs is the use of fluorescent dyes that can be combined with optical imaging (OI). For in vivo imaging, NIR-emitting fluorophores (e.g., IRDye-680RD or IRDye-800CW, Cy5 and AlexaFluor 680) are the label of choice as they provide strong contrast and resolution and enable signal detection at depths ranging from several hundred micrometers (pm) to one centimeter (cm) (Lecocq, 2019). The advantage of OI is its flexibility, simplicity and cost-effective features as it does not require dedicated facilities compared to radioisotope-mediated imaging (Lecocq, 2019). OI is often used to study surface lesions during surgical or endoscopic procedures as OI dyes have limited tissue penetration capacity compared to radioisotope-based imaging (Lecocq, 2019). US can be used as an alternative to radiolabeled VHHs while retaining the ability to provide high-resolution images (Lecocq, 2019). US requires the conjugation of VHHs with US contrast agents, microbubbles or nanobubbles in order to molecularly characterize the vessel wall (after intravenous administration) (Lecocq, 2019). In MRI imaging, VHH-coated superparamagnetic nanoparticles allow antigen detection in xenograft tumors (Lecocq, 2019).

[0148] TfR binding VHHs for therapeutic diagnostics

[0149] An increasingly important modality in precision oncology is the development of theranostics, as it enables patient selection, treatment and monitoring (Lecocq, 2019). In this approach, labeled compounds and imaging techniques are used to diagnose patients and select the best treatment option, while for treatment, relevant compounds are used to target cancer cells or tumor stroma (Lecocq, 2019). In this context, VHHs and VHH-directed therapeutics have attracted interest (Lecocq, 2019). This interest stems from their high antigen specificity, small size, ease of labeling and engineering, allowing specific imaging and design of therapies targeting tumor cells, antigens of immune cells and proteins in the TME (Lecocq, 2019).

[0150] In the field of oncology, there is a growing interest in targeted radionuclide therapy (TRNT), a treatment that can selectively deliver radioactive substances and kill malignant cells while minimizing damage to healthy cells (Ersahin et al., 2011). This therapeutic strategy is gaining more attention due to the wide availability of therapeutic radionuclides (Tomblyn et al., 2012). Radioimmunotherapy (RIT) is a TRNT strategy that uses radiolabeled monoclonal antibodies (mAbs) that interact with tumor-associated proteins expressed on the surface of cancer cells, thus easily accessible by these circulating agents. For the treatment of B-cell non-Hodgkin lymphoma (NHL), RIT consists of radiolabeled anti-CD20 mAbs 90 Y-ibritumomab tiuxetan (Zevalin) and 131 I-tositumomab (Bexxar). Zevalin is now FDA-approved as a late-stage add-on to the unlabeled anti-CD20 mAb rituximab for the treatment of relapsed and refractory NHL. Due to the high radiosensitivity of lymphomas, only a relatively low absorbed dose is required to achieve an objective response. Although recent clinical trials have shown a beneficial effect of the combination of rituximab and Zevalin over rituximab alone (Tomblyn et al., 2012), Zevalin is only approved for late-stage disease (patients with relapsed disease or non-responders to chemotherapy and immunotherapy using rituximab).

[0151] VHHs have superior properties for cell targeting in vivo compared to traditional mAbs and their derived fragments (De Vos et al., 2013). In the context of molecular imaging of cancer, VHHs have been directed against a variety of membrane-bound cancer cell biomarkers, such as CEA, EGFR, HER2 and PSMA (D’Huyvetter et al., 2014). Because VHHs have superior targeting specificity and still function after labeling with radionuclides, VHHs are valuable carriers for nuclear imaging and TRNT (D’Huyvetter et al., 2014).

[0152] Diagnostic tests such as IHC are currently in practice, but cannot depict expression levels throughout the tumor, which is even worse for metastatic lesions (Lecocq, 2019). Indeed, this can explain the failure to accurately predict outcome responses in all patients. Whole-body non-invasive imaging modalities such as PET, SPECT, MRI and OI using VHH-based tracers can remedy these shortcomings and can be implemented repeatedly without the need to collect invasive biopsy samples (Lecocq, 2019). Interestingly, many of the VHHs have the potential to be used as molecular imaging probes as well as therapeutic agents. The term theranostics was initially proposed to describe the development of diagnostic tests and the application of treatments directed to specific molecular features (Lecocq, 2019). Currently, the term theranostics is used in a more strict sense, and more precisely refers to the same or closely related active agents that have potential for both diagnostic and therapeutic purposes (Lecocq, 2019). VHHs targeting cancer-specific membrane proteins such as HER2 have been evaluated for their use in imaging and therapeutic applications (Lecocq, 2019). The clearest example of VHH theranostics is the labeling of both the diagnostic tracer and the therapeutic compound with a radioactive species, i.e. the TRT approach (Lecocq, 2019). The radioactive label can be different (gallium-68 or fluorine-18 for PET imaging, actinium-225 for a-TRT), but sometimes the radioactive label is the same, for example iodine-131 labeled VHHs that are first used in SPECT imaging at low dose for diagnosis and dose estimation, and then at higher dose for TRT (Lecocq, 2019). Importantly, the diagnostic and therapeutic VHH-radio pharmaceuticals have similar pharmacokinetic and biodistribution characteristics (Lecocq, 2019).

[0153] In one embodiment, any of the TfR binding agents of the application are conjugated to a radionuclide. In one embodiment, the TfR binding agent is conjugated or fused to a radionuclide directly or via a conjugation agent and / or linker and / or tag. In a specific embodiment, the TfR binding agent is fused to a radionuclide via a His-tag. Methods for radiolabeling TfR binding agents are all routine methods and known to those skilled in the art. Any available method and chemistry can be used to associate or conjugate a radionuclide to a TfR binding agent. For example, tricarbonyl chemistry can be used for radiolabeling (Xavier et al., 2012). In certain embodiments, the TfR binding agent is conjugated to a radionuclide that is damaging or cytotoxic to cells, and the TfR binding agent targets the radionuclide to cells expressing TfR, preferably to cancer cells. Using a radiolabeled TfR binding agent, for example, but not limited to, to target a damaging radionuclide to cancer tissue, to preferentially damage or kill cancer cells.

[0154] According to specific embodiments, any of the TfR binding agents described herein can be used for targeted radionuclide therapy. As used herein, "targeted radionuclide therapy" refers to the targeted delivery of a radionuclide to a disease site, and subsequent damage of the target cell and neighboring cells (bystander effect). In targeted radiotherapy, also known as systemic targeted radionuclide therapy (STaRT), the biological effect is obtained by the energy absorbed from the radiation emitted by the radionuclide. Non-limiting example radionuclides include iodine-131, astatine-211, bismuth-213, lutetium-177, or yttrium-86. An example radionuclide that can be used to damage cells, such as cancer cells, is a high-energy emitter. For example, a high-energy radionuclide is selected and targeted to cancer cells. The high-energy radionuclide preferably acts over a short distance, such that the cytotoxic effect is localized to the target cell. In this way, the radiotherapy is delivered in a more localized manner to reduce damage to non-cancerous cells.

[0155] The present application also relates to the use of the TfR binding agents described herein for the diagnosis and / or prognosis and / or treatment prediction of a disease in a subject. As a non-limiting example, a subject can be determined to have or be predisposed to cancer based on the level, pattern or profile of expression of TfR in a test sample from the subject compared to a predetermined standard or standard level in a corresponding non-cancer sample. In other words, the TfR polypeptide can be used as a marker to indicate the presence or absence of cancer or the risk of having cancer, as well as to assess the prognosis of cancer and to predict the most appropriate therapy.

[0156] In another related aspect, the application relates to a pharmaceutical composition comprising any of the TfR binding agents described herein and a pharmaceutically acceptable carrier. Thus, the TfR binding agents, alone or coupled to a chemical agent (see above), can be formulated in a physiologically or pharmaceutically acceptable carrier suitable for in vivo administration. In certain embodiments, such compositions are suitable for oral, intravenous, or intraperitoneal administration. In other embodiments, such compositions are suitable for direct local administration to a tumor site. In certain embodiments, such compositions are suitable for subcutaneous administration.

[0157] Therapeutic methods

[0158] In another aspect of the application, there is provided a method of treating a subject, the method comprising the step of administering to the patient a composition comprising one of the TfR binding agents of the application coupled to a cancer drug, wherein the subject is afflicted with a cancer.

[0159] Also provided is a method of binding a TfR binding agent to a cancer tissue, more particularly a cancer tissue expressing TfR, comprising the step of administering to the cancer tissue a composition comprising one of the TfR binding agents of the application. Also provided is a method of directing a compound to a cancer cell or tissue, more particularly a cancer cell or tissue expressing TfR, comprising the step of administering to the cancer cell or tissue a composition comprising a compound coupled to any of the TfR binding agents of the application. In one embodiment, the cancer cell or tissue is present in a mammal, more particularly a human. In another embodiment, the cancer cell or tissue is an in vitro cancer cell or tissue. In another embodiment, the compound is any of the cytotoxic or chemotherapeutic compounds or any of the imaging compounds described herein.

[0160] In one embodiment, there is provided a method of administering or transferring or directing a cancer drug or an imaging compound to a cancer cell expressing TfR, comprising administering to a subject a composition comprising any of the TfR antibodies disclosed herein coupled to the cancer drug or the imaging compound.

[0161] In one embodiment of the above method, the composition is administered to the patient using a route selected from the group consisting of oral administration, nasal administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intradermal administration, topical administration, and enteral administration. In one embodiment, the composition is not administered intracerebrally, or intracerebroventricularly, or epidurally, or not administered to the brain by any other direct means.

[0162] In vivo medical imaging methods

[0163] In another aspect, the present disclosure provides an in vivo medical imaging method. The method comprises administering to a subject (e.g., a human or non-human subject) an effective amount of a labeled TfR binding agent as described herein. An effective amount is an amount sufficient to label the desired cells and tissues so that the labeled structures are detectable during analysis. The method further comprises collecting one or more images of the subject and displaying one or more images of the subject. The images can be taken over a period of time, including multiple images over a period of time. The collection and display of the images is accomplished by commercially available scanners and accompanying computer hardware and software. For example, PET and SPECT scanners can be used. The imaging compound can be any compound that allows for effective in vivo medical imaging. Non-limiting examples are radionuclides, such as technetium (99mTC) or lutetium-177. In addition, to further improve the effectiveness of the images produced, CT, X-ray, or MRI can be used concurrently or sequentially to provide additional information, such as delineation of structural features of the subject. For example, a dual PET / CT scanner can be used to collect the relevant data, and the images are displayed overlaying the data obtained from both modalities. For example, when selecting a radionuclide for in vivo imaging, a gamma or positron emitting radionuclide or a radionuclide that decays by electron transfer can be preferred. Emission can then be readily detected using, for example, positron emission tomography (PET) or single photon emission computed tomography (SPECT). In general, it is desirable for the half-life of the radionuclide to be long enough to prepare and use for testing, but not so long that the radioactivity lingers in the patient for a substantial period of time after testing is performed. In addition, the amount of radioactivity used for labeling can be adjusted so that the desired effect is achieved using a minimum amount of total radiation.

[0164] It is to be understood that while the present cells and methods according to the present application have been discussed in terms of particular embodiments, specific configurations, and materials and / or molecules, that various changes or modifications can be made therein without departing from the scope and spirit of the application. The following examples are provided to better illustrate the particular embodiments and are not to be construed as limiting of the application. The application is to be limited only by the claims.

[0165] Example

[0166] Example 1. Identification of human / cynomolgus TfR binders

[0167] The TfR affinity binders described so far all bind to the apical domain of TfR. However, previous experience from our lab and others has shown that obtaining human and cynomolgus TfR binders is challenging. Despite the fact that the apical domains of human and cynomolgus TfR are 95% homologous, with only 9 amino acids different, it has been shown that this can already result in weak or no binding to the cynomolgus TfR sequence (Kariolis et al. 2020 Sci Transl Med 12). Moreover, we observed that the two residues in the cynomolgus sequence that differ from the human sequence create a glycosylation site that is not present in the human TfR, which can also affect the binding of a biologic to TfR. Therefore, the immunization strategy was reconsidered in this study. To obtain a cross-reactive, BBB-crossing VHH for both human and cynomolgus, a camelid was immunized with DNA encoding llama TfR, in which the sequence encoding the apical region was replaced by the cynomolgus apical region sequence.

[0168] The phage libraries generated after 4 biweekly immunizations of two different camelids were subjected to two rounds of panning on CHO cell lines overexpressing either cynomolgus TfR in the first round or human TfR in the second round. The output libraries were screened with flow cytometry to find binders to human TfR overexpressing cells Figure 1 A). With this strategy, we enriched for human / cynomolgus binders that can bind to TfR in live cells. Two major VHHs, BBB00515 and BBB00533, bound both human and cynomolgus TfR overexpressing cells, while they did not bind mouse TfR overexpressing cells or a control cell line expressing only GFP Figure 1 B-E). The binding kinetics to recombinant human and cynomolgus TfR were further characterized with surface plasmon resonance (SPR). Both BBB00515 and BBB00533 bound immobilized recombinant cynomolgus TfR with similar estimated affinity constants (KD = 63.00 ± 1.20 nM for BBB00515 and KD = 103.77 ± 8.14; Figure 1 F). Both also bound immobilized recombinant human TfR, but with higher KD (KD = 1183.67 ± 423.81 nM for BBB00515 and KD = 207.00 ± 27.84 nM for BBB00533 Figure 1 F).

[0169] Example 2. Anti-human / cynomolgus TfR nanobody shuttles anti-BACE1 mAb into the brain

[0170] BACE1 inhibition in the brain is an example used to evaluate the potential of VHH to cross the BBB and deliver biopharmaceuticals in the brain. BACE1 is responsible for β-secretase cleavage on APP (Sinha et al. 1999 Nature 402, 537-540). A BACE1 inhibitory antibody (Mab 1A11) was able to reduce brain Aβ1-40 levels in vivo without crossing the BBB (Zhou et al. 2011 J Biol Chem 286, 8677-8687). A bispecific antibody with one intact 1A11 arm and VHH on the other arm was engineered and expressed in CHO cells. Figure 2 A). The bispecific antibody BBB00574 carries BBB00515 VHH, while BBB00578 carries BBB00533 VHH. As expected, both bispecific antibodies were still able to bind to hTfR in live cells, but not to the negative control cell line ( Figure 2 BC). Binding to BACE1 was confirmed using biolayer interferometry (BLI), in which biotinylated recombinant human BACE1 protein was immobilized at the tip of a streptavidin-coated biosensor. Figure 2 DF). Two bispecific antibodies bind to human BACE1 at a similar KD of 0.3 nM. Figure 2 F).

[0171] Next, two bispecific antibodies were administered intravenously in a chimeric mouse model, in which the mouse TfR apical domain was replaced with a human sequence (hAPI KI mice) (Wouters et al. 2022 Fluids Barriers CNS 19, 79). The selected injection concentration was 167 nmol / kg, at which no central BACE1 inhibition of mAb 1A11 was observed after peripheral injection (Wouters et al. 2022 Fluids Barriers CNS 19, 79; Zhou et al. 2011 J Biol Chem 286, 8677-8687; Atwal et al. 2011 Sci Transl Med 3). Plasma and brain samples were collected 24 hours later, and Aβ1-40 levels were quantified by ELISA. Compared with mouse samples injected with PBS, the bispecific antibodies BBB00574 and BBB00578 reduced plasma Aβ1-40 levels by 60% and brain Aβ levels by 40%, confirming that the VHHs have the ability to carry biological components across the BBB. Figure 3 ).

[0172] In summary, the results described above clearly demonstrate that the VHHs described herein not only bind to human and NHP TfR, but are also able to deliver biologies across the BBB. Therefore, these VHHs have the potential to be used in the clinic to increase brain penetration of therapeutic and / or diagnostic biologies.

[0173] Example 3. Key optimization of BBB00515 and BBB00533

[0174] Modifications were made to the primary BBB00515 and BBB00533 protein sequences to increase their degree of humanization to human IGHV3 and JH germline consensus sequences, as well as to improve chemical and biophysical stability, while minimizing the impact on target binding and inhibition. To this end, different variants were generated for each primary sequence (variants of BBB00515 are described in SEQ ID NO: 11 to 16, variants of BBB00533 are described in SEQ ID NO: 17 to 31). Figure 6 Sequence alignment results for BBB00515 variants are shown, Figure 7 Sequence alignment results for BBB00533 variants are shown. Residue numbering and CDR partitioning are according to IMGT nomenclature (Lefranc MP and Lefranc G (2023) Computer-aided antibody design 3-59 pages, Springer US). The ability of the variants to compete for binding to human and cynomolgus monkey TfR1 was compared by flow cytometry. The melting (Tm) temperature of the different variants was also determined. Variants were also subjected to temperature stress (1 week at 40°C) in 1 mg / ml PBS solution, followed by analytical size exclusion chromatography (aSEC) to assess the propensity for oligomerization. In addition, they were subjected to long-term temperature stress (1 mg / ml PBS solution, 4 weeks at 40°C) and forced oxidative stress (10 mM H2O2, 3 hours at 37°C), followed by detailed peptidogram mass spectrometry analysis to assess amino acid stability (summary of results obtained is presented in Figure 8 and 9 ). In addition, the following post-translational modifications were evaluated: deamidation (Asn / Gln), isomerization (Asp), oxidation (Met / Trp), N-terminal cyclization (pyroGlu) and C-terminal truncation. Amino acid residues with poor chemical stability (>5% modification after relevant stress) were replaced by suitable substitutes.

[0175] The sequence-optimized variants of BBB00515 with only the HIS6 tag were compared to the reference variant BBB00515_h1 (see Figure 9Accelerated temperature and oxidative stress experiments performed on BBB00515_h1 revealed 10% pyroglutamate cyclization of the N-terminal El residue. For a biological fusion construct with BBB00515 at the N-terminal position, the El D substitution would eliminate this sensitivity without impacting binding. The N82 residue showed minor (6%) deamidation sensitivity. The only other mutation investigated (E84K) was well-tolerated in terms of binding and biophysical characteristics.

[0176] A sequence-optimized variant of BBB00533 with only the HIS6 tag was compared to the reference variant BBB00533_h1 (see Table 1). The sequence-optimized variant of BBB00533 was designated BBB00533_opt. Figure 8 Accelerated temperature and oxidative stress experiments performed on BBB00533_h1 revealed 10% pyroglutamate cyclization of the N-terminal El residue. For a biological fusion construct with BBB00533 at the N-terminal position, the El D mutation would eliminate this sensitivity without impacting binding. Despite the presence of two CDR-based potential aspartate isomerization sites (D62 and D108), accelerated stress experiments revealed no major modifications at these two positions. The N49Q and L68A substitutions had a strong negative impact on binding characteristics, while D82N had only a minor negative impact on binding characteristics. On the other hand, the R72K substitution significantly improved binding characteristics. The L68A substitution improved thermal stability by 7°C.

[0177] Example 4. Comparison with VHH sequences disclosed in WO2020144233

[0178] The main sequence-optimized variants of BBB00515 and BBB00533 were compared to exemplary humanized variants of the TfR1 VHH (described herein as SEQ ID NO: 33, 34, 35, and 36), which are also disclosed in patent application WO2020144233 (Vect-Horus). From a therapeutic biologic development perspective, the Vect-Horus VHHs show a number of serious deficiencies.

[0179] First, good chemical stability characteristics (minimizing drug product heterogeneity from stress-induced post-translational modifications) are a key developable feature. Sequence analysis revealed that the Vect-Horus VHHs have a methionine residue (M106) in their CDR3 sequence. CDR-based methionine residues are known to be susceptible to extensive oxidation. In contrast, the BBB00515 and 533 variants have no CDR-based methionine residues and no major stress-induced post-translational modifications.

[0180] In a next step, we compared the thermal stability (resistance to temperature-induced protein unfolding), another key aspect of biopharmaceutical developability. We found that the Vect-Horus VHH sequences (described here as SEQ ID NO: 33, 34, 35 and 36) have a significantly worse thermal stability profile (Tm values ranging from 64-70°C) compared to the BBB00515 variants (Tm values 84-85°C) and the BBB00533 variants (Tm values 71-77°C).

[0181] Furthermore, non-sticky, good solubility, low oligomerization and low aggregation propensity are also key for developability. In contrast to the sharp and well-defined peaks of BBB00515 and 533, the 4 Vect-Horus humanized variants (VHHA25 (SEQ ID NO: 33), VHHA24 (SEQ ID NO: 34), VHHA22 (SEQ ID NO: 35) and VHHA20 (SEQ ID NO: 36)) show aSCE profiles indicative of stickiness (significant column interactions, leading to longer elution times), product heterogeneity (broad and asymmetric peak shape) and oligomerization (pre-peak) (see Figure 4 ) compared to BBB00533. The Vect-Horus humanized variants have significantly more pronounced Sypro Orange fluorescence spectra at room temperature (see Figure 5 ) compared to BBB00533, indicating higher surface-exposed hydrophobicity in the folded state of the VHHs.

[0182] Example 5 - Engineered variants for improved blood brain barrier shuttling

[0183] The pKa of the histidine side chain is 6.04. Above this pH, histidine has a neutral pH, below this pH, histidine will carry a positive charge. When present in or engineered into antibody CDRs, this can lead to pH-dependent binding of these antibodies. There are many examples in the art showing that antibodies exhibit pH-dependent binding (see Maeda K et al. (2002) J. Control Release 82(1): 71; Klaus T and Deshmukh S (2021 ) J. Biomed. Sci. 28(1): 11 and Schröter et al. (2015) Mabs 7(1): 138). In the context of blood brain barrier shuttling, this can be particularly important, as some blood brain barrier shuttling receptors shuttle this barrier via endosomes (including the transferrin receptor). During this process, endosomes acidify, which can facilitate release of antibodies shuttling molecules bound to such receptors, in turn increasing the efficiency of blood brain barrier crossing, as shown by multiple groups (Yogi A et al. (2022) Pharmaceutics14(7):1452; Edavettal S et al. (2022) MED. 3(12):860 and Esparza Tj et al. (2023) Fluids and Barriers of the CNS 20, 1:64).

[0184] For BBB00515 and BBB00533, individual mutants were generated in which each CDR residue (AbM definition) was replaced by a histidine residue and their pH-dependent binding was assessed. Briefly, DNA was synthesized by Genscript (Piscataway, New Jersey, USA) and cloned into E. coli compatible expression plasmids according to standard methods. E. coli TG1 bacteria were transformed with those plasmids and expression induced. Crude extracts were prepared and binding to human TfR overexpressing cells analyzed by flow cytometry. The top 50% of clones that bound to these cells at neutral pH were selected for re-expression and purification by immobilized metal affinity chromatography. Next, the off-rates of these selected variants were determined on recombinant human TfR by surface plasmon resonance (Biacore, Marlborough, Massachusetts, USA). Biotinylated human TfR was captured on a streptavidin-coated SA sensor chip (Cytiva). Increasing concentrations of nanobodies were sequentially injected at a flow rate of 30 µL / min in a single cycle. The dissociation process was monitored for about 20 min. A reference flow was used as a control to detect non-specific binding and refractive index changes. Double referencing was performed using several buffer blanks. Off-rates were obtained after fitting the experimental data to a 1:1 binding model with Biacore evaluation software. Results are shown in Table 2. Figure 10 and Figure 11 . Figure 12 Detailed information of the histidine mutants of BBB00515 and BBB00533 is shown.

[0185] Dissociation rates were compared to those of BBB00736 and BBB00677 (variants of BBB00515 and BBB00533, respectively, which include some mutations in the framework that do not affect VHH affinity). For BBB00515, the following variants were identified that have improved dissociation rate ratio at pH 7.4 / pH 5.5 compared to BBB00736: BBB00697, BBB00698, BBB00704, BBB00709, BBB00710, BBB00718, and BBB00729, with dissociation rate ratios ranging from 1.79 to 2.84. For BBB00533, the following variants were identified that have improved dissociation rate ratio at pH 7.4 / pH 5.5 compared to BBB00677: BBB00739, BBB00741, BBB00756, BBB00758, BBB00763, BBB00768, and BBB00770, with dissociation rate ratios ranging from 2.63 to 7.24.

[0186] These variants with improved ratios can potentially have better BBB penetration efficiency compared to variants with the corresponding wild-type amino acids at those positions. As shown in Esparza et al. (2023) Fluids and barriers of the CNS 20, no. 1: 64), combining two or more mutations in a single VHH can result in even improved dissociation rates, and thus even more improved BBB penetration.

[0187] Experimental procedures

[0188] Materials and methods

[0189] Animals

[0190] All animal experiments were performed according to protocols approved by the local ethical committee for experimental animals of the University of Leuven (government license LA1210579, ECD project number P213 / 2020) and following government and EU guidelines. Humanized Tfrc mice, which express a chimeric mouse TfR with a human apical domain under the endogenous promoter, were used for this study (Wouters et al. 2022 Fluid Barriers CNS 17, 62).

[0191] Immunization and nanobody library preparation

[0192] A targeting VHH library was obtained in collaboration with VIB Nanobodies core (VIB, Belgium). Three llamas were immunized four times every two weeks with a recombinant pVAX1 plasmid DNA (Thermo Fisher Scientific) encoding a chimeric llama TfR with a cynomolgus monkey apical domain (synthesized at Twist Biosciences) intradermally at multiple sites on the fore and hind limbs near the draining lymph nodes, followed by electroporation. Blood samples were collected at day 4 and day 8 after the last immunization, pooled, and total RNA from peripheral blood lymphocytes was isolated to recover nanobody-encoding genes. Phagemid libraries were prepared following a previous method (Pardon et al. 2014 Nat Protoc 9, 674-693). Briefly, total RNA was used as a template for first-strand cDNA synthesis with oligodT primers. This cDNA was used to amplify by PCR the open reading frames encoding nanobodies, digested with Pstl and Notl, and cloned into a phagemid vector (pBDS001, a modified pMECS vector with a 3xFlag / 6xHis tag insertion at the C-terminus of the nanobody insertion site). Electrocompetent E. coli TGI cells were transformed to obtain nanobody libraries.

[0193] Cell line generation

[0194] A Flp-In™-CHO™ system (Thermo Fisher Scientific) was used to generate stable CHO cell lines overexpressing cynomolgus or human TfR. DNA encoding cynomolgus or human TfR followed by a HA tag and IRES-GFP was synthesized and subcloned by Twist Bioscience into a pcDNA™5 / FRT mammalian expression vector (Thermo Fisher Scientific). Flp-In™-CHO™ cells were maintained with Gibco™ Ham's F-12 Nutrient Mix Medium supplemented with GlutaMAX™ (Thermo Fisher Scientific) and 10% FBS and 100 pg / mL Zeocin™ selection antibiotic (Invivogen) until the day of transfection. Cells were transfected with TransIT-PRO® transfection kit (Mirus) and maintained in Gibco™ Ham's F-12 Nutrient Mix Medium supplemented with GlutaMAX™ (Thermo Fisher Scientific) and 10% FBS and Hygromycin B Gold (Invivogen) to select stable transfectants. Stable transfectants were then expanded and frozen with 10% DMSO for further use.

[0195] VHH selection, expression and purification

[0196] M13 phage libraries displaying VHH were prepared according to standard protocols (Pardon et al 2014 Nat Protoc 9, 674-693) and subjected to two rounds of selection on TfR overexpressing cells. Briefly, 6 x 10 11 cfu of phage and incubated for one hour with aliquots of 5 million cells containing CHO-cynomolgus TfR overexpressing cells for the first selection round or CHO-human TfR overexpressing cells for the second selection round. Five consecutive washing steps with PBS / 10% FBS were performed to discard non-binding phage, while bound phage were eluted by trypsin digestion. The phage library output of the second selection round was subcloned into an expression vector (pBDS119, a modified pHEN6 vector with an OmpA signal peptide and a C-terminal 3xFlag / 6xHis tag) and transformed into E. coli TG1 cells. Individual clones were picked, sequenced and clustered according to sequence homology. In addition, small scale expression of sequenced clones was performed and periplasmic extracts were prepared as previously described (Pardon et al 2014 Nat Protoc 9, 674-693) to screen for direct binding to CHO-human TfR overexpressing cells. Key VHHs were expressed and purified according to the protocol of Pardon et al (Nat Protoc 9, 674-693) by immobilized metal affinity chromatography (IMAC).

[0197] Flow cytometry-based binder screening and validation

[0198] Periplasmic extracts diluted 1 :10 in PBS 2% FBS, or dilution ranges of different VHH or bispecific antibody concentrations prepared in PBS 2% FBS, were incubated with 0.1 million CHO cells overexpressing human, cynomolgus or mouse TfR for 30 minutes at 4°C. As a control for background binding, periplasmic extracts, VHH or bispecific antibodies were also incubated with 0.1 million CHO cells overexpressing GFP. Binding of VHH was next resolved by a second step of incubation with anti-FLAG-iFluor647 antibody (A01811, Genscript) diluted 1 :500 for screening and 1 :250 for validation assays, or anti-human IgG Fc-AlexaFluor647 antibody (410714, Biolegend) diluted 1 :200 for 30 minutes at 4°C. Dead cells were stained with live dye eFluor™780 (1 :2000; 65-0865-14, ThermoFisher Scientific) for 30 minutes at 4°C before analysis after fixation of cells with 4% paraformaldehyde. Flp-In™-CHO™ cells used as un-stained control and single-stained control for the assay were used to determine the cut-off between background fluorescence and positive population. Single-stained controls for anti-FLAG-iFluor647 antibody and anti-human IgG Fc-Alexa Fluor647 antibody were generated using UltraComp eBeads™ compensation microspheres (ThermoFisher Scientific). Data were acquired with an Attune Nxt flow cytometer (Invitrogen) and analyzed by FCS Express 7 Research Edition.

[0199] Surface plasmon resonance

[0200] Surface plasmon resonance (SPR) was used to measure the interaction between VHHs and human or cynomolgus TfR receptors. Human TfR (2474-TR, R&D Systems) and cynomolgus TfR (90253-C07H, Sino Biological) were biotinylated with the EZ-Link NHS-PEG4-biotinylation kit (ThermoFischer Scientific) according to the manufacturer’s instructions. Binding experiments were performed at 25°C in a Biacore T200 instrument (Cytiva, Uppsala, Sweden) in HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v / v Surfactant P20). Biotinylated human TfR and cynomolgus TfR were captured on a SA sensor chip (Cytiva) at a density of about 250 RU. Increasing concentrations of VHHs were injected sequentially at a flow rate of 30 μl / min in a single cycle. Dissociation was monitored for 20 minutes. No special regeneration was necessary. A reference flow liquid was used as a control to detect non-specific binding and refractive index changes. Double referencing was performed using several buffer blanks. After fitting the experimental data to a 1:1 binding model using the single-cycle kinetics method with Biacore T200 evaluation software 3.1, the binding affinity (KD) and kinetic rate constants (kon, koff) were obtained. Each interaction was repeated at least three times.

[0201] Bio-layer interferometry

[0202] Binding of bispecific antibodies to BACE1 was evaluated with Octet RED96 (Forté Bio / Molecular Devices). Briefly, streptavidin (SA) biosensors (18-5020, FortéBio / Molecular Devices) were pre-wetted in kinetics buffer for at least 10 minutes. Next, the biosensors were immersed in biotinylated BACE1 (5 μg / ml in kinetics buffer). BACE1 (Protein Service Facility, VIB) was biotinylated with the EZ-Link NHS-PEG4-biotinylation kit (ThermoFischer Scientific) according to the manufacturer’s instructions. The biosensors were then sequentially immersed in baseline wells containing kinetics buffer, wells containing bispecific antibodies diluted in kinetics buffer, and finally back in baseline wells to evaluate dissociation. Data were recorded using Forté Bio Octet RED analysis software (Forté Bio / Molecular Devices) and sensorgrams were generated using Graphpad.

[0203] Bispecific engineering and expression

[0204] 1A11WT-2xNb62 and 1A11WT are custom-made by GenScript Biotech. Figure 1 A), where 1A11 is our internally developed anti-BACE1 antibody (Zhou et al. 2011 J Biol Chem 286, 8677-8687). In short, the heavy chain (mouse IgG2a) and light chain (mouse κ) were cloned into the mammalian expression vector pcDNA3.4, expressed from Expi293F™ human cells (A14527, ThermoFischer Scientific), and purified using a HiTrap® MabSelect™ column (GE11-0034-93, Cytiva). Purity was assessed by densitometric analysis of an SDS-PAGE gel stained with Coomassie Brilliant Blue under non-reducing conditions, yielding 75% (1A11WT-2xNb62). DNA encoding 1A11AM-Nb62 (1A11AM is a humanized version of 1A11WT), 1A11AM-Nb188, and 1A11AM-aGFP (aGFP is an anti-green fluorescent protein (GFP) nanobody) was synthesized by Twist Bioscience (CA, USA) and cloned into its pTwist CMV BetaGlobin WPRE Neo vector: Nb62-Fc, Nb188-Fc, and aGFP-Fc (human IgG1, L234A, L235A, P329G, T350V, T366L, K392L, T394W), 1A11AM heavy chain (human IgG1, L234A, L235A, P329G, T350V, L351Y, F405A, Y407V), and 1A11AM light chain (human κ). 1A11AM and 1A11WT bind to BACE1 with similar affinity. Antibodies were expressed in Hek293F cells using X-tremeGENE™ HP DNA transfection reagent (6366546001, Merck) and purified according to the protocol of Nesspor et al. (2020 Sci Rep10, 7557). The purification protocol consisted of protein A purification followed by purification on a CaptureSelect™ CH1-XL pre-packed column (494346205, ThermoFischer Scientific).

[0205] Sample collection, Aβ extraction and ELISA

[0206] Mice were euthanized by intraperitoneal overdose injection of Dolethal (150-200 mg / kg). To harvest plasma, blood was collected by cardiac puncture with a pre-filled heparin syringe. Next, blood samples were centrifuged at 2000 g for 10 minutes and plasma was collected. Brains were harvested after transcardial perfusion with heparinized PBS.

[0207] Mouse Abeta1-40 samples from brain and plasma were prepared according to Serneels et al. (2020 Mol Neurodegener 15, 60). In short, the cerebral hemispheres of each mouse were homogenized in a buffer containing 20 mM Tris, 250 mM sucrose, 0.5 mM EDTA, 0.5 mM EGTA (pH 7.4 HC1) supplemented with cOmplete™ protease inhibitor cocktail (Roche) using a bead beater. Next, soluble Abeta1-40 was extracted by treatment with 0.4% diethylamine for 30 minutes at 4°C, high-speed centrifugation (100000 g, 1 h, 4°C) and neutralization with 0.5 M Tris-HCl (pH 6.8). Abeta1-40 levels were quantified by ELISA using MesoScale Discovery (MSD) 96-well plates and antibodies provided by Janssen Pharmaceutica. MAb JRFcAβ40 / 28, which recognizes the C-terminus of Abeta1-40, was used as capture antibody, while JRF / rAβ / 2, labeled with sulfoTAG, was used as detection antibody.

[0208] Protein thermal stability measurements

[0209] Two techniques were used to determine the melting temperature (Tm) of the proteins.

[0210] Sypro Orange (Invitrogen; Waltham, MA, USA) binding during temperature-induced unfolding was monitored in an Uncle or QuantStudio 5 qPCR (Thermo Fisher; Waltham, MA, USA) instrument. 10 mΐ (Uncle) or 20 mΐ (qPCR) samples were tested at 0.75 mg / ml (Uncle) or 0.2 mg / ml (qPCR) concentration and 10x Sypro Orange concentration. Linear ramping from 25 °C to 95 °C (Uncle) or 25 °C to 99 °C (qPCR) was initiated at a rate of 0.5 °C / min (Uncle) or 0.05 °C / min (qPCR). A pre-run incubation of 180 s (Uncle) or 15 s (qPCR) was performed and excitation occurred at 473 nm (Uncle) or 520 ± nm (qPCR). The area under the curve of the fluorescence emission spectrum (Uncle) or 558 ± 11 nm (qPCR) signal was plotted against temperature. The inflection point of protein semi-unfolding (Infliction point) = Tm was derived from the local minimum / maximum of the first derivative curve.

[0211] Intrinsic tryptophan fluorescence during temperature-induced protein unfolding was monitored using an Uncle instrument (Unchained Labs; Pleasanton, CA, USA). Here, 10 mΐ samples were loaded into the sample cell at 1 mg / mL, linear ramping from 25 °C to 95 °C was initiated at a rate of 0.5 °C / min, a pre-run incubation of 180 s was performed, and the back center mean (BCM) and static light scattering (SLS, at 266 nm and 473 nm) signals were plotted against temperature to obtain Tm and the onset of aggregation temperature (T agg ), respectively.

[0212] Protein surface hydrophobicity measurements

[0213] The relative surface hydrophobicity of the proteins in the folded state was assessed as follows (Munch and Bertolotti, 2010). Sypro Orange (Invitrogen; Waltham, MA, USA) binding to hydrophobic patches was measured on an Uncle instrument (Unchained Labs; Pleasanton, CA, USA) at 25 °C. 10 pL of sample at a concentration of 1 mg / ml were tested at a 10x Sypro Orange concentration. After a pre-incubation of 180 s, a linear temperature ramp between 25 °C and 26 °C at a rate of 0.1 °C / min was applied. The fluorescence signal was detected in the wavelength range between 250 nm and 727 nm, and the measurement at 25 °C was reported.

[0214] Aggregation assays

[0215] Protein samples at a concentration of 20 pL at 1 mg / mL were injected onto an Agilent SEC3 (4.6 mm x 300 mm) (Mw 500-150,000) column on an Agilent HPLC system (Agilent, Santa Clara, CA, USA) for analytical size exclusion chromatography (aSEC). The samples were run in PBS at a flow rate of 0.4 mL / min. The outlet of the column was coupled to a UV detector. Retention time, recovery, and percent front were measured by UV280.

[0216] Chemical stability

[0217] Samples were stored at -80°C for reference, while temperature-stressed samples were stored at 40°C for 4 weeks. To forcedly oxidized samples (1 mg / mL in PBS) hydrogen peroxide was supplemented to a final concentration of 10 mM, followed by incubation at 37°C for three hours, final buffer exchange to phosphate buffered saline (PBS) using PD MidiTrap G-25 columns (GE Healthcare; Chicago, IL, USA) according to the manufacturer’s instructions, and storage at -80°C. Peptide mass analysis was performed at the Research Institute for Chromatography (RIC, Kortrijk, Belgium). Samples were reduced with dithiothreitol, alkylated with iodoacetamide, followed by proteolytic digestion with trypsin and LysC (overnight at 25°C). Digested samples were analyzed on RPLC-MS using C18 RPLC columns. RPLC was performed with formic acid (FA) as additive, H2O and acetonitrile as mobile phases. Analysis was performed on a 1290 Infinity UHPLC system (Agilent Technologies) coupled to a 6545 Q-TOF mass spectrometer (Agilent Technologies) operating in MS and MS / MS mode. Data processing was performed using BioConfirm 10.0 and MassHunter 7.0 (Agilent Technologies). Measured signals were matched to the sequence. Identification was based on MS-only data. The specified enzymes were trypsin (cleavage at lysine or arginine C-termini) or LysC (cleavage at lysine C-termini), and 0-2 missed cleavage sites were allowed. N-terminal cyclization (pyroglutamic acid from E), D-isomerization, N / Q deamidation and M / W oxidation were considered as variable modifications, while cysteine carbamidomethylation (related to sample preparation) was considered as a fixed modification. Peak areas of extracted ion chromatograms (EIC) were used for quantification of modifications.

[0218] Sequence

[0219] SEQ ID No. 2 (BBB00533 full length sequence)

[0220] SEQ ID No. 3 (CDR1 of BBB00533)

[0221] GRTFNYAMG

[0222] SEQ ID No. 4 (CDR2 of BBB00533)

[0223] TIDWKDGSSY

[0224] SEQ ID No. 5 (CDR3 of BBB00533)

[0225] GDGDYCSTYTCAAEVEYDY

[0226] SEQ ID No. 6 (full length sequence of BBB00515)

[0227] SEQ ID No. 7 (CDR1 of BBB00515)

[0228] GSIFSINAMG

[0229] SEQ ID No. 8 (CDR2 of BBB00515)

[0230] VITSGGSTI

[0231] SEQ ID No. 9 (CDR3 of BBB00515)

[0232] HVGLKVPTIQELSLGFGS

Claims

1. A transferrin receptor (TfR) binding agent capable of binding human and non-human primate TfR, which facilitates uptake of a chemical entity into the central nervous system (CNS) when coupled to said chemical entity across the blood brain barrier (BBB), wherein the TfR binding agent comprises a CDR3 sequence consisting of an amino acid sequence that differs from SEQ ID No. 5 or 9 by at most two amino acids, and / or wherein the TfR binding agent comprises a CDR2 sequence consisting of an amino acid sequence that differs from SEQ ID No. 4 or 8 by at most two amino acids, and / or wherein the TfR binding agent comprises a CDR1 sequence consisting of an amino acid sequence that differs from SEQ ID No. 3 or 7 by at most two amino acids.

2. The TfR binding agent according to claim 1, wherein the CDR3 sequence is as set forth in SEQ ID No. 5 or 9 and / or wherein the CDR2 sequence is as set forth in SEQ ID No. 4 or 8 and / or wherein the CDR1 sequence is as set forth in SEQ ID No. 3 or 7.

3. The TfR binding agent according to any one of the preceding claims, comprising an amino acid sequence that is at least 90% identical to SEQ ID No. 2 or 6 over the full length of the sequence.

4. The TfR binding agent according to claim 3, comprising an amino acid sequence as set forth in SEQ ID No. 11, 12, 13, 14... to SEQ ID No. 31 or a sequence as set forth in SEQ ID No. 37, 38, 39... to SEQ ID No. 68 or a sequence as set forth in SEQ ID No. 2 or a sequence as set forth in SEQ ID No.

6.

5. The TfR binding agent according to any one of the preceding claims, wherein the chemical entity is a biological, a small molecule, a therapeutic agent, a radionuclide, an antisense oligonucleotide, an imaging agent or a test compound.

6. The TfR binding agent according to any one of the preceding claims, wherein the chemical entity is a neurotensin, a neurotensin analogue or an anti-BACE1 antibody.

7. The TfR binding agent according to any one of the preceding claims, wherein the binding agent comprises or consists of an antibody or an antibody fragment, more particularly an immunoglobulin single variable domain or VHH.

8. A blood-central nervous system (CNS)-barrier shuttle comprising a TfR binding agent according to any one of the preceding claims, further comprising a molecule to be transported to the CNS, more particularly across the BBB.

9. The blood CNS-barrier shuttle according to claim 8, wherein the molecule is a neurological disorder drug, a cancer drug or an imaging compound.

10. The blood CNS-barrier shuttle according to any one of claims 8-9, wherein the blood CNS-barrier shuttle is a BBB shuttle.

11. The TfR binding agent according to any one of claims 1-7 or the blood CNS-barrier shuttle according to any one of claims 8-10 for use as a medicament.

12. The TfR binding agent according to any one of claims 1-7 or the blood CNS barrier shuttle according to any one of claims 8-10 for use in transporting one or more compounds to the CNS, more particularly across the BBB.

13. The TfR binding agent according to any one of claims 1-7 or the blood CNS barrier shuttle according to any one of claims 8-10 for use in the treatment of a neurological disorder.

14. The TfR binding agent according to any one of claims 1-7 or the blood CNS barrier shuttle according to any one of claims 8-10 for use according to claim 13, wherein the neurological disorder is selected from the group consisting of Alzheimer’s disease, stroke, dementia, muscular dystrophy, multiple sclerosis, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, dystonia, Parkinson’s disease, viral or microbial infection, inflammation, brain cancer, neuropathic pain, and traumatic brain injury.

15. A nucleic acid molecule encoding the TfR binding agent or blood CNS barrier shuttle of any one of the preceding claims.

16. A vector comprising the nucleic acid molecule according to claim 15.

17. A host cell comprising the nucleic acid molecule according to claim 15 or the vector according to claim 16.

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