Anti-transferrin receptor compositions and methods thereof

Abstract

The application describes anti-TfR antibodies and antigen-binding fragments thereof for delivering an agent to the brain of a subject in need thereof are described. Also described are conjugates and fusion constructs containing the anti-TfR antibody or antigen-binding fragment thereof coupled to a therapeutic or diagnostic agent, such as a second antibody and antigen- binding fragment thereof, for treating or detecting a neurological disorder and / or delivering a therapeutic or diagnostic agent across the blood-brain barrier. Also described are nucleic acids.

Description

[0001] ANTI- TRANSFERRIN RECEPTOR COMPOSITIONS AND METHODS THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority of US Patent Application No. 63 / 553,699, filed on February 15, 2024, and US Patent Application No. 63 / 645,431, filed on May 10, 2024, which are incorporated by reference herein, in their entireties and for all purposes.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a blood-brain barrier shuttle that binds to the transferrin receptor (TfR) and methods of using the same.

[0006] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0007] This application contains a sequence listing, which is submitted electronically. The information contained in the electronic sequence listing (Sequence listing_JBI6864WOPCT_SL.xml; size: 2.127MB; and date of creation: February 11, 2025) is incorporated herein by reference in its entirety.

[0008] BACKGROUND

[0009] While the blood-brain barrier (BBB) prevents harmful substances from entering the brain and is essential for brain homeostasis, it presents a formidable obstacle for efficiently delivering drugs to the brain. Large molecules, such as monoclonal antibodies and other biotherapeutics, have great therapeutic / diagnostic potential for treating / detecting pathology in the central nervous system (CNS). However, their route into the brain is prevented by the BBB. Previous studies have illustrated that only a very small percentage (approximately 0.1%) of an IgG injected in the bloodstream are able to penetrate the BBB into the CNS compartment (Felgenhauer, Klin. Wschr. 52: 1158-1164, 1974)). This will limit any pharmacological effect due to the low concentration within the CNS of the antibody.

[0010] Numerous approaches have been studied to improve the brain delivery of therapeutic monoclonal antibodies (mAbs), including the use of receptor-mediated transcytosis (RMT). RMT utilizes abundantly expressed receptors on the luminal side of the BBB for transport through brain endothelial cells. Previous efforts to generate a clinically feasible platform for delivery of therapeutic mAbs into the brain have been focused on antibody engineering to increase the efficiency of transcytosis, with gains made through observations on valency of binding, pH dependency and affinity (reviewed in Goulatis et al., 2017, Curr Opin Struct Biol 45: 109-115). However, translation into NHPs and the clinic has been limited by rapid peripheral clearance from target-mediated drug disposition (TMDD) and safety from acute reticulocyte depletion (Gadkar, 2016, Eur J Pharm Biopharm. 2016 Apr;101 : 53-61 ). Transferrin receptor (TfR), particularly TfRl, mediates the transport of iron-loaded transferrin (Tf) from blood to brain and the return of iron-depleted Tf to the blood (Kawabata, Free Radical Biology & Medicine, 133, 46-54, 2019). Anti-TfRl monoclonal antibodies have been used to deliver drugs to the brain (see, e.g., Burkhart, et al. Progress in neurobiology, 181, 101665, 2019; US 2023 / 0174646). However, safety liabilities and poor pharmacokinetics (PK) of anti-TfRl monoclonal antibodies may hamper their clinical development as BBB carriers.

[0011] Therefore, there is still a need for an anti-TfR monoclonal antibody or antigen binding fragment thereof that can be used to shuttle drugs into the brain efficiently with improved safety and PK.

[0012] SUMMARY OF THE INVENTION

[0013] In one general aspect, the application describes an anti-TfR antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL) comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, and a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein:

[0014] (1) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 222-308 and 1456-1485;

[0015] (2) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 309-359 and 1486-1511;

[0016] (3) the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 360-518 and 1512-1561;

[0017] (4) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 519-645 and 1562-1606;

[0018] (5) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 646-792 and 1607-1661; and (6) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 793-980 and 1662-1717.

[0019] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the LCDR1 , LCDR2 and LCDR3 have the amino acid sequences of the LCDR1, LCDR2 and LCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

[0020] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1198-1420 and 1776-1832.

[0021] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the VL comprises the amino acid sequence of the light chain variable region of any one of the antigen binding proteins specified in Table 6.

[0022] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the HCDR1 , HCDR2 and HCDR3 have the amino acid sequences of the HCDR1, HCDR2 and HCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

[0023] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an ammo acid sequence selected from the group consisting of SEQ ID NOs: 981-1197 and 1718-1775.

[0024] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the VH comprises the amino acid sequence of the heavy chain variable region of any one of the antigen binding proteins specified in Table 6.

[0025] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the LCDR1, LCDR2 LCDR3, HCDR1, HCDR2, and HCDR3 have the amino acid sequences of the LCDR1, LCDR2 LCDR3, HCDR1, HCDR2, and HCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

[0026] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the VL and VH comprise the amino acid sequences of the light chain variable region and the heavy chain variable region, respectively, of any one of the antigen binding proteins specified in Table 6.

[0027] In other embodiments, the application relates to an anti-TfR antibody or antigen-binding fragment thereof, having a single-chain variable fragment (scFv) comprising the VH and VL of the anti-TfR antibody or antigen-binding fragment thereof.

[0028] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the scFv further comprises a linker that covalently links the VH and VL, such as a linker having the amino acid sequence selected from the group consisting of SEQ ID NO: 1421 and SEQ ID NOs: 1424-1455.

[0029] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the scFv comprises one or more disulfide bonds formed between a cysteine (Cys) in the linker and a surface exposed Cys in VH and / or VL, preferably a surface exposed Cys in a framework region of the VH and / or VL.

[0030] In another general aspect, the application describes an anti-TfR antibody or antigenbinding fragment thereof having a single variable domain on a heavy chain (VHH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein:

[0031] (1) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24;

[0032] (2) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-46; and

[0033] (3) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-70.

[0034] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, wherein the HCDR1 , HCDR2 and HCDR3 have the amino acid sequences of the HCDR1, HCDR2 and HCDR3, respectively, of any one of the VHH antigen binding proteins specified in Table 3.

[0035] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-221. In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof comprises one or more amino acid modifications that increases its stability. The one or more amino acid modifications can be made using site-directed mutagenesis, for example, as described in Manning, M.C., Chou, D.K., Murphy, B.M. et al. Stability of Protein Pharmaceuticals: An Update. Pharm Res 27, 544-575 (2010). Forced degradation studies such as described in Nowak C, K Cheung J, M Dellatore S, Katiyar A, Bhat R, Sun J, Ponniah G, Neill A, Mason B, Beck A, Liu H. Forced degradation of recombinant monoclonal antibodies: A practical guide. MAbs. 2017 Nov / Dec;9(8): 1217-1230 can be used to identify risks (e.g., risks associated with post-translational modifications) that may contribute to lower stability. Site-directed mutagenesis to reduce or eliminate such risks may be employed. The relevant disclosures of all such references are incorporated herein by reference.

[0036] In certain embodiments, the application relates to an anti-TfR antibody or antigenbinding fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-221.

[0037] In another aspect, the application relates to a conjugate comprising the anti-TfR antibody or antigen-binding fragment thereof of the application coupled to a therapeutic or diagnostic agent, preferably, the conjugate is a multi-specific antibody comprising a first antigen binding region which binds the TfR and comprises the anti-TfR antibody or antigen-binding fragment thereof and a second antigen binding region which binds a brain target. In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof of the application is coupled to a therapeutic or diagnostic agent, where the therapeutic or diagnostic agent is a polynucleotide.

[0038] In certain embodiments, the application relates to a fusion construct comprising the anti- TfR antibody or antigen-binding fragment thereof of the application covalently linked to a second antibody or an antigen binding fragment thereof that binds to a brain target, such as a brain target selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, 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. In certain embodiments, the application relates to a fusion construct, wherein the anti-TfR antibody or antigen-binding fragment thereof is covalently linked to the carboxy terminus of only one of the two heavy chains of the second antibody or antigen binding fragment thereof via a linker, preferably the linker has the amino acid sequence of SEQ ID NO: 1422 or SEQ ID NO: 1423.

[0039] In certain embodiments, each of the two heavy chains of the second antibody or antigen binding fragment thereof comprises a modified constant heavy chain 3 (CH3) domain as compared to a wild-type CH3 domain to facilitate the formation of a heterodimer between the two heavy chains. Any mutation that facilitates the formation of a heterodimer between the two heavy chains can be used. Preferably, the modified CH3 domain of the first heavy chain comprises amino acid modifications at positions T350, L351, F405, and Y407, and the modified CH3 domain of the second heavy chain comprises amino acid modifications at positions T350, T366, K392 and T394. Preferably, the amino acid modification at position T350 is T350V, T350I, T350L or T350M; the amino acid modification at position L351 is L351 Y; the amino acid modification at position F405 is F405A, F405V, F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W. More preferably, the modified heterodimeric CH3 domain of the first heavy chain comprises mutations T350V, L351Y, F405A and Y407V, and the modified heterodimeric CH3 domain of the second heavy chain comprises mutations T350V, T366L, K392L and T394W. The numbering of amino acid residues in the antibody throughout the specification is performed according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), unless otherwise explicitly stated.

[0040] In certain embodiments, the fragment crystallizable region (Fc region) of the second antibody or antigen binding fragment thereof contains substitutions that alter (increase or diminish), preferably eliminate, effector function, such as antibody dependent cellular cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC). Preferably, the Fc region of the second antibody or antigen binding fragment thereof comprises one or more amino acid modifications that decrease or abolish the binding of the second antibody or antigen binding fragment thereof to Fc gamma receptors (FcyR) and avoid effector function mediated toxicity. For example, the Fc region of the second antibody or antigen binding fragment thereof can comprise one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0041] In certain embodiments, the Fc region of the second antibody or antigen binding fragment thereof contains substitutions that alter (increase or diminish), preferably increase, the binding of the second antibody or antigen binding fragment thereof to neonatal Fc receptor (FcRn). Preferably the one or more mutations enhance the binding at an acidic pH, more preferably the Fc has the M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0042] Another general aspect of the application relates to an isolated nucleic acid encoding the antibody or antigen-binding fragment, a conjugate, or a fusion construct of the application. Also provided is a vector comprising the isolated nucleic acid of the application, a host cell comprising the nucleic acid or the vector of the application.

[0043] Another general aspect of the application relates to a method of producing the antibody or antigen-binding fragment, a conjugate, or a fusion construct of the application. The method comprises culturing a cell comprising a nucleic acid of the application under conditions to produce the antibody or antigen-binding fragment, the conjugate, or the fusion construct, and recovering the antibody or antigen-binding fragment, the conjugate or the fusion construct from the cell or cell culture.

[0044] Further provided is a pharmaceutical composition comprising a conjugate or a fusion construct of the application and a pharmaceutically acceptable carrier.

[0045] Another general aspect of the application relates to a method of treating or detecting a disorder, preferably a neurological disorder, in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment, a conjugate, a fusion construct, or a pharmaceutical composition, preferably, the neurological disorder is selected from the group consisting of neurodegenerative diseases (such as Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (such as Alzheimer’s disease and supranuclear palsy), prion diseases (such as bovine spongiform encephalopathy, scrapie, Creutz-feldt- Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (such as Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden- Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (such as Pick's disease, and spinocerebellar ataxia), and cancer of the CNS and / or brain (such as brain metastases resulting from cancer elsewhere in the body).

[0046] Preferably, the antibody or antigen-binding fragment thereof, the conjugate, or the pharmaceutical composition is administered intravenously.

[0047] Also described is a method of delivering a therapeutic or diagnostic agent across the blood- brain barrier (BBB) of a subject in need thereof, comprising administering to the subject a complex comprising the therapeutic or diagnostic agent coupled to, preferably covalently conjugated to, the antibody or antigen-binding fragment thereof of the application. Preferably, the administration of the therapeutic or diagnostic agent coupled to an anti-TfR antibody or antigen-binding fragment thereof of the application to the brain of a subject results in reduced Fc-mediated effector function and / or does not induce rapid reticulocyte depletion, as compared to the administration of the therapeutic or diagnostic agent not coupled to the anti-TfR antibody or antigen-binding fragment thereof.

[0048] Yet another general aspect of the invention relates to a method of inducing antibodydependent phagocytosis (ADP) without stimulating secretion of a pro-inflammatory cytokine in a subject in need thereof, comprising administering to the subject a complex comprising a therapeutic antibody or antigen binding fragment thereof coupled to, preferably covalently conjugated to, an antigen-binding fragment thereof according to an embodiment of the invention, wherein the therapeutic antibody or antigen binding fragment thereof does not have effector function, for example, the therapeutic antibody or antigen binding fragment thereof comprises one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. Preferably, the therapeutic antibody or antigen binding fragment thereof binds specifically to tau aggregates. Further aspects, features and advantages of the present invention will be better appreciated upon a reading of the following detailed description of the invention and claims.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0051] Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present application, exemplary materials and methods are described herein.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0053] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0054] Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a dosage of 10 mg includes 9 mg to 11 mg. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0055] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0056] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.”

[0057] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the invention can be replaced with the term “consisting of’ or “consisting essentially of’ to vary scopes of the disclosure.

[0058] The term “antibody” herein is used in the broadest sense and specifically includes full- length monoclonal antibodies, polyclonal antibodies, and, unless otherwise stated or contradicted by context, antigen-binding fragments, antibody variants, and multispecific molecules thereof, so long as they exhibit the desired biological activity. Generally, a full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. General principles of antibody molecule structure and various techniques relevant to the production of antibodies are provided in, e.g., Harlow and Lane, ANHBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1988).

[0059] Depending on the amino acid sequence of the constant domain of their heavy chains, full length antibodies can be assigned to different “classes”. There are five major classes of full- length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0060] An “antibody” can also be a single variable domain on a heavy chain (VHH) antibody, also referred to as a heavy chain only antibody (HcAb), which are devoid of light chains and can be naturally produced by camelids or sharks. The antigen binding portion of the HcAb is comprised of a VHH fragment.

[0061] The term “recombinant antibody”, as used herein, refers to an antibody (e.g. a chimeric, humanized, or human antibody or antigen-binding fragment thereof) that is expressed by a recombinant host cell comprising nucleic acid encoding the antibody. Examples of “host cells” for producing recombinant antibodies include: (1) mammalian cells, for example, Chinese Hamster Ovary (CHO), COS, myeloma cells (including YO and NSO cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect cells, for example, sf9, sf21 and Tn5; (3) plant cells, for example plants belonging to the genus Nicotiana (e.g. Nicotiana tabacum),' (4) yeast cells, for example, those belonging to the genus Saccharomyces (e.g. Saccharomyces cerevisiae) or the genus Aspergillus (e.g. Aspergillus niger),' (5) bacterial cells, for example Escherichia, coli cells or Bacillus subtilis cells, etc. “Antibody-dependent phagocytosis” or “ADP” refers to the mechanism of elimination of antibody-coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells.

[0062] “Antigen” refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable of being bound by an antigen binding region or a T-cell receptor that is capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells, such as T cells, B cells or NK cells. Antigens may be expressed by genes, synthetized, or purified from biological samples such as a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins / antigens, killed or inactivated whole cells or lysates.

[0063] An “antigen binding region” or “antigen binding fragment” or “antigen binding domain” each refers to a portion of a full-length antibody that binds an antigen. An antigen binding region can be synthetic, enzymatically obtainable or genetically engineered polypeptides. An antigen binding region typically comprises one or more portions of at least the VH region. Antigen-binding fragments include multivalent molecules comprising one, two, three, or more antigen-binding portions of an antibody, and single-chain constructs wherein the VL and VH regions, or selected portions thereof, are joined by synthetic linkers or by recombinant methods to form a functional, antigen-binding molecule. Antigen-binding fragments can also be a singledomain antibody (sdAb), also known as a nanobody, which is an antibody fragment consisting of a single monomeric variable antibody domain (VHH). Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv, see e.g., Bird et al., Science 1988; 242:423-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (typically the VH and CHI domain), and dAb (typically a VH domain) fragments; VH, VL, VHH, and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, where the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and / or the HCDR3 and the LCDR1, the LCDR2 and / or the LCDR3, alternative scaffolds that bind an antigen, and multispecific antigen-binding constructs comprising the antigen binding regions. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23: 1126-1136; W02005040219, and published U.S. Patent Applications 20050238646 and 20020161201. Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and the fragments can be screened for antigen-binding or other function in the same manner as are intact antibodies.

[0064] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of full-length antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 1993 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibody fragments can be monospecific or bispecific.

[0065] The term "antibody derivative" as used herein refers to a molecule comprising a full- length antibody or an antigen-binding fragment thereof, wherein one or more amino acids are chemically modified or substituted. Chemical modifications that can be used in antibody derivative includes, e.g., alkylation, PEGylation, acylation, ester formation or amide formation or the like, e.g., for linking the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine- PEGylation), biotinylation, radiolabeling, and conjugation with a second agent (such as a cytotoxic agent).

[0066] Antibodies herein include “amino acid sequence variants” with altered antigen-binding or biological activity. Examples of such amino acid alterations include antibodies with enhanced affinity for antigen (e.g. “affinity matured” antibodies), and antibodies with altered Fc region, if present, e.g. with altered (increased or diminished) antibody dependent cellular cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) (see, for example, WO 00 / 42072, Presta, L. and WO 99 / 51642, Iduosogie et al); and / or increased or diminished serum half-life (see, for example, WO00 / 42072, Presta, L.).

[0067] A “multispecific molecule” comprises an antibody, or an antigen-binding fragment thereof, which is associated with or linked to at least one other functional molecule (e.g. another peptide or protein such as another antibody or ligand for a receptor) thereby forming a molecule that binds to at least two different binding sites or target molecules. Exemplary multispecific molecules include bi-specific antibodies and antibodies linked to soluble receptor fragments or ligands.

[0068] The term “human antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from (i.e., are identical or essentially identical to) human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is “derived from” human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in viva). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0069] A “humanized” antibody is a human / non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), WO 92 / 02190, US Patent Application 20060073137, and U.S. Pat. Nos. 6,750,325, 6,632,927, 6,639,055, 6,548,640, 6,407,213, 6,180,370, 6,054,297, 5,929,212, 5,895,205, 5,886,152, 5,877,293, 5,869,619, 5,821,337, 5,821,123, 5,770,196, 5,777,085, 5,766,886, 5,714,350, 5,693,762, 5,693,761, 5,530,101, 5,585,089, and 5,225,539.

[0070] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity-determining region” or “CDR” (residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and / or those residues from a “hypervariable loop” (residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987; 196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as “Kabat position”, “variable domain residue numbering as in Kabat” and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0071] The term “administering” with respect to the methods of the invention, means a method for therapeutically or prophylactically preventing, treating or ameliorating a syndrome, disorder or disease as described herein by using a conjugate of the invention or a form, composition or medicament thereof. Such methods include administering an effective amount of said antibody, antigen-binding fragment thereof, or conjugate, or a form, composition or medicament thereof at different times during the course of a therapy or concurrently in a combination form. The methods of the invention are to be understood as embracing all known therapeutic treatment regimens.

[0072] The ability of a target antibody to “block” the binding of a target molecule to a natural target ligand, means that the antibody, in an assay using soluble or cell-surface associated target and ligand molecules, can detectably reduce the binding of a target molecule to the ligand in a dose-dependent fashion, where the target molecule detectably binds to the ligand in the absence of the antibody.

[0073] The “blood-brain barrier” or “BBB” refers a physiological barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain. The BBB can restrict the transport of even very small molecules such as urea (60 Daltons) into the brain. Examples of the BBB include the BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina, all of which are contiguous capillary barriers within the CNS. The BBB also encompasses the blood-CSF barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.

[0074] A “blood-brain barrier receptor” (abbreviated “R / BBB” herein) is an extracellular membrane-linked receptor protein expressed on brain endothelial cells which is capable of transporting molecules across the BBB or be used to transport exogenous administrated molecules. Examples of R / BBB include, but are not limited to, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low density lipoprotein receptors including without limitation low density lipoprotein receptor-related protein 1 (LRP1) and low density lipoprotein receptor-related protein 8 (LRP8), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). An exemplary R / BBB herein is transferrin receptor (TfR).

[0075] The “central nervous system” or “CNS” refers to the complex of nerve tissues that control bodily function and includes the brain and spinal cord.

[0076] “Complementarity determining regions” (CDR) are antibody regions that bind an antigen. There are three CDRs in the VH (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (LCDR1, LCDR2, LCDR3). CDRs may be defined using various delineations such as Kabat (Wu et al. (1970) J Exp Med 132: 211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77), and AbM (Martin and Thornton J Bmol Biol 263: 800-15, 1996). The correspondence between the various delineations and variable region numbering is described (see e.g., Lefranc et al. (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun (2001), J Mol Biol 309:657-70; International ImMunoGeneTics (IMGT) database; Web resources, http: / / www_imgt_org). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2” and “LCDR3” as used herein include CDRs defined by any of the methods described supra, Kabat, Chothia, IMGT or AbM, unless otherwise explicitly stated in the specification. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra; Chothia, supra; Martin, supra; Lefranc et al., supra).

[0077] Table 1: Relationship Between CDR Numbering Schemes

[0078] A “conjugate” as used herein refer to an anti-TfR antibody or antigen-binding fragment thereof of the application covalently linked to one or more heterologous molecule(s). Examples of heterologous molecule(s) that can be used in a conjugate of the application include, but are not limited to, a therapeutic peptide or protein, an antibody, a nucleic acid (e.g., DNA or RNA), a label, or a neurological disorder drug. In certain embodiments, an antibody or antigen-binding fragment of the application can be conjugated to one or more heterologous molecule(s) through a non-peptide linker. In certain embodiments, an antibody or antigen-binding fragment of the application can be conjugated to one or more heterologous molecule(s) through a peptide linker. When one protein is conjugated to another protein via a peptide linker, it is also referred to that the two proteins are fused together. By way of a non-limiting example, an antibody or antigenbinding fragment of the application can be conjugated to another polypeptide to form a fusion protein. In certain embodiments, an antibody or antigen-binding fragment of the application can be conjugated to another polypeptide through a non-peptide linker.

[0079] As used herein the term “coupled” refers to the joining or connection of two or more objects together. When referring to chemical or biological compounds, coupled can refer to a covalent connection between the two or more chemical or biological compounds. By way of a non-limiting example, an antibody of the invention can be coupled with a peptide of interest to form an antibody coupled peptide. An antibody coupled peptide can be formed through specific chemical reactions designed to conjugate the antibody to the peptide. In certain embodiments, an antibody of the invention can be covalently coupled with a peptide of the invention through a linker. The linker can, for example, be first covalently connected to the antibody or the peptide, then covalently connected to the peptide or the antibody.

[0080] An “effective amount” or “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0081] “Encode” or “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0082] “Epitope” refers to a portion of an antigen to which an antibody, or the antigen binding portion thereof, specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. For a discontinuous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3 -dimensional space through the folding of the protein molecule. Antibody “epitope” depends on the methodology used to identify the epitope.

[0083] “Expression vector” or “vector” refers to a biotic or abiotic agents that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0084] “Fv” or “Fv fragment” refers to an antibody fragment composed of the VH and the VL domains from a single arm of the antibody. Fv fragments lack the constant regions of Fab (CHI and CL) regions. The VH and VL in Fv fragments are held together by non-covalent interactions.

[0085] “Framework region” or “FR” residues are those VH or VL residues other than the CDRs as herein defined.

[0086] “Host cell” refers to any cell that contains a heterologous nucleic acid. An exemplary heterologous nucleic acid is a vector (e.g., an expression vector).

[0087] A “linker” as used herein refers to a chemical linker or a single chain peptide linker that covalently connects two different entities. A linker can be used to connect any two of an antibody or a fragment thereof, a blood brain barrier shuttle, a fusion protein and a conjugate of the present invention. The linker can connect, for example, the VH and VL in scFv, or the monoclonal antibody or antigen-binding fragment thereof with a therapeutic molecule, such as a second antibody. In some embodiment, if the monovalent binding entity comprises an scFv directed to TfR, preferably huTfRl, and the therapeutic molecule comprises an antibody directed to a CNS target, such as Tau, then the linker can connect the scFv to the antibody directed to Tau. Single chain peptide linkers, comprised of from 1 to 25 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 ammo acids, joined by peptide bonds, can be used. In certain embodiments, the amino acids are selected from the twenty naturally occurring amino acids. In certain other embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. Chemical linkers, such as a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a hybrid linker consisting of PEG and an embedded heterocycle, and a hydrocarbon chain can also be used.

[0088] A “neurological disorder” as used herein refers to a disease or disorder which affects the CNS and / or which has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease. For the purposes of this application, the CNS will be understood to include the eye, which is normally sequestered from the rest of the body by the blood-retina barrier. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (including, but not limited to, Alzheimer’s disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutz-feldt- Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease, and spinocerebellar ataxia), cancer (e.g. of the CNS and / or brain, including brain metastases resulting from cancer elsewhere in the body).

[0089] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to a composition comprises an active ingredient and a pharmaceutically acceptable carrier. A “pharmaceutical formulation” or “pharmaceutical composition” is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no components which are unacceptably toxic to a subject to which the formulation would be administered.

[0090] “Pharmaceutically acceptable carrier” or “excipient” refers to an ingredient in a pharmaceutical composition, other than the active ingredient, which is nontoxic to a subject. Exemplary pharmaceutically acceptable carriers include a buffer, stabilizer or preservative.

[0091] “Polynucleotide” or “nucleic acid” refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. Polynucleotide may be a DNA or a RNA molecule.

[0092] “Protein” or “polypeptide” are used interchangeably herein and refers to a molecule that comprises one or more polypeptides each comprised of at least two amino acid residues linked by a peptide bond. Protein may be a monomer, or may be protein complex of two or more subunits, the subunits being identical or distinct. Small polypeptides of less than 50 amino acids may be referred to as “peptides”. Protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation or biotinylation. Protein may be recombinantly expressed.

[0093] The phrases “sequence identity” or “percent (%) sequence identity” or “% identity” or “% identical to” when used with reference to an amino acid sequence describe the number of matches (“hits”) of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences. In other terms, using an alignment, for two or more sequences the percentage of amino acid residues that are the same (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full-length of the amino acid sequences) may be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids. Suitable programs for aligning protein sequences are known to the skilled person. The percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g., using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

[0094] The term “substantially identical” in the context of two amino acid sequences means that the sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 50 percent sequence identity. Typically sequences that are substantially identical will exhibit at least about 60, at least about 70, at least about 80, at least about 90, at least about 95, at least about 98, or at least about 99 percent sequence identity. "Single chain Fv" or "scFv" refers to a single chain protein comprising a VH, a VL and a linker between the VH and the VL. The scFv may have the VL and VH variable regions in either orientation, e.g., with respect to the N- to C-terminal order of the VH and the VL. The scFv may thus be in the orientation VL-linker-VH or VH-linker-VL. scFv may be engineered to 20 comprise disulfide bonds between the VH, the VL and the linker.

[0095] “Specific binding” or “specifically binds” or “binds” refer to antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the antibody binds to the antigen or the epitope within the antigen with a dissociation constant (KD) of about IxlO'8M or less, for example about IxlO'9M or less, about IxlO'10M or less, about IxlO'11M or less, or about IxlO'12M or less, typically with a KD that is at least one hundred fold less than its KD for binding to a non-specific antigen (e.g., BSA, casein). KD is the equilibrium dissociation constant, a ratio of kOff / kOn, between the antibody and its antigen. KD and affinity are inversely related. The “on-rate” (kon) is a constant used to characterize how quickly the antibody binds to its target. The “off-rate” (kOff) is a constant used to characterize how quickly an antibody dissociates from its target. The dissociation constant KD can be measured using standard procedures. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system. The smaller the value of the KD of an antibody, the higher affinity that the antibody binds to a target antigen. Antibodies that specifically bind to the antigen or the epitope within the antigen can, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.

[0096] The term “subject” as used herein refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. When the subject is human, they can also be referred to as a “patient”. The term “transferrin receptor” or “TfR,” as used herein, refers to a cell surface receptor necessary for cellular iron uptake by the process of receptor-mediated endocytosis. carrier protein for transferrin. A TfR is involved in iron uptake in vertebrates and is regulated in response to intracellular iron concentration. It imports iron by internalizing the transferrin-iron complex through receptor-mediated endocytosis. Two transferrin receptors in humans, transferrin receptor 1 and transferrin receptor 2, have been characterized. Both these receptors are transmembrane glycoproteins. TfRl is a high affinity ubiquitously expressed receptor. TfR2 binds to transferrin with a 25-30-fold lower affinity than TfRl. The expression of TfR2 is restricted to certain cell types and is unaffected by intracellular iron concentrations. In one embodiment, the TfR is a human TfR comprising the amino acid sequence as in Schneider et al. Nature 311: 675-678 (1984), for example. It can have a molecular weight of about 180,000 Dalton, having two subunits each of apparent molecular weight of about 90,000 Dalton. Preferably, the TfR is a human TfRl.

[0097] A “target antigen” or “brain target,” as used herein, refers to an antigen and / or molecule expressed in the CNS, including the brain, which can be targeted with an antibody or small molecule. Examples of such antigens and / or molecules include, without limitation: beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, 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. In some embodiments, the target antigen is BACE1. In some embodiments, the target antigen is Tau.

[0098] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease. “VHH” refers to a single-domain antibody or nanobody, exclusively composed of the antigen binding region of a heavy chain. A VHH single domain antibody lacks the light chain and the CHI domain of the heavy chain of conventional Fab region.

[0099] The numbering of amino acid residues of the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of 30 Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

[0100] Anti-TfR Antibodies and antigen binding fragments thereof

[0101] In one general aspect, the application relates to an anti-TfR antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL) comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, and a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein:

[0102] (7) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 222-308 and 1456-1485;

[0103] (8) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 308-359 and 1486-1511;

[0104] (9) the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 360-518 and 1512-1561;

[0105] (10) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 519-645 and 1562-1606;

[0106] (11) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 646-792 and 1607-1661; and

[0107] (12) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 793-980 and 1662-1717.

[0108] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a LCDR1 , LCDR2 and LCDR3 having the amino acid sequences of the LCDR1 , LCDR2 and LCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

[0109] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a VL having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1198-1420 and 1776-1832.

[0110] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a VL having the amino acid sequence of the light chain variable region of any one of the antigen binding proteins specified in Table 6.

[0111] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a HCDR1, HCDR2 and HCDR3 having the amino acid sequences of the HCDR1, HCDR2 and HCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

[0112] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 981-1197 and 1718-1775.

[0113] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a VH having the amino acid sequence of the heavy chain variable region of any one of the antigen binding proteins specified in Table 6.

[0114] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a LCDR1, LCDR2 LCDR3, HCDR1, HCDR2, and HCDR3 having the amino acid sequences of the LCDR1, LCDR2 LCDR3, HCDR1, HCDR2, and HCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

[0115] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a VL and a VH having the amino acid sequences of the light chain variable region and the heavy chain variable region, respectively, of any one of the antigen binding proteins specified in Table 6.

[0116] In other embodiments, the antibody or antigen-binding fragment thereof is single-chain variable fragment (scFv) comprising the heavy chain variable region (Hv) covalently linked to the light chain variable region (Lv) via a flexible linker. The scFv can retain the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. In a scFv, the order of the domains can be either Hv-linker- Lv, or Lv-linker- Hv. The linker can be designed de novo or derived from known protein structure to provide a compatible length and conformational in bridging the variable domains of a scFv without serious steric interference. The linker can have 10 to about 25 amino acids in length. Preferably, the linker is a peptide linker spanning about 3.5 nm (35 A) between the carboxy terminus of the variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site (Huston et al., Methods in Enzymology, vol. 203, pp. 46-88, 1991, which is incorporated herein by reference in its entirety). The linker preferably comprises a hydrophilic sequence in order to avoid intercalation of the peptide within or between the variable domains throughout the protein folding (Argos, Journal of Molecular Biology, vol. 211, no. 4, pp. 943-958, 1990). For example, the linker can comprise Gly and Ser residues and / or together with the charged residues such as Glu, Thr and Lys interspersed to enhance the solubility. In one embodiment, the linker has the amino acid sequence of SEQ ID NO: 1421 (GTEGKSSGSGSESKST). Any other suitable linker can also be used in view of the present disclosure. For example, the linker can also comprise one or more cysteine (Cys) residues to help stabilize the scFv, such as the linkers described for example in Int. Pat. Publ. No. W02021 / 030657.

[0117] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof of, comprises a scFv further comprises a linker that covalently links the VH and VL, such as a linker having the amino acid sequence selected from the group consisting of SEQ ID NO: 1421 and SEQ ID NOs: 1424-1455.

[0118] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof of the application, comprises an scFv having one or more disulfide bonds formed between a cysteine (Cys) in the linker and a surface exposed Cys in VH and / or VL, preferably a surface exposed Cys in a framework region of the VH and / or VL.

[0119] In some embodiments, the VH Cys is at Hl 05 and the VL Cys is at L42, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0120] In some embodiments, the VH Cys is at H43 and the VL Cys is at a LI 00, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0121] In some embodiments, the VH Cys is at H3 and the VL Cys is at L3, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0122] In some embodiments, the VH Cys is at H3 and the VL Cys is at L5, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0123] In some embodiments, the VH Cys is at H3 and the VL Cys is at L39, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0124] In some embodiments, the VH Cys is at H3 and the VL Cys is at L42, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0125] In some embodiments, the VH Cys is at H3 and the VL Cys is at L45, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0126] In some embodiments, the VH Cys is at H3 and the VL Cys is at LI 00, wherein the residue numbering of the VH and the VL regions are according to Chothia. In some embodiments, the VH Cys is at H3 and the VL Cys is at LI 02, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0127] In some embodiments, the VH Cys is at H5 and the VL Cys is at L3, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0128] In some embodiments, the VH Cys is at H5 and the VL Cys is at L5, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0129] In some embodiments, the VH Cys is at H5 and the VL Cys is at L39, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0130] In some embodiments, the VH Cys is at H5 and the VL Cys is at L45, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0131] In some embodiments, the VH Cys is at H5 and the VL Cys is at LI 00, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0132] In some embodiments, the VH Cys is at H5 and the VL Cys is at LI 02, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0133] In some embodiments, the VH Cys is at H40 and the VL Cys is at L3, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0134] In some embodiments, the VH Cys is at H40 and the VL Cys is at L5, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0135] In some embodiments, the VH Cys is at H40 and the VL Cys is at L39, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0136] In some embodiments, the VH Cys is at H40 and the VL Cys is at L42, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0137] In some embodiments, the VH Cys is at H40 and the VL Cys is at L45, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0138] In some embodiments, the VH Cys is at H40 and the VL Cys is at LI 00, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0139] In some embodiments, the VH Cys is at H40 and the VL Cys is at LI 02, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0140] In some embodiments, the VH Cys is at H43 and the VL Cys is at L3, wherein the residue numbering of the VH and the VL regions are according to Chothia. In some embodiments, the VH Cys is at H43 and the VL Cys is at L5, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0141] In some embodiments, the VH Cys is at H43 and the VL Cys is at L39, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0142] In some embodiments, the VH Cys is at H43 and the VL Cys is at L42, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0143] In some embodiments, the VH Cys is at H43 and the VL Cys is at L45, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0144] In some embodiments, the VH Cys is at H43 and the VL Cys is at LI 02, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0145] In some embodiments, the VH Cys is at H46 and the VL Cys is at L3, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0146] In some embodiments, the VH Cys is at H46 and the VL Cys is at L5, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0147] In some embodiments, the VH Cys is at H46 and the VL Cys is at L39, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0148] In some embodiments, the VH Cys is at H46 and the VL Cys is at L42, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0149] In some embodiments, the VH Cys is at H46 and the VL Cys is at L45, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0150] In some embodiments, the VH Cys is at H46 and the VL Cys is at LI 00, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0151] In some embodiments, the VH Cys is at H46 and the VL Cys is at L102, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0152] In some embodiments, the VH Cys is at Hl 05 and the VL Cys is at L3, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0153] In some embodiments, the VH Cys is at H105 and the VL Cys is at L5, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0154] In some embodiments, the VH Cys is at H105 and the VL Cys is at L39, wherein the residue numbering of the VH and the VL regions are according to Chothia. In some embodiments, the VH Cys is at H105 and the VL Cys is at L45, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0155] In some embodiments, the VH Cys is at H105 and the VL Cys is at LI 00, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0156] In some embodiments, the VH Cys is at Hl 05 and the VL Cys is at LI 02, wherein the residue numbering of the VH and the VL regions are according to Chothia.

[0157] In another general aspect, the application describes an anti-TfR antibody or antigenbinding fragment thereof having a single variable domain on a heavy chain (VHH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein:

[0158] (4) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24;

[0159] (5) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-46; and

[0160] (6) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-70.

[0161] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises a HCDR1, HCDR2 and HCDR3 having the amino acid sequences of the HCDR1, HCDR2 and HCDR3, respectively, of any one of the VHH antigen binding proteins specified in Table 3.

[0162] Preferably, the anti-TfR antibody or antigen-binding fragment thereof, comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-221.

[0163] In certain embodiments, the anti-TfR antibody or antigen-binding fragment thereof, comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-221.

[0164] An anti-TfR antibody or antigen-binding fragment thereof (such as a VHH or scFv fragment) can be produced using suitable methods in the art in view of the present disclosure. For example, a VHH or scFv fragment can be recombinantly produced by growing a recombinant host cell (such as a bacterial, yeast or mammalian cell) under suitable conditions for the production of the antibody fragment and recovering the fragment from the cell culture. Brain shuttle construct

[0165] An optimized receptor-mediated transcytosis (RMT) brain delivery platform is developed using the transferrin receptor (TfR) by enhancing the intrinsic transcytosis efficiency, extending peripheral pharmacokinetics, and engineering for an acceptable safety profile while maintaining efficacy of the therapeutic mAb.

[0166] In one general aspect, the application relates to an antibody-targeted brain delivery system comprising an anti-TfR antibody or antigen binding fragment thereof of the application. The anti-TfR antibody or antigen binding fragment thereof can be used to deliver a therapeutic or diagnostic agent into a cell (e.g., a cancer cell) or a BBB system. Agents that can be delivered include any neurological disorder drug or agent that can be used to detect or analyze a neurological disorder drug. For example, such agent can be neurotrophic factors, including, but not limited to, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line neurotrophic factor (GDNF) and insulin-like growth factor (IGF); neuropeptides, including, but not limited to, Substance P, neuropeptide Y, vasoactive intestinal peptide (VIP), gamma-amino-butyric acid (GABA), dopamine, cholecystokinin (CCK), endorphins, enkephalins and thyrotropin releasing hormone (TRH); cytokines; anxiolytic agents; anticonvulsants; polynucleotides and transgenes, including, for example, small interfering RNAs and / or antisense oligos; or antibodies or antigen binding fragments thereof that bind to a brain target. An anti-TfR antibody or antigen binding fragment thereof of the application can be an effective means to enhance the delivery of an agent of interest from the blood into the brain and function there.

[0167] In particular, an agent of interest can be delivered in a combined form or linked to an anti-TfR antibody or antigen binding fragment thereof of the application, parenterally, e.g., intravenously. For example, the agent can be non-covalently attached to the anti-TfR antibody or antigen binding fragment thereof. The agent can also be covalently attached to the anti-TfR antibody or antigen binding fragment thereof to form a conjugate. In certain embodiments, the conjugation is by construction of a protein fusion (i.e., by genetic fusion of the two genes encoding an anti-TfR antibody or antigen binding fragment thereof and a neurological disorder drug and expression as a single protein). Known methods can be used to link an agent to an antibody or antigen binding fragment thereof in view of the present disclosure. See, for example, Wu et al., Nat Biotechnol., 23(9): 1137-46, 2005; Trail et al., Cancer Immunol Immunother., 52(5):328-37, 2003; Saito et al., Adv Drug Deliv Rev., 55(2): 199-215, 2003; Jones et al., Pharmaceutical Research, 24(9):1759-1771, 2007.

[0168] In some embodiments, a therapeutic or diagnostic agent to be delivered to the brain and an anti-TfR antibody or antigen binding fragment thereof can be covalently linked together (or conjugated) via a non-peptide linker or a peptide linker. Examples of non-peptide linkers include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymer, polymerized lipid, chitins, and hyaluronic acid, or derivatives thereof, or combinations thereof. A peptide linker can be a peptide chain consisting of 1 to 50 amino acids linked by peptide bonds or a derivative thereof, whose N- terminus and C-terminus can be covalently linked to an anti-TfR antibody or an antigen binding fragment thereof.

[0169] In certain embodiments, a conjugate of the application is a multi-specific antibody comprising a first antigen binding region which binds a TfR and a second antigen binding region which binds a brain antigen, such as beta-secretase 1 (BACE1), tau, and the other brain antigens disclosed herein. Techniques for making multi-specific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537, 1983), WO 93 / 08829, and Traunecker et al, EMBO J. 10: 3655, 1991), and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies can also be made by engineering electrostatic steering effects (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al, Science, 229: 81, 1985); using leucine zippers (see, e.g., Kostelny et al, J. Immunol., 148(5): 1547-1553,1992)); using “diabody” technology (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993)); using single-chain Fv (sFv) dimers (see, e.g. Gruber et al, J. Immunol, 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60, 1991. A multi-specific antibody of the application also encompasses antibodies having three or more functional antigen binding sites, including “Octopus antibodies” or “dual-variable domain immunoglobulins” (DVDs) (see, e.g., US 2006 / 0025576A1, and Wu et al. Nature Biotechnology, 25(11): 1290-7, 2007). A multispecific antibody of the application also encompasses a “Dual Acting Fab” or “DAF” comprising an antigen binding region that binds to TfR as well as the brain antigen (e.g., BACE1 or Tau) (see, US 2008 / 0069820, for example). In one embodiment, the antibody is an antibody fragment, various such fragments being disclosed herein.

[0170] In one embodiment, a multi-specific antibody of the application is a fusion construct comprising an anti-TfR antibody or antigen-binding fragment thereof of the application covalently linked (or fused) to a second antibody or antigen binding fragment thereof. Preferably, the second antibody or antigen binding fragment thereof binds to a brain target, such as BACE, tau or other brain antigens, such as those described herein. The anti-TfR antibody or antigen-binding fragment thereof can be fused to the carboxy- and / or amino- terminus of a light and / or heavy chain of the second antibody or antigen binding fragment thereof, directly or via a linker.

[0171] In one embodiment, the anti-TfR antibody or antigen-binding fragment thereof is fused to the carboxy-terminus of a light chain of the second antibody or antigen binding fragment thereof, directly or via a linker.

[0172] In another embodiment, the anti-TfR antibody or antigen-binding fragment thereof is fused to the amino-terminus of a light chain of the second antibody or antigen binding fragment thereof, directly or via a linker.

[0173] In another embodiment, the anti-TfR antibody or antigen-binding fragment thereof is fused to the carboxy-terminus of a heavy chain of the second antibody or antigen binding fragment thereof, directly or via a linker.

[0174] In another embodiment, the anti-TfR antibody or antigen-binding fragment thereof is fused to the amino-terminus of a heavy chain of the second antibody or antigen binding fragment thereof, directly or via a linker.

[0175] In a preferred embodiment, a fusion construct of the application comprises an anti-TfR antibody or antigen-binding fragment thereof, preferably an anti-huTfRl VHH or scFv fragment, of the application covalently linked, via a linker, to the carboxy terminus of only one of the two heavy chains of a second antibody or antigen binding fragment thereof that binds to a brain target. Preferably, the linker has the amino acid sequence of SEQ ID NO: 1422 or SEQ ID NO: 1423.

[0176] To facilitate the formation of a heterodimer between the two heavy chains, e.g., one with a fusion of the anti-TfR antibody or antigen-binding fragment thereof and one without, or one containing the Fc for the anti-TfR arm and one for the anti-brain target arm, heterodimeric mutations introduced into the Fc of the two heavy chains. Examples of such Fc mutations include, but are not limited to, the Zymework mutations (see, e.g., US 10,457,742) and the “knob in hole” mutations (see, e.g., Ridgway et al., Protein Eng., 9(7): 617-621, 1996). Other heterodimer mutations can also be used in the invention. In some embodiment, a modified CH3 as described herein is used to facilitate the formation of a heterodimer between the two heavy chains.

[0177] In addition to the heterodimeric mutations, other mutations can also be introduced. In some embodiment, the Fc region of the fusion construct or bispecific antibody further comprises one or more mutations that alter (increase or diminish), preferably eliminate ADCC / CDC (such as the AAS mutations described herein), and / or one or more mutations that alter (increase or diminish), preferably increase, the binding of the fusion construct or bispecific antibody to FcRn (such as the YTE mutations described herein). In some embodiment, one or more cysteine residues in the fusion construct or bispecific antibody are substituted with other amino acids, such as serine.

[0178] A conjugate, such as a multi-specific antibody or fusion construct, of the application can be produced by any of a number of techniques known in the art in view of the present disclosure. For example, it can be expressed from a recombinant host cells, wherein expression vector(s) encoding the heavy and light chains of the fusion construct or multi-specific antibody is (are) transfected into a host cell by standard techniques. The host cells can be prokaryotic or eukaryotic host cells.

[0179] In an exemplary system, one or more recombinant expression vectors encoding the heterodimeric two heavy chains and the light chains of a fusion construct of the application is / are introduced into host cells by transfection or electroporation. The selected transformant host cells are cultured to allow for expression of the heavy and light chains under conditions sufficient to produce the fusion construct, and the fusion construct is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the protein construct from the culture medium.

[0180] The application provides an isolated nucleic acid encoding the amino acid sequence of an anti-TfR antibody or antigen binding fragment thereof alone or as part of a fusion construct or multispecific antibody in any of the embodiments described herein or any of the claims. The isolated nucleic acid can be part of a vector, preferably an expression vector.

[0181] In another aspect, the application relates to a host cell transformed with the vector disclosed herein. In an embodiment, the host cell is a prokaryotic cell, for example, E. coli. In another embodiment, the host cell is a eukaryotic cell, for example, a protist cell, an animal cell, a plant cell, or a fungal cell. In an embodiment, the host cell is a mammalian cell including, but not limited to, CHO, COS, NSO, SP2, PER.C6, or a fungal cell, such as Saccharomyces cerevisiae, or an insect cell, such as Sf9.

[0182] Pharmaceutical comuosition and related methods

[0183] The invention also relates to pharmaceutical compositions, methods of preparation and methods for use thereof.

[0184] In another general aspect, the invention relates to a pharmaceutical composition, comprising an anti-TfR antibody or antigen binding fragment thereof or a conjugate thereof of the invention and a pharmaceutically acceptable carrier. The anti-TfR antibody or antigen binding fragment thereof or conjugate (such as a multi-specific antibody or fusion construct) of the invention is also useful in the manufacture of a medicament for therapeutic applications mentioned herein. The pharmaceutically acceptable carrier can be any suitable excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.

[0185] Accordingly, in one embodiment, the application relates to a method of transporting a therapeutic or diagnostic agent across the blood-brain barrier (BBB) comprising exposing an anti-TfR antibody or antigen binding fragment thereof coupled to the therapeutic or diagnostic agent to the blood- brain barrier such that the antibody or antigen binding fragment thereof transports the agent coupled thereto across the blood- brain barrier. In one embodiment, the agent is a neurological disorder drug. In another embodiment, the agent is an imaging agent or an agent for detecting a neurological disorder. Preferably, the anti-TfR antibody or antigen binding fragment thereof or conjugate thereof does not impair the binding of the TfR to its native ligand transferrin. The antibody specifically binds to TfR in such a manner that it does not inhibit binding of the TfR to transferrin. In some embodiment, the BBB is in a mammal, preferably a primate, such as a human, more preferably a human having a neurological disorder. In one embodiment, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury.

[0186] In one embodiment, an anti-TfR antibody or antigen binding fragment thereof, or a conjugate thereof of the application, is used to detect a neurological disorder before the onset of symptoms and / or to assess the severity or duration of the disease or disorder. The antibody, antigen binding fragment or conjugate thereof permits detection and / or imaging of the neurological disorder, including imaging by radiography, tomography, or magnetic resonance imaging (MRI).

[0187] In another embodiment, an anti-TfR antibody or antigen binding fragment thereof, or a conjugate thereof, is used in treating a neurological disorder (e.g., Alzheimer's disease), comprising administering to a subject in need of the treatment an effective amount of anti-TfR antibody or antigen binding fragment thereof, or a conjugate thereof. In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent.

[0188] In another embodiment, the application relates to the use of an anti-TfR antibody or antigen binding fragment or conjugate thereof of the application in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of neurological disease or disorder. In a further embodiment, the medicament is for use in a method of treating neurological disease or disorder comprising administering to an individual having neurological disease or disorder an effective amount of the medicament.

[0189] Another general aspect of the application relates to a method of inducing antibody dependent phagocytosis (ADP) without stimulating secretion of a pro-inflammatory cytokine in a subject in need thereof, comprising administering to the subject a complex comprising a therapeutic antibody or antigen binding fragment thereof coupled to, preferably covalently conjugated to, the antigen-binding fragment thereof of an anti-TfR antibody binding fragment according to an embodiment of the application, wherein the therapeutic antibody or antigen binding fragment thereof does not have effector function. For example, the therapeutic antibody or antigen binding fragment thereof can comprise one or more amino acid modifications that reduces or eliminates the effector function, such as the ADCC or CDC, such as mutations that reduce or abolish the binding to Fc gamma receptor. Such mutations can be at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. In one embodiment, the therapeutic antibody or antigen binding fragment thereof binds specifically to tau aggregates.

[0190] In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is a therapeutic agent effective to treat the same or a different neurological disorder as the anti-TfR antibody or antigen binding fragment or conjugate thereof is being employed to treat. Exemplary additional therapeutic agents include, but are not limited to: the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rovastigmine, and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, y-secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy, PPARy agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicrotinic receptor modulators, active or passive amyloid beta peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists and anti-amyloid beta peptide antibodies. In certain embodiments, the at least one additional therapeutic agent is selected for its ability to mitigate one or more side effects of the neurological drug. The additional therapeutic agent can be administered in the same or separate formulations and administered together or separately with the anti-TfR antibody or antigen binding fragment or conjugate thereof. The anti-TfR antibody or antigen binding fragment or conjugate of the application can be administered prior to, simultaneously with, and / or following, the administration of the additional therapeutic agent and / or adjuvant. The anti- TfR antibody or antigen binding fragment or conjugate thereof of the application can also be used in combination with other interventional therapies such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and appropriate for the neurological disorder to be treated or prevented.

[0191] The anti-TfR antibody or antigen binding fragment or conjugate thereof of the application (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time- points, bolus administration, and pulse infusion are contemplated herein.

[0192] For the prevention or treatment of a disease, the appropriate dosage of an anti-TfR antibody or antigen binding fragment or conjugate thereof of the application (when used alone or in combination with one or more other additional therapeutic agents) will depend on various factors, such as the type of disease to be treated, the type of antibody or conjugate, the severity and course of the disease, whether the antibody, antigen binding fragment or conjugate thereof is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, the physiological state of the subject (including, e.g., age, body weight, health), and the discretion of the attending physician. Treatment dosages are optimally titrated to optimize safety and efficacy. The antibody, antigen binding fragment or conjugate thereof is suitably administered to the patient at one time or over a series of treatments.

[0193] According to particular embodiments, a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the disease, disorder or condition to be treated, or a symptom associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0194] In another aspect, the application relates to an article of manufacture (such as a kit) containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody, antigen binding fragment thereof or a conjugate of the application. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can include (a) a first container with a composition contained therein, wherein the composition comprises an antibody, antigen binding fragment thereof or a conjugate of the application; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention can further include a package insert indicating that the compositions can be used to treat a particular condition. Optionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0195] The following examples of the invention are to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and the scope of the invention is to be determined by the appended claims.

[0196] EXAMPLES

[0197] Example 1. Anti-TfR Antibodies

[0198] Anti-TfR antibodies or antigen binding fragments of the invention are illustrated in this Example. Table 3 lists the HCDRs of exemplary anti-TfR VHHs of the invention. Table 4 lists the VHH sequences of exemplary anti-TfR VHHs of the invention. Table 5 shows the LCDRs and HCDRs of exemplary anti-TfR antibodies or antigen binding fragments of the invention. Table 6 provides the VL and VH sequences of exemplary anti-TfR antibodies or antigen binding fragments of the invention. The CDRs listed in the tables herein are according to IMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77).

[0199] The anti-TfR antibodies or antigen binding fragments can be made and tested using methods known in the art in view of the present disclosure.

[0200] Example 2: Binding characterization by Surface Plasmon Resonance

[0201] The binding interaction of test articles against recombinant human, rat, cynomolgus, and murine transferrin receptor (TfR; TFRW2, TFRW1, TFRW3, TFRW5) are studied by SPR using a Biacore 8k+ instrument at 25°C with pH 7.4 buffer, supplemented with 0.01% BSA, and 0.05% Tween 20. The biosensor surface is prepared by coupling anti-human IgG Fcy-fragment specific antibody to the surface of a Cl sensor chip using vendor recommended protocol for amine-coupling chemistry (>400 response units (RU)). The coupling buffer is 10 mM sodium acetate, pH 4.5. The test articles are diluted in the running buffer and injected over the antihuman IgG to obtain enough capture to enable detection of antigen binding. Capture of test articles is followed by separate injection of the four recombinant antigens at the same concentration series in single-cycle kinetics mode (200 nM to 7.41 nM series at 3-fold dilutions). The association was monitored for 2 minutes and dissociation for 20 minutes at a 50 pL / min flow rate. Regeneration of the sensor surface is performed with 0.85% H3PO4. The binding sensorgrams are fitted using the 1 : 1 Langmuir binding model to obtain on-rates, off-rates, and affinities.

[0202] Example 3: Creation of therapeutic antibody - anti-transferrin receptor (TfR) fusion expression constructs

[0203] Generation of therapeutic monoclonal antibodies (mAbs) in mammalian expression cell lines incorporates the use of the pEE6.4 and pEE12.4 Chinese hamster ovary (CHO) expression vector system (Lonza). Each vector contains a human cytomegalovirus (huCMV-MIE) promoter to drive the expression of the heavy chain (HC) or light chain (LC) of the mAb and contains the ampicillin resistance gene. The pEE12.4 vector also includes the gene encoding the glutamine synthetase (GS) enzyme. Growth conditions which require glutamine synthetase activity places selective pressure on the cells to maintain the expression vector (GS Gene Expression System Manual Version 4.0). pEE6.4 is used to clone the HC gene and pEE12.4 to clone the LC gene as single gene vectors.

[0204] The DNA encoding the anti-TfR antibody HC and LC or VHH is synthesized and subcloned into the Lonza expression vector backbones pEE6.4 and pEE12.4, respectively, by the In-Fusion Cloning method (Takara Bio) using Type IIS restriction enzyme Aarl. The open reading frames of subcloned genes are sequence confirmed at Genewiz, Azenta Life Sciences (NJ, USA).

[0205] The constructs can be analyzed, e.g., for affinity, binding specificity, internalization, pharmacokinetics and pharmacodynamics, etc., using methods known in the art, such as those described in US 2023 / 0174646, the relevant content of which is incorporated herein by reference in its entirety.

[0206] Example 4: SPR binding analysis method

[0207] The anti-transferrin receptor (TfR) antibodies binding to human or cynomolgus transferrin receptor was tested by Surface Plasmon Resonance (SPR) using Carterra LSA instrument at 25°C. The analyses were performed using HBS buffer supplemented with 0.05% Tween-20 and 0.01% BSA as the running buffer. Briefly, the biosensor was prepared by amine coupling of Goat anti -Human IgGFcy fragment-specific antibody to the surface of a CMDP sensor chip using vendor recommended protocol for amine-coupling chemistry (-1000 RUs). The Goat anti-Human IgG reagent was prepared in 10 mM sodium acetate, pH 4.5. The anti-TfR antibody solutions and various concentrations of antigens (human or Cyno TfR) were prepared in the running buffer (0.1 nM to 100 nM). The antibodies were captured on the sensor chip with capture levels of -100 RUs, followed by series of antigen injections in a non-regenerative multicycle kinetics mode. The association and dissociation of antigens were measured for 5 and 15 minutes, respectively. The surface of the sensor chip was regenerated using 0.85 % H3PO4. The data was evaluated using the Carterra Kinetics software and fitted using 1 : 1 Langmuir model to obtain binding kinetics (on- and off-rates) and affinities. Table 2 lists the dissociation constant (Ka) of the anti-TfR antibodies or antigen binding fragments according to embodiments of the invention.

[0208] Table 2: SPR Binding to Human and Cynomolgus Transferrin Receptor (TfR)

[0209] Example 5: Differential Scanning Fluorimetry (DSF) of Protein Therapeutics

[0210] Conformational stability of monovalent Fab-Fc proteins with unique TfR binding arms were measured using advanced differential scanning fluorimetry (nanoDSF) technology, by monitoring the intrinsic fluorescence of tryptophan upon thermal unfolding. The unfolding was measured by loading each sample into 24 well capillary (NanoTemper, Cat# PR-AC002) from a 384 well sample plate (ThermoNunc, Cat# 264573), with a heating ramp of l°C / minute between 20°C to 95°C using the Prometheus NT.48 instrument (NanoTemper Technologies GmbH). Each sample was measured at 0.5 mg / ml in phosphate buffer saline (PBS) in duplicates. The intrinsic fluorescence of each sample at 330 and 350 nm was used to monitor unfolding during temperature ramp and recorded as changes in fluorescence intensity over time. Data was collected and saved as projects, processed using the PR. Stability Analysis vl.0.2 software. The processed data contained integrated thermal melting profiles, first derivatives for fluorescence at 330nm, 350nm, ratio 330 / 350, and light scattering data for all the samples. Thermal melting midpoint (Tm) values as well as onset of aggregation (Tagg) were identified and reported.

[0211] Various anti-TfR antibodies or antigen binding fragments according to embodiments of the invention were analyzed using the above method, with the determined Tm values ranging from about 50°C to 73°C and Tagg from about 69°C to 79°C.

[0212] Example 6: Cell binding

[0213] Panels of antibodies were screened for specific TfR binding on 3 cell lines expressing human or cyno TfR and 1 negative control cell line not expressing human or cyno TfR. The 4 cell lines were stained separately with fluorescent cell dyes, to be differentiated through the assay. LIVE / DEAD™ Fixable Near-IR Stain (Thermo Fisher Scientific Cat# L34976) was also added to all the cells to select only live cells for the analysis. Human TfR transfected Canine kidney cells (MDCK huTfR clone 2C5) were stained with CellTrace™ CFSE (Thermo Fisher Scientific Cat# C34554). Cyno TfR transfected cells (MDCK cynoTfR stable pool) were stained with CellTrace™ Violet (Thermo Fisher Scientific Cat# C34557). MDCK parental cells received no stains. Human brain endothelial cells (hCMEC / D3) were stained with CellTrace CFSE and CellTrace Violet.

[0214] All four cell lines were mixed 1 : 1 : 1 : 1 together and 50,000 mixed cells were distributed to each well of 96-well assay plates. Fc x Fab-Fc or Fc x VHH-Fc monovalent anti-TfR mAbs were diluted in staining buffer and added to each well. Each antibody was tested in an 11 -point doseresponse starting at lOOnM with 3 -fold serial dilutions. Negative control wells received no primary antibody.

[0215] After 1 hour incubation at 4°C, cells were washed and stained with Alexa Fluor® 647 F(ab’)2 anti-human Fc (Jackson Immunosciences cat# 109-606-098) for 30min at 4°C. Cells were washed again with staining buffer, and data were acquired by IntelliCyt® iQue Screeners (Sartorius).

[0216] Cell lines were identified by fluorescent cell dyes. Live singlets of each cell lines were gated and the associated RL1H Geomean (signal of Alexa Fluor® 647) was measured. Then the signal over Background was calculated using the Forecyt software (S / B = RL1H Geomean for each cell line in each well / averaged RL1H Geomean for the same cell line from negative control wells). This metric characterizes the antibody binding to each cell line. S / B results were exported and analyzed with Genedata Screener for curve fitting and affinity determination. Max binding activity of each mAb was defined as the highest S / B reached in the tested concentration range.

[0217] Various anti-TfR antibodies or antigen binding fragments according to embodiments of the invention were analyzed using the above method. It was shown that apparent binding EC50s ranged from high nM to about 0.7 nM on hCMEC / D3 and MDCK huTfR cells. Apparent binding EC50s ranged from high nM to about 0.8 nM on MDCK cynoTfR cells. No significant binding was detected on MDCK parental cells.

[0218] Docket No. : JBI6864WOPCT 1

[0219] SEQUENCE LIST le 3: CDR SEQ ID NOs of VHH clones

[0220] Docket No. : JBI6864WOPCT 1

[0221] Docket No. : JBI6864WOPCT 1

[0222] Docket No. : JBI6864WOPCT 1

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[0224] Table 4: VHH SEQ ID NOs

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[0233] Table 5: CDR SEQ ID NOs of Fv clones

[0234] No.: JBI6864WOPCT1

[0235] No.: JBI6864WOPCT1

[0236] No.: JBI6864WOPCT1

[0237] No.: JBI6864WOPCT1

[0238] No.: JBI6864WOPCT1

[0239] No.: JBI6864WOPCT1

[0240] No.: JBI6864WOPCT1

[0241] No.: JBI6864WOPCT1

[0242] No.: JBI6864WOPCT1

[0243] No.: JBI6864WOPCT1

[0244] No.: JBI6864WOPCT1

[0245] No.: JBI6864WOPCT1

[0246] No.: JBI6864WOPCT1

[0247] No.: JBI6864WOPCT1

[0248] Table 6: VH / VL SEQ ID NOs of Fv clones

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[0250] Docket No. : JBI6864WOPCT 1

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[0255] Docket No. : JBI6864WOPCT 1

[0256] Docket No. : JBI6864WOPCT 1

[0257] Docket No. : JBI6864WOPCT 1

[0258] Docket No. : JBI6864WOPCT 1

[0259] Docket No. : JBI6864WOPCT 1

[0260] Docket No. : JBI6864WOPCT 1

[0261] Docket No. : JBI6864WOPCT 1

[0262] Docket No. : JBI6864WOPCT 1

[0263] Docket No. : JBI6864WOPCT 1

[0264] Docket No. : JBI6864WOPCT 1

[0265] Docket No. : JBI6864WOPCT 1

[0266] Docket No. : JBI6864WOPCT 1

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[0268] Docket No. : JBI6864WOPCT 1

[0269] Docket No. : JBI6864WOPCT 1

[0270] Docket No. : JBI6864WOPCT 1

[0271] Table 7: Linker SEQ ID NOs

Claims

CLAIMSWe claim:

1. An anti-TfR antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL) comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, and a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein:(i) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 222-308 and 1456-1485;(ii) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 309-359 and 1486-1511;(iii) the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 360-518 and 1512-1561;(iv) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 519-645 and 1562-1606;(v) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 646-792 and 1607-1661; and(vi) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 793-980 and 1662-1717.

2. The anti-TfR antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR1 , LCDR2 and LCDR3 have the amino acid sequences of the LCDR1 , LCDR2 and LCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

3. The anti-TfR antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1198-1420 and 1776-1832.

4. The anti-TfR antibody or antigen-binding fragment thereof of claim 3, wherein the VL comprises the amino acid sequence of the light chain variable region of any one of the antigen binding proteins specified in Table 6.

5. The anti-TfR antibody or antigen-binding fragment thereof of any one of the previous claims, wherein the HCDR1, HCDR2 and HCDR3 have the amino acid sequences of theHCDR1, HCDR2 and HCDR3, respectively, of any one of the antigen binding proteins specified in Table 5.

6. The anti-TfR antibody or antigen-binding fragment thereof of claim 5, wherein the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 981-1197 and 1718-1775.

7. The anti-TfR antibody or antigen-binding fragment thereof of claim 6, wherein the VH comprises the amino acid sequence of the heavy chain variable region of any one of the antigen binding proteins specified in Table 6.

8. The anti-TfR antibody or antigen-binding fragment thereof of any one of the previous claims, wherein the LCDR1, LCDR2 LCDR3, HCDR1, HCDR2, and HCDR3 have the amino acid sequences of the LCDR1, LCDR2 LCDR3, HCDR1, HCDR2, and HCDR3, respectively, of any one of the antigen binding proteins specified in Table 3.

9. The anti-TfR antibody or antigen-binding fragment thereof of claim 8, wherein the VL and VH comprise the amino acid sequences of the light chain variable region and the heavy chain variable region, respectively, of any one of the antigen binding proteins specified in Table 4.

10. The anti-TfR antibody or antigen-binding fragment thereof of any of the previous claims, having a single-chain variable fragment (scFv) comprising the VH and VL of the anti-TfR antibody or antigen-binding fragment thereof.

11. The anti-TfR antibody or antigen-binding fragment thereof of claim 10, wherein the scFv further comprises a linker that covalently links the VH and VL, such as a linker having the amino acid sequence selected from the group consisting of SEQ ID NO: 1421 and SEQ ID NOs:1424- 1455.

12. The anti-TfR antibody or antigen-binding fragment thereof of claim 11 , wherein the scFv comprises one or more disulfide bonds formed between a cysteine (Cys) in the linker and a surface exposed Cys in VH and / or VL, preferably a surface exposed Cys in a framework region of the VH and / or VL.

13. An anti-TfR antibody or antigen-binding fragment thereof having a single variable domain on a heavy chain (VHH) comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein:(i) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24;(ii) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-46; and(iii) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-70.

14. The anti-TfR antibody or antigen-binding fragment thereof of claim 13, wherein the HCDR1 , HCDR2 and HCDR3 have the amino acid sequences of the HCDR1 , HCDR2 and HCDR3, respectively, of any one of the VHH antigen binding proteins specified in Table 3.

15. The anti-TfR antibody or antigen-binding fragment thereof of claim 13 or 14, comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-221.

16. The anti-TfR antibody or antigen-binding fragment thereof of claim 15, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-221.

17. A conjugate comprising the anti-TfR antibody or antigen-binding fragment thereof of any one of claims 1-16 coupled to a therapeutic or diagnostic agent, preferably, the conjugate is a multi-specific antibody comprising a first antigen binding region which binds the TfR and comprises the anti-TfR antibody or antigen-binding fragment thereof of any one of claims 1-16 and a second antigen binding region which binds a brain target, such as a brain target selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, 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.

18. A fusion construct comprising the anti-TfR antibody or antigen-binding fragment thereof of any one of claims 1 to 16 covalently linked to a second antibody or an antigen binding fragment thereof that binds to a brain target, such as a brain target selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, 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.

19. The fusion construct of claim 18, wherein the anti-TfR antibody or antigen-binding fragment thereof is covalently linked to the carboxy terminus of only one of the two heavy chains of the second antibody or antigen binding fragment thereof via a linker, preferably the linker has the amino acid sequence of SEQ ID NO: 1422 or SEQ ID NO: 1423.

20. The fusion construct of claim 19, wherein each of the two heavy chains of the second antibody or antigen binding fragment thereof comprises one or more heterodimeric mutations, such as a modified heterodimeric CH3 domain, or one or more knob and hole mutations, as compared to a wild-type CH3 domain polypeptide.

21. The fusion construct of claim 20, wherein the heterodimeric mutations comprise the modified heterodimeric CH3 domain of the first heavy chain comprises amino acid modifications at positions T350, L351, F405, and Y407, and the modified heterodimeric CH3 domain of the second heavy chain comprises amino acid modifications at positions T350, T366, K392 and T394, wherein the amino acid modification at position T350 is T350V, T350I, T350L or T350M; the amino acid modification at position L351 is L351 Y; the amino acid modification at position F405 is F405A, F405V, F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W, and wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

22. The fusion construct of claim 20, wherein the modified heterodimeric CH3 domain of the first heavy chain comprises mutations T350V, L351 Y, F405A and Y407V, and the modified heterodimeric CH3 domain of the second heavy chain comprises mutations T350V, T366L, K392L and T394W.

23. The fusion of any one of claims 18-22, wherein the second antibody or antigen binding fragment thereof comprises one or more mutations in the Fc domain that enhance binding of the fusion to the neonatal Fc receptor (FcRn), preferably the one or more mutations enhance the binding at an acidic pH, more preferably the Fc has the M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

24. The fusion of any one of claims 18-23, wherein the second antibody or antigen binding fragment thereof comprises one or more mutations in the Fc domain that reduce or eliminate the effector function, preferably the Fc has one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

25. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-16, the conjugate of claim 17 or the fusion construct of any one of claims 18-24.

26. A vector comprising the isolated nucleic acid of claim 25.

27. A host cell comprising the nucleic acid of claim 25 or the vector of claim 26.

28. A method of producing the antibody or antigen-binding fragment of any one of claims 1- 16, the conjugate of claim 17, or the fusion construct of any one of claims 18-24, comprising culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment, the conjugate or the fusion construct under conditions to produce the antibody or antigen-binding fragment, the conjugate or the fusion construct, and recovering the antibody or antigen-binding fragment, the conjugate or the fusion construct from the cell or cell culture.

29. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-16, the conjugate of claim 17, or the fusion construct of any one of claims 18-24, and a pharmaceutically acceptable carrier.

30. A method of treating or detecting a disorder, preferably a neurological disorder, in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment of any one of claims 1-16, the conjugate of claim 17, or the fusion construct of any one of claims 18-24, or the pharmaceutical composition of claim 29, preferably, the neurological disorder is selected from the group consisting of neurodegenerative diseases (such as Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (such as Alzheimer's disease and supranuclear palsy), prion diseases (such as bovine spongiform encephalopathy, scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (such as Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis,Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (such as Pick's disease, and spinocerebellar ataxia), and cancer of the CNS and / or brain (such as brain metastases resulting from cancer elsewhere in the body).

31. The method of claim 30, wherein the antibody or antigen-binding fragment thereof, the conjugate, or the pharmaceutical composition is administered intravenously.

32. A method of delivering a therapeutic or diagnostic agent across the blood-brain barrier (BBB) of a subject in need thereof, comprising administering to the subject a complex comprising the therapeutic or diagnostic agent coupled to, preferably covalently conjugated to, the antibody or antigen-binding fragment thereof of any one of claims 1 to 16.

33. The method of any one of claims 30 to 32, wherein the administration reduces Fc- mediated effector function and / or does not induce rapid reticulocyte depletion.

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