Anti-transferrin receptor antibody with customized affinity

Abstract

Disclosed herein is an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor and the rhesus monkey transferrin receptor, comprising i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO:01, and ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO:26, wherein the dissociation rate of the antibody for the human transferrin receptor is equal to or less than (i.e., at most) the dissociation rate of anti-transferrin receptor antibody 128.1 for the rhesus monkey transferrin receptor, wherein the dissociation rate is determined by surface plasmon resonance, and anti-transferrin receptor antibody 128.1 has a heavy chain variable domain of SEQ ID NO:64 and a light chain variable domain of SEQ ID NO:65.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680037169.1 (PCT / EP2016 / 064460), with the filing date of June 22, 2016 and the invention title of "Anti-transferrin receptor antibodies with customized affinity". Field of the Invention

[0002] The present invention relates to anti-transferrin receptor antibodies having a designed dissociation rate against the human transferrin receptor and their use as a blood-brain barrier shuttle module. Background Art

[0003] The brain penetration of drugs for neurological disorders (e.g., large biotherapeutic drugs or small molecule drugs with low brain permeability) is strictly limited by the extensive and impermeable blood-brain barrier (BBB) and other cellular components in the neurovascular unit (NVU). Many strategies to overcome this hurdle have been tested and one of them is to utilize the transcytosis pathway mediated by endogenous receptors (blood-brain barrier receptors) expressed on brain capillary endothelium. Recombinant proteins, such as monoclonal antibodies or peptides, have been designed to target these receptors so that biotherapeutic agents can be delivered to the brain via receptor-mediated means. However, strategies to maximize brain uptake while minimizing miss-sorting in brain endothelial cells (BECs) and the extent of accumulation within certain organelles of BECs (especially those leading to degradation of biotherapeutic agents) have still not been developed.

[0004] Monoclonal antibodies and other biotherapeutic agents have great therapeutic potential for the treatment of central nervous system (CNS) disorders. However, their access to the brain is blocked by the BBB. Previous studies have shown that a very small proportion (about 0.1%) of IgG injected into the bloodstream can penetrate into the CNS compartment (Felgenhauer, Klin. Wschr. 52 (1974) 1158 - 1164). Due to the low concentration of antibodies within the CNS, this will surely limit any pharmacological effect.

[0005] Previously, it has been found that the percentage of antibodies distributed to the CNS can be increased by utilizing BBB receptors (i.e., transferrin receptor, insulin receptor, etc.) (see, for example, WO 95 / 02421).

[0006] Therefore, there is a need for a drug delivery system for neurological disorders across the BBB to effectively shuttle drugs into the brain.

[0007] In WO 2014 / 033074, blood-brain barrier shuttles were reported.

[0008] In WO 2014 / 189973, anti-transferrin receptor antibodies and methods of use were reported. It was further reported that targeting BBB receptors with conventional highly specific high-affinity antibodies generally results in limited improvement in BBB transport. It was then found that among the anti-BBB antibodies studied, the level of antibody uptake into and distribution within the CNS was negatively correlated with its binding affinity for the BBB receptor. For example, a low-affinity antibody to the transferrin receptor (TfR) greatly enhanced the BBB transport and CNS residence of the anti-TfR antibody when administered at therapeutic dose levels, relative to a higher-affinity anti-TfR antibody, which allowed for easier attainment of therapeutic concentrations in the CNS (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43). Evidence for this BBB transport was obtained using a bispecific antibody that binds both TfR and the amyloid precursor protein (APP) cleavage enzyme (β-secretase (BACE1)). Single systemic administration of a bispecific anti-TfR / BACE1 antibody engineered with a low-affinity antibody resulted in not only significant antibody uptake in the brain but also a marked reduction in brain Aβ1-40 levels, compared to a monospecific anti-BACE1 alone, indicating that BBB permeability affects the efficacy of anti-BACE1 (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43; Yu et al., Sci. Transl. Med. 3 (2011) 84ra44).

[0009] Due to the variability introduced by the increasing complexity of antibody engineering and the diversity of recombinant production cell systems used for antibody production, it is very important to conduct a further thorough non-clinical safety evaluation of monoclonal antibodies (mAbs) intended for therapeutic applications. In addition, apart from the concerns arising from the extended clinical use of mAbs for the treatment of chronic diseases, the complex structure, unique biological function, and longer half-life of mAbs, compared to traditional small molecule drugs, also increase the safety considerations (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Kim, S.J. et al., Mol. Cells 20 (2005) 17-29).

[0010] The overall goal of nonclinical studies for mAbs is to determine the toxicological properties of the mAb under investigation and to provide information for product development. The primary objectives of nonclinical evaluation are (1) to identify the target organs of toxicity and determine whether the toxicity is reversible after treatment, (2) to identify the safe starting dose for human Phase I clinical trials and subsequent dose escalation regimens, (3) to provide information for monitoring safety parameters in clinical trials, and (4) to provide safety data to support claims on the product label. To achieve these objectives, in vitro and in vivo nonclinical studies were conducted to define and understand the pharmacological properties of the antibody (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Cavagnaro, J.A., in: Cavagnaro, J.A. (ed.) "Preclinical safety evaluation of biopharmaceuticals"; Hoboken, NJ: Wiley 2008; 45-65).

[0011] For the successful nonclinical safety evaluation of mAbs, the most relevant animal species should be selected for toxicity testing (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Chapman, K. et al., Nat. Rev. Drug Discov. 6 (2007) 120-126). A relevant species is one in which the antibody has pharmacological activity, the target antigen should be present or expressed, and the tissue cross-reactivity profile should be similar to that of humans (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Chapman, K. et al., Nat. Rev. Drug Discov. 6 (2007) 120-126; Subramanyam, M. and Mertsching, E., in: Cavagnaro J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205; Hall, W.C. et al., in: Cavagnaro, J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240). The relevant animal species expressing the desired epitope and demonstrating tissue cross-reactivity profiles similar to human tissues can be identified using immunochemical or functional tests (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Hall, W.C. et al., in: Cavagnaro, J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240). Species cross-reactivity studies useful in this approach involve immunohistochemical studies of tissues from various species using commercially available multi-species tissue microarrays (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Hall, W.C. et al., in: Cavagnaro, J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240).Alternatively, the binding of the antibody to cells from these animals can be evaluated by fluorescence-activated cell sorting (FACS), which is generally more sensitive than immunohistochemical analysis of tissue sections (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., in: Cavagnaro J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205). The DNA and amino acid sequences of the target antigen should be compared across species; the homology between species should be determined (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., in: Cavagnaro J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205).

[0012] In addition, the biodistribution, function, and structure of the antigen should be comparable between relevant animal species and humans to allow for the evaluation of toxicity caused by antibody binding to the target antigen, which is referred to as on-target toxicity (Lynch, C.M. et al., mAbs 1 (2009) 2-11; 19, 20). In addition, a strong similarity in the tissue distribution of the target antigen between animal species and humans makes it more likely to predict potential toxicity in humans by the target organs of toxicity identified in animals. The lack of similarity in the tissue distribution of the antigen between animal species and humans does not completely rule out the use of the animal species for toxicity studies, but these differences must be considered for risk assessment in humans. With regard to antigen density or affinity, similarly, an absolute equivalence between the animal model and humans is not required. The rationale for the relevance of the species selected for toxicity testing should be included in the regulatory submission. If only one species is used for safety evaluation, the experimental summary should state the absence of other relevant species (Lynch, C.M. et al., mAbs 1 (2009) 2-11)

[0013] If a monoclonal antibody intended for therapeutic use does not have species cross-reactivity, an alternative antibody or a different species must be used for the model. Thus, alternative antibodies are a potential solution to the limited safety testing (possible when using humanized monoclonal antibodies with restricted species cross-reactivity). However, there are currently no clear criteria for determining a potential alternative antibody before it can be used to address safety issues of a clinical drug (Regulatory Toxicology and Pharmacology Volume 40, Issue 3, December 2004, pp. 219-226).

[0014] Therefore, in order to identify an animal model for a specific mAb, the above considerations must be made. However, the mAb under discussion must have cross-reactivity with the target antigen of the test species. Otherwise, even the most suitable test species cannot be used. Thus, an mAb is needed that does not have intra-species cross-reactivity but has inter-species cross-reactivity with its target in humans and the species intended for non-clinical trials.

[0015] In EP 2 708 560, antibodies that specifically recognize the transferrin receptor are reported. In FR 2 953 841, antibodies against the transferrin receptor and their use for immunotherapy of iron-dependent tumors are reported. In US 2009 / 162359, bivalent, bispecific antibodies are reported. Summary of the Invention

[0016] It has been found that the anti-transferrin receptor antibodies reported herein can be used as a blood-brain barrier shuttle module to deliver a brain effector entity across the blood-brain barrier into the brain. In certain embodiments, the blood-brain barrier shuttle module is a monovalent binding entity that specifically binds to the transferrin receptor. When used as a blood-brain barrier shuttle module, the anti-transferrin antibodies reported herein are useful, for example, for the diagnosis or treatment of neurological disorders such as Alzheimer's disease, Parkinson's disease, and comorbidities of Alzheimer's disease and Parkinson's disease.

[0017] Anti-transferrin receptor antibodies that specifically bind to the human transferrin receptor (huTfR) and the cynomolgus transferrin receptor (cyTfR) are reported herein. In certain embodiments, the anti-transferrin receptor antibody

[0018] · binds to the human transferrin receptor (huTfR) and the cynomolgus transferrin receptor (cyTfR);

[0019] ·The dissociation rate of the antibody to the human transferrin receptor is equal to or less than (i.e., at most) the dissociation rate of anti-transferrin receptor antibody 128.1 to the rhesus monkey transferrin receptor, wherein the dissociation rate is determined by surface plasmon resonance, and anti-transferrin receptor antibody 128.1 has a heavy chain variable domain of SEQ ID NO:64 and a light chain variable domain of SEQ ID NO:65;

[0020] ·Bind to the human transferrin receptor with a dissociation rate of from 0.1 1 / s to 0.005 1 / s (including 0.1 1 / s and 0.005 1 / s).

[0021] One aspect reported herein is an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor and the rhesus monkey transferrin receptor, which comprises

[0022] i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO:01, and

[0023] ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO:26,

[0024] wherein the dissociation rate of the antibody to the human transferrin receptor is equal to or less than (i.e., at most) the dissociation rate of anti-transferrin receptor antibody 128.1 to the rhesus monkey transferrin receptor,

[0025] wherein the dissociation rate is determined by surface plasmon resonance, and

[0026] anti-transferrin receptor antibody 128.1 has a heavy chain variable domain of SEQ ID NO:64 and a light chain variable domain of SEQ ID NO:65.

[0027] In one embodiment, the dissociation rate of the antibody to the human transferrin receptor is from 0.1 1 / s to 0.005 1 / s (including 0.1 1 / s and 0.005 1 / s).

[0028] In one embodiment, the antibody has a proline amino acid residue (P) at position 80 in the light chain variable domain (according to Kabat numbering).

[0029] In one embodiment, the antibody has an asparagine amino acid residue (N) at position 91 in the light chain variable domain (according to Kabat numbering).

[0030] In one embodiment, the antibody has an alanine amino acid residue (A) at position 93 in the light chain variable domain (according to Kabat numbering).

[0031] In one embodiment, the antibody has a serine amino acid residue (S) at position 100g in the heavy chain variable domain (according to Kabat numbering).

[0032] In one embodiment, the antibody has a glutamine amino acid residue (Q) at position 100g in the heavy chain variable domain (according to Kabat numbering).

[0033] In one embodiment, the antibody has a serine amino acid residue (S) at position 65 in the heavy chain variable domain (according to Kabat numbering).

[0034] In one embodiment, the antibody has a glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (according to Kabat numbering).

[0035] In one embodiment, the antibody has a proline amino acid residue (P) at position 80 in the light chain variable domain, an asparagine amino acid residue (N) at position 91 in the light chain variable domain, an alanine amino acid residue (A) at position 93 in the light chain variable domain, a serine amino acid residue (S) at position 100g in the heavy chain variable domain, a serine amino acid residue (S) at position 65 in the heavy chain variable domain, and a glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (according to Kabat numbering).

[0036] In one embodiment, the antibody has a proline amino acid residue (P) at position 80 in the light chain variable domain, an asparagine amino acid residue (N) at position 91 in the light chain variable domain, an alanine amino acid residue (A) at position 93 in the light chain variable domain, a glutamine amino acid residue (Q) at position 100g in the heavy chain variable domain, a serine amino acid residue (S) at position 65 in the heavy chain variable domain, and a glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (according to Kabat numbering).

[0037] One aspect reported herein is an anti-transferrin receptor antibody, comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 71, 72 or 73; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 75; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 76; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0038] In a preferred embodiment, the anti-transferrin receptor antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 75; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 76; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0039] One aspect reported herein is an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor (huTfR), which comprises

[0040] i) a heavy chain variable domain (VH) sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 24, and

[0041] ii) a light chain variable domain (VL) sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 37,

[0042] wherein the antibody has an approximately the same dissociation rate as an antibody comprising the heavy chain variable domain (VH) sequence of SEQ ID NO: 24 and the light chain variable domain (VL) sequence of SEQ ID NO: 37.

[0043] In one embodiment, the dissociation rate for the human transferrin receptor is from 0.1 1 / s to 0.005 1 / s (including 0.1 1 / s and 0.005 1 / s).

[0044] One preferred aspect reported herein is an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor (huTfR), which comprises

[0045] i) a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 24, and

[0046] ii) a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 37.

[0047] In one embodiment in all aspects, the antibody is a multispecific antibody having at least one binding specificity for the transferrin receptor and at least one binding specificity for a therapeutic target. In one embodiment, the antibody comprises a first antigen-binding site that binds the transferrin receptor and a second antigen-binding site that binds a brain antigen. In a further embodiment, the brain antigen is selected from human Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human α-synuclein, human tau protein (which is phosphorylated on tyrosine or serine residues), human CD20, amyloid precursor protein (APP), and human glucocerebrosidase. In a preferred embodiment, the multispecific antibody binds:

[0048] i) both the transferrin receptor and Aβ, or

[0049] ii) both the transferrin receptor and CD20, or

[0050] iii) both the transferrin receptor and α-synuclein, or

[0051] iv) both the transferrin receptor and phospho-tau protein, or

[0052] v) both the transferrin receptor and HER2, or

[0053] vi) both the transferrin receptor and glucocerebrosidase.

[0054] In one embodiment, the antibody is a bispecific antibody that comprises at least one pair of the heavy chain variable domain of SEQ ID NO:24 and the light chain variable domain of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of the heavy chain variable domain of SEQ ID NO:81 and the light chain variable domain of SEQ ID NO:82 that form a human Aβ binding site.

[0055] In one embodiment, the antibody is a bispecific antibody that comprises at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of heavy chain variable domains of SEQ ID NO:79 and light chain variable domains of SEQ ID NO:80 that form a human CD20 binding site. In one embodiment, the heavy chain variable region comprises an amino acid residue substitution at Kabat position 11 with any amino acid other than leucine. In one embodiment, the substitution comprises replacing the amino acid residue at Kabat position 11 with a non-polar amino acid. In a preferred embodiment, the substitution comprises replacing the amino acid residue at Kabat position 11 in the heavy chain variable domain of SEQ ID NO:79 with an amino acid residue selected from valine, leucine, isoleucine, serine, and phenylalanine.

[0056] In one embodiment, the antibody is a bispecific antibody that comprises at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of heavy chain variable domains of SEQ ID NO:83 and light chain variable domains of SEQ ID NO:84 that form a human α-synuclein binding site.

[0057] In one embodiment, the antibody is a bispecific antibody that comprises at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:85 and humanized light chain variable domains derived from SEQ ID NO:86 that form a human α-synuclein binding site.

[0058] In one embodiment, the antibody is a bispecific antibody that comprises at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:87 and humanized light chain variable domains derived from SEQ ID NO:88 that form a human α-synuclein binding site.

[0059] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:89 and humanized light chain variable domains derived from SEQ ID NO:90 that form a human α-synuclein binding site.

[0060] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:91 and humanized light chain variable domains derived from SEQID NO:92 that form a human α-synuclein binding site.

[0061] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:93 and humanized light chain variable domains derived from SEQ ID NO:94 that form a human α-synuclein binding site.

[0062] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and binding sites for: i) glucocerebrosidase having the amino acid sequence of SEQ ID NO:97, or ii) a functional variant of SEQ ID NO:97 having at least 70% sequence identity, or iii) a functional variant of SEQ ID NO:97 having one or more amino acid mutations, deletions or insertions, or iv) a truncated functional variant of SEQ ID NO:97 having at least one amino acid residue deletion at the N-terminus or C-terminus or within the amino acid sequence, or v) a combination of iii) and iv).

[0063] In one embodiment in all respects, the antibody comprises

[0064] i) a homodimeric Fc-region of the human IgG1 subclass, optionally having the mutations P329G, L234A and L235A, or

[0065] ii) a homodimeric Fc-region of the human IgG4 subclass, optionally having the mutations P329G, S228P and L235E, or

[0066] iii) Heterodimeric Fc-region, wherein

[0067] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0068] b) one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0069] c) one Fc-region polypeptide comprises the mutations T366W and S354C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C,

[0070] or

[0071] iv) Heterodimeric Fc-region of the human IgG4 subclass, wherein both Fc-region polypeptides comprise the mutations P329G, L234A and L235A, and

[0072] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0073] b) one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0074] c) one Fc-region polypeptide comprises the mutations T366W and S354C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C,

[0075] or

[0076] v) Heterodimeric Fc-region of the human IgG4 subclass, wherein both Fc-region polypeptides comprise the mutations P329G, S228P and L235E, and

[0077] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0078] b) one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0079] c) One Fc-region polypeptide contains the mutations T366W and S354C, while another Fc-region polypeptide contains the mutations T366S, L368A, Y407V, and Y349C.

[0080] In one embodiment in all respects, the antibody is a CrossMab.

[0081] One aspect reported herein is an anti-transferrin receptor antibody that comprises

[0082] i) a heavy chain variable domain selected from SEQ ID NO:52, 53, 54, 55, 56, 57, and 58 and a light chain variable domain selected from SEQ ID NO:60, 61, 62, and 63, or

[0083] ii) a heavy chain variable domain selected from SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 and a light chain variable domain selected from SEQ ID NO:27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.

[0084] One aspect reported herein is a pharmaceutical formulation comprising the antibody reported herein and a pharmaceutically acceptable carrier.

[0085] One aspect reported herein is the use of the antibody reported herein as a medicament.

[0086] One aspect reported herein is the use of the antibody reported herein in the preparation of a medicament for treating a neurological disorder.

[0087] In one embodiment, the neurological disorder is selected from neuropathy, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, CNS inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, CD20-positive cancer with brain metastases, and Her2-positive cancer with brain metastases.

[0088] One aspect reported herein is the use of the antibody reported herein in the preparation of a medicament for transporting one or more compounds across the blood-brain barrier (BBB). Brief Description of the Drawings

[0089] Figure 1 : Schematic diagram of the transcytosis assay.

[0090] Figure 2 : Dissociation rates of different anti-transferrin receptor antibodies measured using BIAcore at 25 °C; 1: 128.1; 2: 128.1 fused with anti-pTau antibody mAb86; 3: 567; 4: 932; 5: 567 fused with anti-pTau antibody mAb86; 6: 1026; 7: 1027; squares: binding to macaque transferrin receptor; circles: binding to human transferrin receptor; y-axis: dissociation rate [1 / s].

[0091] Figure 3 : Dissociation rates of different anti-transferrin receptor antibodies measured using BIAcore at 37 °C; 1: 128.1; 2: 932; 3: 1026; 4: 1027; squares: binding to macaque transferrin receptor; circles: binding to human transferrin receptor; y-axis: dissociation rate [1 / s].

[0092] Detailed Description of the Invention Embodiments

[0093] A humanized variant of rabbit antibody 299 that showed high transcytosis in the transcytosis assay according to Example 8 is reported herein. It has cross-reactivity with human and macaque transferrin receptors, i.e., specifically binds these two orthologs of transferrin, shows good cell staining, and has a half-life (reflected by the dissociation rate) similar to that of murine antibody 128.1 for both macaque transferrin receptor and human transferrin receptor.

[0094] One aspect reported herein is a humanized antibody that specifically binds to the human transferrin receptor, wherein the antibody comprises the HVRs of SEQ ID NO: 66, 68, and 72 in the heavy chain variable domain and the HVRs of SEQ ID NO: 75, 76, and 78 in the light chain variable domain.

[0095] In one embodiment, the humanized antibody comprises the heavy chain variable domain of SEQ ID NO: 24 and the light chain variable domain of SEQID NO: 37.

[0096] In one embodiment, the humanized antibody is effector function silent.

[0097] In one embodiment, the humanized antibody specifically binds to the human transferrin receptor and the macaque transferrin receptor.

[0098] In one embodiment, the humanized antibody is

[0099] a) a full-length antibody of human subclass IgG1, or

[0100] b) a full-length antibody of human subclass IgG4, or

[0101] c) a full-length antibody of human subclass IgG1 having mutations L234A, L235A and P329G,

[0102] d) a full-length antibody of human subclass IgG4 having mutations S228P, L235E and optionally P329G,

[0103] e) a full-length antibody of human subclass IgG1 having mutations L234A, L235A and P329G in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the corresponding other heavy chain, or

[0104] f) a full-length antibody of human subclass IgG4 having mutations S228P, L235E and optionally P329G in both heavy chains, and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the corresponding other heavy chain.

[0105] One aspect reported herein is a bispecific antibody that comprises

[0106] i) a first binding site comprising the heavy chain variable domain of SEQ ID NO:24 and the light chain variable domain of SEQ ID NO:37, and

[0107] ii) a second binding site selected from

[0108] a) the heavy chain variable domain of SEQ ID NO:81 and the light chain variable domain of SEQ ID NO:82, or

[0109] b) the heavy chain variable domain of SEQ ID NO:83 and the light chain variable domain of SEQ ID NO:84, or

[0110] c) the heavy chain variable domain of SEQ ID NO:85 and the light chain variable domain of SEQ ID NO:86, or

[0111] d) the heavy chain variable domain of SEQ ID NO:87 and the light chain variable domain of SEQ ID NO:88, or

[0112] e) The heavy chain variable domain of SEQ ID NO:91 and the light chain variable domain of SEQ ID NO:92, or

[0113] f) The heavy chain variable domain of SEQ ID NO:89 and the light chain variable domain of SEQ ID NO:90, or

[0114] g) The heavy chain variable domain of SEQ ID NO:93 and the light chain variable domain of SEQ ID NO:94, or

[0115] h) The heavy chain variable domain of SEQ ID NO:79 and the light chain variable domain of SEQ ID NO:80.

[0116] One aspect reported herein is a pharmaceutical formulation comprising an antibody reported herein and an optional pharmaceutically acceptable carrier.

[0117] One aspect reported herein is the use of an antibody reported herein as a medicament.

[0118] One aspect reported herein is the use of an antibody reported herein for the treatment of neurological disorders.

[0119] One aspect reported herein is the use of an antibody reported herein in the preparation of a medicament.

[0120] One aspect reported herein is a method of treatment comprising administering an antibody reported herein to treat a neurological disorder.

[0121] I. Definitions

[0122] For the purposes of the present invention, a "receptor human framework" is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework that contains a human immunoglobulin framework or a human consensus framework as defined below. A receptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence as it, or may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL receptor human framework sequence is the same as the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0123] "Affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0124] An "affinity matured" antibody is an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parental antibody that does not have such an alteration, which results in an increase in the affinity of the antibody for an antigen.

[0125] The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0126] An "antibody fragment" is a molecule that is not a full antibody and that comprises a portion of a full antibody that binds an antigen to which the full antibody binds. Examples of antibody fragments include (but are not limited to) Fv, Fab, Fab', Fab'-SH, F(ab')2; bispecific antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0127] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0128] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0129] "Effector function" refers to those biological activities attributable to the Fc-region of an antibody, which vary with antibody class. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as the B-cell receptor); and B-cell activation.

[0130] An effective amount of an agent (e.g., a pharmaceutical formulation) is an amount that is effective to achieve the desired therapeutic or prophylactic result at the required dosage and for the required duration.

[0131] The term "Fc region" as used herein defines the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc-regions and variant Fc-regions. In one embodiment, the human IgG heavy chain Fc-region extends from Cys226 or from Pro230 of the heavy chain to the carboxyl terminus. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0132] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0133] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having a heavy chain that includes an Fc-region as defined herein.

[0134] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical, in nucleic acid content, to the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as that screened for or selected for in the originally transformed cell are included herein.

[0135] "Human consensus framework" is a framework that represents the amino acid residues that occur most frequently in a selected human immunoglobulin VL or VH framework sequence. Generally, the selected human immunoglobulin VL or VH sequence is from a subgroup of variable domain sequences. Generally, the sequence subgroup is a subgroup such as that in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI as in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0136] A "humanized" antibody is an antibody in which amino acid residues from a non-human HVR and amino acid residues from a human FR are included. In certain embodiments, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0137] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementary determining region" or "CDR") and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). Generally, an antibody comprises six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3).

[0138] The HVRs herein include

[0139] (a) Hypervariable loops present at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia, C. and Lesk, A.M., J. Mol. Biol. 196 (1987) 901 - 917);

[0140] (b) Complementary determining regions (CDRs) present at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91 - 3242);

[0141] (c) Antigen - contacting points present at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996)); and

[0142] (d) Combinations of (a), (b), and / or (c), including HVR amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1), 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3).

[0143] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to the Kabat EU index numbering system (Kabat et al., supra).

[0144] "Individual" or "subject" is 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.

[0145] An "isolated" antibody is one that has been separated from components in its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, which is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0146] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components in its natural environment. Isolated nucleic acids include the nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0147] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody constituting the population is identical and / or binds the same epitope, except for possible variant antibodies that are present in minor amounts (e.g., antibodies containing natural mutations or variant antibodies that arise during the production of the monoclonal antibody preparation). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against one determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals that contain all or part of the human immunoglobulin locus, and these methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0148] A "natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of antibodies can be assigned to one of two types (referred to as kappa (κ) and lambda (λ)) based on the amino acid sequence of their constant domains.

[0149] The term "package insert" is used to denote the instructions that are customarily included in the commercial packaging of a therapeutic product and that contain information on indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of the therapeutic product.

[0150] The "percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence when the candidate sequence is aligned with the reference sequence and gaps are introduced, if necessary, to achieve the maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in a variety of ways in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 is used to generate the % amino acid sequence identity value. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington, D.C., 20559, under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including Digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are not changed.

[0151] In the case of amino acid sequence comparison using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A relative to, with, or against a given amino acid sequence B (which can alternatively be stated as a given amino acid sequence A having or containing a specific % amino acid sequence identity relative to, with, or against a given amino acid sequence B) can be calculated as follows:

[0152] 100 × fraction X / Y

[0153] Wherein X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in an ALIGN-2 program alignment of A and B, and wherein Y is the total number of amino acid residues in B. It should be understood that in the case where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. Unless otherwise specifically stated, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0154] The term "pharmaceutical composition" refers to a composition that is presented in a form in which the biological activity of the active ingredient contained therein is effective and that does not contain other components that have unacceptable toxicity to the subject to which the composition is to be administered.

[0155] "Pharmaceutically acceptable carrier" refers to the components of a pharmaceutical composition other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0156] As used herein, "treatment" (and its grammatical variants such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated and can be performed for prophylaxis or during a clinical pathologic process. Desired therapeutic effects include, but are not limited to: preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathologic consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some embodiments, the antibodies of the invention are used to delay the development or slow the progression of a disease.

[0157] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that is involved in binding of the antibody to its antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of a native antibody generally have a similar structure, each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening a library of complementary VL or VH domains using the VH or VL domain from an antibody that binds the antigen (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628). Numbering of amino acid residues in the variable regions (variable regions of the light and heavy chains) will be according to Kabat (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3).

[0158] The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0159] The term "blood-brain barrier" (BBB) refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight junctions within the plasma membrane of brain capillary endothelial cells, forming a tight barrier that restricts the transport of molecules, even very small molecules such as urea (60 daltons), into the brain. The BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina are continuous capillary barriers within the CNS and are collectively referred to herein as the blood-brain barrier or BBB. The BBB also encompasses the blood-CSF barrier (choroid plexus), where the barrier is composed of ependymal cells rather than capillary endothelial cells.

[0160] The term "central nervous system" (CNS) refers to the complex of nerve tissue that controls the body's functions and includes the brain and spinal cord.

[0161] The term "blood-brain barrier receptor" (BBBR) refers to an extracellular membrane-linked receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the BBB or can be used to transport exogenously administered molecules. Examples of BBBRs include, but are not limited to, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low density lipoprotein receptor (including, but not limited to, 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 BBBR is transferrin receptor (TfR).

[0162] The term "brain effector entity" refers to a molecule to be transported across the BBB. Effector entities typically have the desired characteristic therapeutic activity to be delivered to the brain. Effector entities include drugs for neurological disorders and cytotoxic agents such as polypeptides and antibodies, particularly monoclonal antibodies or fragments thereof directed against brain targets.

[0163] The term "monovalent binding entity" refers to a molecule capable of specifically binding to a BBBR in a monovalent binding mode. As reported herein, the blood-brain shuttle module and / or conjugate is characterized by the presence of a single unit of the monovalent binding entity, i.e., the blood-brain shuttle module and / or conjugate of the present invention contains only one unit of the monovalent binding entity. Monovalent binding entities include, but are not limited to, polypeptides, full-length antibodies, antibody fragments, including Fab, Fab’, Fv fragments, single-chain antibody molecules, e.g., single-chain Fab, scFv. Monovalent binding entities can be, for example, scaffold proteins engineered using prior art such as phage display or immunization. Monovalent binding entities can also be polypeptides. In certain embodiments, the monovalent binding entity comprises a CH2-CH3 Ig domain and a single-chain Fab (scFab) directed against a blood-brain barrier receptor. The scFab is conjugated to the C-terminus of the CH2-CH3 Ig domain via a linker. In certain embodiments, the scFab is directed against the transferrin receptor.

[0164] The term "monovalent binding mode" refers to specific binding to a BBBR, wherein the interaction between the monovalent binding entity and the BBBR occurs through a single epitope. This monovalent binding mode can prevent any dimerization / polymerization of the BBBR due to the single epitope interaction point. The monovalent binding mode prevents alteration of the intracellular sorting of the BBBR.

[0165] The term "epitope" refers to any polypeptide determinant capable of specifically binding an antibody. In certain embodiments, the epitope determinant includes the chemically reactive surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural features and / or specific charge features. An epitope is the antigenic region to which an antibody binds.

[0166] "Transferrin receptor" (TfR) is a transmembrane glycoprotein (with a molecular weight of approximately 180,000 Da), which consists of two subunits bound by disulfide bonds (the apparent molecular weight of each subunit is approximately 90,000 Da) and is involved in iron uptake in vertebrates. In one embodiment, the TfR herein is human TfR comprising the amino acid sequence reported in Schneider et al. (Nature 311 (1984) 675–678).

[0167] The term "imaging agent" refers to a compound having one or more properties that allow direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins and small molecule compounds conjugated with labeled entities that allow detection.

[0168] The terms "CNS antigen" and "brain target" refer to antigens and / or molecules expressed in the CNS (including the brain) that can be targeted by antibodies or small molecules. Examples of such antigens and / or molecules include, but are not limited to: beta-secretase 1 (BACE1), amyloid-beta protein (Aβ), 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), glucocerebrosidase, and caspase 6.

[0169] The term "specifically binds" means that an antibody selectively or preferentially binds to an antigen. Standard tests, such as Scatchard analysis or surface plasmon resonance techniques (e.g., using ), are typically used to determine the binding affinity.

[0170] The term "CH2-CH3 Ig entity" as used herein refers to a protein entity derived from the immunoglobulin CH2 or CH3 domain. The "CH2-CH3 Ig entity" comprises two "CH2-CH3" polypeptides that form a dimer. The immunoglobulin can be IgG, IgA, IgD, IgE, or IgM. In one embodiment, the CH2-CH3 Ig entity is derived from an IgG immunoglobulin and is referred to herein as a "CH2-CH3 IgG entity". The term includes the native sequence of the CH2-CH3 domain and variant CH2-CH3 domains. In one embodiment, the "CH2-CH3 Ig entity" is derived from the human heavy chain CH2-CH3 IgG domain, which extends from Cys226 or from Pro230 of the heavy chain to the carboxyl terminus. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the CH2-CH3 domain region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0171] A "conjugate" is a fusion protein of the invention conjugated to one or more heterologous molecules, including but not limited to a label, a neurological disorder drug, or a cytotoxic agent.

[0172] The term "linker" refers to a chemical linker or a single-chain peptide linker that covalently connects different entities of the blood-brain barrier shuttle module and / or fusion polypeptide and / or conjugate reported herein. The linker can, for example, connect a brain effector entity to a monovalent binding entity. For example, if the monovalent binding entity comprises a CH2-CH3 Ig entity and an scFab against a blood-brain barrier receptor, then the linker can conjugate the scFab to the C-terminus of the CH2-CH3 Ig entity. The linker that conjugates the brain effector entity to the monovalent binding entity (the first linker) and the linker that connects the scFab to the C-terminus of the CH2-CH3 Ig domain (the second linker) can be the same or different.

[0173] A single-chain peptide linker consisting of one to twenty amino acid residues linked by peptide bonds can be used. In certain embodiments, the amino acids are selected from the twenty natural amino acids. In certain other embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is a chemical linker. In certain embodiments, the linker is a single-chain peptide linker having an amino acid sequence with a length of at least 25 amino acid residues, and in a preferred embodiment, having a length of 32 to 50 amino acid residues. In one embodiment, the peptide linker is a (GxS)n linker, where G = glycine, S = serine, (x = 3, n = 8, 9, or 10) or (x = 4 and n = 6, 7, or 8), in one embodiment, x = 4, n = 6 or 7, and in a preferred embodiment, x = 4, n = 7. In one embodiment, the linker is (G4S)4 (SEQ ID NO:95). In one embodiment, the linker is (G4S)6G2 (SEQ ID NO:96).

[0174] A variety of chemical linkers can be used for conjugation. For example, a variety of bifunctional protein crosslinking agents can be used to conjugate a monovalent binding entity or a fusion polypeptide and a brain effector entity, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazo compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). The linker can be a "cleavable linker" that facilitates the release of the effector entity upon delivery to the brain. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52(1992)127-131; US 5,208,020).

[0175] Covalent conjugation can be direct or via a linker. In certain embodiments, direct conjugation is the construction of a polypeptide fusion (e.g., a gene fusion encoding two genes for a monovalent binding entity and an effector entity targeting a BBBR and expressed as a single polypeptide (chain)). In certain embodiments, direct conjugation is the formation of a covalent bond between a reactive group on one of the two moieties of a monovalent binding entity targeting a BBBR and a corresponding group or receptor on a brain effector entity. In certain embodiments, direct conjugation is the modification (i.e., genetic modification) of one of the two molecules to be conjugated to contain a reactive group (as a non-limiting example, a thiol group or a carboxyl group), which forms a covalent linkage with the other molecule to be conjugated under suitable conditions. As a non-limiting example, a molecule (i.e., an amino acid) having a desired reactive group (i.e., a cysteine residue) can be introduced into, for example, a monovalent binding entity of an antibody targeting a BBBR and form a disulfide bond with a neuropharmaceutical. Methods for covalently conjugating nucleic acids to proteins are also known in the art (i.e., photocrosslinking, see, e.g., Zatsepin et al., Russ. Chem. Rev. 74 (2005) 77-95). Conjugation can also be carried out using various linkers. For example, various bifunctional protein coupling agents can be used to conjugate a monovalent binding entity and an effector entity, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazo compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Peptide linkers consisting of one to twenty amino acid residues linked by peptide bonds can also be used. In certain such embodiments, the amino acid residues are selected from the twenty natural amino acids. In certain other embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker can be a "cleavable linker" that facilitates the release of the effector entity after delivery to the brain. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52 (1992) 127-131; US 5,208,020).

[0176] II. Compositions and Methods

[0177] Equilibrium dissociation constant (K D)It is usually used to describe molecular interactions. It serves as a measure of the strength of the interaction between two molecules (e.g., affinity). Thus, the K D value is a measure of the strength of biomolecular interactions.

[0178] However, the K D value does not describe the kinetics of molecular interactions. That is, on the one hand, how fast two molecules bind to each other (association rate constant or "on rate") cannot be inferred from the K D value, and on the other hand, how fast the molecules dissociate (dissociation rate constant or "off rate") cannot be inferred from the K D value. Characterizing biomolecular interactions solely by the K D value would ignore the fact that the same K D value can be composed of very different (orders of magnitude different) association rates and dissociation rates, because the K D value is the ratio of the association rate and the dissociation rate.

[0179] However, the association rate and the dissociation rate are important for characterizing the binding behavior of molecules. The dissociation rate is particularly important because it characterizes, for example, the binding duration of an antibody to its antigen. A long dissociation rate corresponds to slow dissociation of the formed complex, while a short dissociation rate corresponds to fast dissociation.

[0180] To have long-lasting (i.e., low-frequency dosing requirement) or customized (e.g., according to the surrounding situation) interactions, the dissociation rate must be determined experimentally. This is even more important because it is almost impossible to predict the dissociation rate. Additionally, as mentioned above, the correlation between the dissociation rate and the binding affinity is poor. For example, since the K D value is the ratio of the association rate and the dissociation rate, even a weak binder can maintain long-term binding to its target, while a strong binder can dissociate quickly.

[0181] A typical bottleneck in antibody production projects is the ranking of many candidates obtained after panning based on antibody binding strength. Ideally, such a method would be carried out without prior antigen labeling and using crude bacterial lysates. Ylera, F. et al. (Anal. Biochem. 441 (2013) 208-213) reported a dissociation rate screening method for selecting high-affinity anti-drug antibodies in crude Escherichia coli lysates containing monovalent Fab fragments. They chose the dissociation rate as the ranking parameter because of reasons such as the dissociation rate being concentration-independent. They selected the monovalent form to avoid the influence of avidity during the dissociation rate ranking and affinity determination processes (avidity effects would be observed when using intact IgG). Ylera et al. have found that clones with the best koff-rate were identified through the koff ranking step, while this clone would not be identified using only the ELISA signal intensity as the selection criterion.

[0182] Murray, J.B. et al. (J. Med. Chem. 57 (2014) 2845-2850) reported Off-Rate Screening (ORS) By Surface Plasmon Resonance as An Efficient Method to Kinetically Sample Hit to Lead Chemical Space from Unpurified Reaction Products. It was outlined that the off-rate constant kd (dissociation rate) is the most prominent factor that is likely to enhance compound efficacy in ligand-protein binding. The authors pointed out that kinetic measurements of affinity provide more information than steady-state affinity equilibrium determinations throughout the drug development project. For example, a compound with a 10-fold slower association rate and dissociation rate may not be considered different if evaluated by affinity equilibrium measurements. In addition, the authors have found that data measured with a BIAcore T200 instrument showed an average kd difference of 19% between crude and pure samples. Similarly, data measured with an older BIAcore T100 instrument had a 15% difference. When comparing between instruments and over time, the authors observed an average kd difference of only 30%. This demonstrates that carryover contamination, long-term storage, and different equipment have a modest impact on the observed kd. In fact, these small deviations in kd closely reflect the differences observed in multi-laboratory studies, where depending on the system, observed variability has been reported to be between 14% and 40% (Murray, J.B. et al., J. Med. Chem. 57 (2014) 2845-2850; Katsamba, P.S. et al., Anal. Biochem. 352 (2006) 208-221).

[0183] In WO 2014 / 189973, anti-transferrin receptor antibodies and methods of use were reported. It was further reported that targeting BBB receptors with traditional highly specific and high-affinity antibodies generally results in limited improvement in BBB transport. It was then found that among the anti-BBB antibodies studied, the level of antibody uptake into the CNS and distribution within the CNS was inversely correlated with its binding affinity for the BBB receptor. For example, a low-affinity antibody to the transferrin receptor (TfR) administered at a therapeutic dose level resulted in a substantial increase in BBB transport and CNS residence of the anti-TfR antibody, relative to a higher-affinity anti-TfR antibody, and enabled easier attainment of therapeutic concentrations within the CNS (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43). Proof of such BBB delivery was obtained using a bispecific antibody that binds both TfR and the amyloid precursor protein (APP) cleaving enzyme (β-secretase (BACE1)). A single systemic dose of the bispecific anti-TfR / BACE1 antibody engineered with a low-affinity antibody resulted in not only significant antibody uptake in the brain but also a marked reduction in brain Aβ1-40 levels, compared to monospecific anti-BACE1 alone, indicating that BBB penetration affects the efficacy of anti-BACE1 (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43; Yu et al., Sci. Transl. Med. 3 (2011) 84ra44).

[0184] Available data and experiments point to several causative mechanisms underlying the use of low-affinity antibody approaches to enhance antibody uptake into the CNS.

[0185] First, high-affinity anti-BBB-receptor (BBB-R) antibodies (e.g., the anti-TfR antibodies from Atwal and Yu et al. cited above) can limit brain uptake by rapidly saturating the BBB-R in the cerebral vasculature, thereby reducing the total amount of antibody taken up into the brain and also limiting its distribution within the vasculature. Notably, reducing the affinity for the BBB-R can enhance brain uptake and distribution, and robust migration from the vasculature to neurons and the associated neuropil distributed within the CNS has been observed. It has been found that the affinity must be less than a certain upper limit and greater than a certain lower limit.

[0186] Second, it has been proposed that the lower affinity of the antibody for the BBB-R weakens the ability of the antibody to return from the CNS side of the membrane to the vascular side of the BBB via the BBB-R, because the overall affinity of the antibody for the BBB-R is low and the local concentration of antibody on the CNS side of the BBB is unsaturated due to rapid dispersion of the antibody into the CNS compartment.

[0187] Third, antibodies with a lower affinity for BBB-R, in vivo and as observed for the TfR system, are not cleared from the system as effectively as antibodies with a higher affinity for BBB-R and will therefore retain a higher circulating concentration compared to their higher affinity counterparts. This is advantageous because the circulating antibody levels of the lower affinity antibody will be maintained at therapeutic levels for a longer period of time than the higher affinity antibody, thereby improving antibody uptake in the brain for a longer time. Further, this improvement in both plasma and brain exposure can reduce the clinical dosing frequency, which has potential benefits not only for patient compliance and convenience, but also for reducing any potential side effects or off-target effects of the antibody and / or the therapeutic compound to which it binds.

[0188] These prior studies utilized murine antibodies that specifically bind murine TfR but do not specifically recognize primate or human TfR. Accordingly, antibodies and functional portions thereof that specifically recognize both primate (especially macaque) and human TfR are provided herein to facilitate safety and efficacy studies in primates prior to human therapeutic or diagnostic applications.

[0189] Due to the increasing complexity in antibody engineering and the variability introduced by the diversity of recombinant production cell systems used for antibody generation, a thorough non-clinical safety evaluation of monoclonal antibodies (mAbs) intended for therapeutic applications is of great importance. In addition, aside from the issues arising from the long-term clinical use of mAbs for the treatment of chronic diseases, the complex structure, unique biological function, and longer half-life of mAbs also add to the safety considerations compared to traditional small molecule drugs (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Kim, S.J. et al., Mol. Cells 20 (2005) 17-29).

[0190] The overall objective of nonclinical studies for mAbs is to determine the toxicological properties of the mAb under investigation and to provide information for product development. The primary objectives of nonclinical evaluation are (1) to identify the target organs of toxicity and determine whether the toxicity is reversible following treatment, (2) to identify a safe starting dose for human Phase I clinical trials and subsequent dose escalation regimens, (3) to provide information for monitoring safety parameters in clinical trials and (4) to provide safety data to support claims on the product label. To achieve these objectives, in vitro and in vivo nonclinical studies were conducted to define and understand the pharmacological properties of the antibody (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Cavagnaro, J.A., in: Cavagnaro, J.A. (ed.) "Preclinical safety evaluation of biopharmaceuticals"; Hoboken, NJ: Wiley 2008; 45-65).

[0191] For the successful nonclinical safety evaluation of mAbs, the most relevant animal species should be selected for toxicity testing (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Chapman, K. et al., Nat. Rev. Drug Discov. 6 (2007) 120-126). A relevant species is one in which the antibody has pharmacological activity, the target antigen should be present or expressed, and the tissue cross-reactivity profile should be similar to that of humans (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Chapman, K. et al., Nat. Rev. Drug Discov. 6 (2007) 120-126; Subramanyam, M. and Mertsching, E., in: Cavagnaro J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205; Hall, W.C. et al., in: Cavagnaro, J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240). The relevant animal species expressing the desired epitope and demonstrating tissue cross-reactivity profiles similar to human tissues can be identified using immunochemical or functional tests (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Hall, W.C. et al., in: Cavagnaro, J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240). Species cross-reactivity studies useful in this approach involve immunohistochemical studies of tissues from various species using commercially available multi-species tissue microarrays (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Hall, W.C. et al., in: Cavagnaro, J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240).Alternatively, the binding of the antibody to cells from these animals can be evaluated by fluorescence-activated cell sorting (FACS), which is generally more sensitive than immunohistochemical analysis of tissue sections (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., in: Cavagnaro J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205). The DNA and amino acid sequences of the target antigen should be compared across species; the homology between species should be determined (Lynch, C.M. et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., in: Cavagnaro J.A. (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205).

[0192] In addition, the biodistribution, function, and structure of the antigen should be comparable between relevant animal species and humans to allow for the evaluation of toxicity caused by antibody binding to the target antigen, which is referred to as on-target toxicity (Lynch, C.M. et al., mAbs 1 (2009) 2-11; 19, 20). In addition, a strong similarity in the tissue distribution of the target antigen between animal species and humans makes it more likely to predict potential toxicity in humans through the target organs of toxicity identified in animals. A lack of similarity in the tissue distribution of the antigen between animal species and humans does not completely rule out the use of the animal species for toxicity studies, but these differences must be considered when conducting human risk assessments. As for antigen density or affinity, similarly, an absolute equivalence between the animal model and humans is not required. The rationale for the relevance of the species selected for toxicity testing should be included in the regulatory submission. If only one species is used for safety evaluation, it is necessary to state in the experimental summary that no other relevant species exist (Lynch, C.M. et al., mAbs 1 (2009) 2-11)

[0193] If a monoclonal antibody intended for therapeutic use does not have species cross - reactivity, an alternative antibody or a different species must be used for the model. Thus, alternative antibodies are a potential solution to the limited safety testing (which may be limited when using humanized monoclonal antibodies with restricted species cross - reactivity). However, there are currently no clear criteria for judging a potential alternative antibody before it is used to determine the safety issues of a clinical drug (Regulatory Toxicology and Pharmacology Volume 40, Issue 3, December 2004, pp. 219 - 226).

[0194] Therefore, in order to identify an animal model for a specific mAb, the above considerations must be made. However, in any case, the mAb under discussion must have cross - reactivity with the target antigen of the test species. Otherwise, even the most suitable test species cannot be used. Thus, an mAb is needed that does not have intra - species cross - reactivity but has inter - species cross - reactivity with its target in humans and the species intended for non - clinical trials.

[0195] A. Exemplary anti - transferrin antibodies

[0196] Anti - transferrin receptor antibodies are reported herein that have a dissociation rate within a certain range for binding to the human transferrin receptor to ensure appropriate BBB transport. It has been found that one end of this range is defined by the dissociation rate of the murine anti - transferrin receptor antibody 128.1 (amino acid sequences of the variable domains given in SEQ ID NO: 64 and 65) measured by surface plasmon resonance against the rhesus macaque transferrin receptor, and the other end is defined by 5% of this dissociation rate (i.e., a dissociation 20 times slower). In one embodiment, the dissociation rate for the human transferrin receptor is between 0.1 1 / s and 0.005 1 / s (including 0.1 1 / s and 0.005 1 / s).

[0197] Using standard humanization techniques, a humanized antibody of clone 229 reported herein cannot be obtained. Non - standard mutations need to be introduced into the amino acid sequence to obtain a humanized antibody with a transferrin receptor - binding dissociation rate within the expected range (including 0.1 1 / s and 0.005 1 / s). This is particularly important because the antibodies reported herein were developed to cross the human blood - brain barrier to transport a therapeutic payload to the brain.

[0198] It has been found that, in order to obtain suitable and developable humanized antibodies, two cysteine amino acid residues in the light chain of the parental rabbit antibody must be replaced with proline and asparagine amino acid residues, respectively. In addition, serine residues present in the middle of rabbit CDRL3 must be replaced with alanine residues within a given dissociation rate range.

[0199] It has further been found that it is advantageous to alter three amino acid residues at positions 65, 100g, and 105 (according to Kabat numbering) in the heavy chain.

[0200] All numbering used herein is based on the Kabat variable domain numbering scheme.

[0201] Rabbit anti-transferrin antibody clone 299 shows characteristics comparable to those of anti-transferrin receptor antibody 128.1. This can be seen in the table below.

[0202]

[0203] In the table below, the dissociation rates of humanized variants of the rabbit light chain variable domain of clone 299 in combination with humanized variants of the rabbit heavy chain variable domain are shown. The binding partner is human transferrin receptor (measured at 25 °C).

[0204]

[0205]

[0206] The combination of VH23 and VL9 was selected as the starting point for further engineering to develop such a binding site that more closely reflects the binding characteristics of antibody 128.1 to the macaque transferrin receptor with respect to binding to the human transferrin receptor.

[0207] In the table below, the dissociation rates (measured at 25 °C according to Example 14) of different exemplary variants of VH23 and VL9 and other different variable domain humanized variants against the human transferrin receptor are compared.

[0208]

[0209] Reference: 128.1 = 7.78E-02 (measured against macaque transferrin receptor).

[0210] In the table below, the kinetic data of different exemplary variants of VH23 and VL9 are compared (measured according to Example 13)

[0211] BIAcore assay @ 25 °C TfR <![CDATA[kd[s -1 > <![CDATA[ka[s -1 M -1 > kD [M] mAb 128.1 Rhesus monkey 7.33E-02 5.41E+05 1.36E-07 VH23-DASG / VL09-NYA Human 3.95E-02 8.83E+04 4.47E-07 VH23-DAQG / VL09-NYA Human 1.37E-02 1.21E+05 1.13E-07 VH23-DANG / VL09-NYA Human 8.83E-03 1.55E+05 5.72E-08

[0212] In the table below, the dissociation rates of humanized variants of the murine light chain variable domain of clone 494 and humanized variants of the murine heavy chain variable domain of clone 494 are shown. The binding partner is the human transferrin receptor.

[0213]

[0214] More specifically, in one aspect, the present invention is in part based on the discovery that the anti-transferrin receptor reported herein can be used as a blood-brain barrier shuttle module to deliver brain effector entities across the blood-brain barrier into the brain. In certain embodiments, the blood-brain barrier shuttle module is a monovalent binding entity that specifically binds to the transferrin receptor. The anti-transferrin receptor antibodies reported herein are useful when used as blood-brain barrier shuttle modules, for example, for the diagnosis or treatment of neurological disorders such as Alzheimer's disease, Parkinson's disease, and the comorbidity of Alzheimer's disease and Parkinson's disease.

[0215] It has been found that with respect to the binding dissociation rate, an antibody comprising the heavy chain variable domain of SEQ ID NO:24 and the light chain variable domain of SEQ ID NO:37 reflects the binding characteristics of murine antibody 128.1 with respect to the cynomolgus monkey transferrin receptor relative to the human transferrin receptor.

[0216] Accordingly, one aspect reported herein is an isolated antibody that binds to the human transferrin receptor (huTfR) and the cynomolgus monkey transferrin receptor (cyTfR), wherein the antibody has a dissociation rate of 0.1 1 / s to 0.005 1 / s with respect to the human transferrin receptor as determined by surface plasmon resonance.

[0217] Another aspect reported herein is the use of an antibody or antibody fragment that binds to the human transferrin receptor (huTfR) and the cynomolgus monkey transferrin receptor (cyTfR) for delivering a therapeutic entity across the blood-brain barrier, wherein the antibody has a dissociation rate of 0.1 1 / s to 0.005 1 / s with respect to the human transferrin receptor as determined by surface plasmon resonance.

[0218] In one embodiment, the dissociation rate is measured at 500, 250, 125, 62.5, 31.25, 15.625, and 0 nM.

[0219] In one embodiment, a surface plasmon resonance chip having a biotin surface and a 1×PBS running buffer supplemented with 250 mM sodium chloride are used, and the dissociation rate is measured at a flow rate of 10 μL / min.

[0220] In one embodiment, binding is monitored for 180 seconds and dissociation is monitored for 600 seconds.

[0221] In one embodiment, the dissociation rate is determined on a BIAcore T200.

[0222] In one embodiment, the dissociation rate is from 0.08 l / s to 0.008 l / s.

[0223] In one embodiment in all respects, the dissociation rate is determined at 25 °C.

[0224] In one embodiment in all respects, the dissociation rate is the dissociation rate determined at 25 °C.

[0225] One aspect reported herein is an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor (huTfR) and the cynomolgus transferrin receptor (cyTfR), comprising i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO:01 and ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO:26, wherein the light chain variable domain has a proline amino acid residue (P) at position 80, an asparagine amino acid residue (N) at position 91, and an alanine amino acid residue (A) at position 93 (according to Kabat numbering).

[0226] In one embodiment, the antibody further has a serine residue (S) at position 100g in the heavy chain variable domain (numbered according to Kabat).

[0227] In one embodiment, the antibody further has a serine residue (S) at position 65 in the heavy chain variable domain (numbered according to Kabat).

[0228] In one embodiment, the antibody has a glutamine amino acid residue (Q) at position 105 in the heavy chain variable domain (according to Kabat numbering).

[0229] One aspect reported herein is an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor (huTfR) and the cynomolgus transferrin receptor (cyTfR), comprising i) a humanized heavy chain variable domain derived from the heavy chain variable domain of SEQ ID NO:01 and ii) a humanized light chain variable domain derived from the light chain variable domain of SEQ ID NO:26, wherein the antibody has a dissociation rate in units of 1 / s for the human transferrin receptor, the dissociation rate being equal to or less than (i.e., at most) the dissociation rate in units of 1 / s of anti-transferrin receptor antibody 128.1 for the cynomolgus transferrin receptor, wherein the dissociation rate is determined by surface plasmon resonance, and wherein anti-transferrin receptor antibody 128.1 has a heavy chain variable domain of SEQ ID NO:64 and a light chain variable domain of SEQ ID NO:65.

[0230] In one embodiment, the antibody has a dissociation rate in units of 1 / s for the human transferrin receptor, and the dissociation rate i) is equal to or less than (i.e., at most) the dissociation rate in units of 1 / s of anti-transferrin receptor antibody 128.1 for the cynomolgus monkey transferrin receptor, and ii) is equal to or greater than (i.e., at least) 5% of the dissociation rate in units of 1 / s of anti-transferrin receptor antibody 128.1 for the cynomolgus monkey transferrin receptor.

[0231] One aspect reported herein is an anti-transferrin receptor antibody that specifically binds to the human transferrin receptor (huTfR) and the cynomolgus monkey transferrin receptor (cyTfR). In certain embodiments, the anti-transferrin receptor antibody

[0232] · binds to the human transferrin receptor (huTfR) and the cynomolgus monkey transferrin receptor (cyTfR);

[0233] · has a dissociation rate in units of 1 / s for the human transferrin receptor, the dissociation rate being equal to or less than (i.e., at most) the dissociation rate in units of 1 / s of anti-transferrin receptor antibody 128.1 for the cynomolgus monkey transferrin receptor, wherein the dissociation rate is determined by surface plasmon resonance, and anti-transferrin receptor antibody 128.1 has a heavy chain variable domain of SEQ ID NO:64 and a light chain variable domain of SEQ ID NO:65;

[0234] · binds to the human transferrin receptor with a dissociation rate of from 0.1 1 / s to 0.005 1 / s (including 0.1 1 / s to 0.005 1 / s).

[0235] In one aspect, provided herein is an anti-transferrin receptor antibody that comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:71, 72, or 73; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:76; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:78.

[0236] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VHHVR sequences selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 71, 72, or 73. In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72. In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the following: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 73. In one embodiment, the antibody comprises HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 71 or 72 or 73. In another embodiment, the antibody comprises HVR-H3 and HVR-L3, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 71 or 72 or 73, and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 78. In a further embodiment, the antibody comprises HVR-H3, HVR-L3, and HVR-H2, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 71 or 72 or 73, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 78, and HVR-H2 comprises the amino acid sequence of SEQ ID NO: 68. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 68; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 72.

[0237] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:76; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:78. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:76; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:78.

[0238] In another aspect, the antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:66; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:68; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:71 or 72 or 73; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:76; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:78.

[0239] In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:72; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:76; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:78.

[0240] In any of the above embodiments, the anti-transferrin receptor antibody is humanized. In one embodiment, the anti-transferrin receptor antibody comprises the HVRs of any of the above embodiments and further comprises a human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0241] In another aspect, the anti-transferrin receptor antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:24 and having a dissociation rate substantially the same as that of an antibody comprising the heavy chain variable domain (VH) sequence of SEQ ID NO:24. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-transferrin receptor antibody comprising the sequence retains the ability to bind the transferrin receptor at the same dissociation rate. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO:24. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-transferrin receptor antibody comprises the VH sequence of SEQ ID NO:24, including post-translational modifications of the sequence. In some embodiments, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:66; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:68; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:72.

[0242] In another aspect, an anti-transferrin receptor antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:37 and has a dissociation rate substantially the same as that of an antibody comprising the light chain variable domain (VL) sequence of SEQ ID NO:37. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the reference sequence contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-transferrin receptor antibody comprising the sequence retains the ability to bind to the transferrin receptor at the same dissociation rate. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO:37. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVRs (i.e., in the FRs). Optionally, the anti-transferrin receptor antibody comprises the VL sequence of SEQ ID NO:37, including post-translational modifications of the sequence. In some embodiments, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:75; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:76; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:78.

[0243] In another embodiment, an anti-transferrin receptor antibody is provided, wherein the antibody comprises a VH from any of the embodiments provided above and a VL from any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:24 and SEQ ID NO:37, respectively, including post-translational modifications of these sequences.

[0244] In yet another aspect of the invention, the anti-transferrin receptor antibody according to any of the embodiments above is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-transferrin receptor antibody is an antibody fragment, e.g., an Fv, Fab, Fab’, scFv, diabody or F(ab’)2 fragment. In another embodiment, the antibody is a full-length antibody, e.g., a complete IgG1 antibody or other antibody class or isotype as defined herein.

[0245] In one embodiment of all aspects, the antibody is conjugated to a therapeutic compound.

[0246] In another embodiment of all aspects, the antibody is conjugated to an imaging agent or a label.

[0247] In another embodiment, the antibody is a multispecific antibody and the therapeutic compound optionally forms part of the multispecific antibody. In one such embodiment, the multispecific antibody comprises a first antigen-binding site that binds to TfR and a second antigen-binding site that binds to a brain antigen. In one such aspect, the brain antigen is selected from: beta-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), 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), glucocerebrosidase, and caspase 6. In another embodiment, the multispecific antibody binds both TfR and BACE1. In another embodiment, the multispecific antibody binds both TfR and Aβ. In another embodiment, the multispecific antibody binds both TfR and alpha-synuclein. In another embodiment, the multispecific antibody binds both TfR and CD20. In another embodiment, the multispecific antibody binds both TfR and glucocerebrosidase. In another embodiment, the therapeutic compound is a neurological disorder drug.

[0248] In one aspect of the above embodiments, the present invention provides a pharmaceutical formulation comprising any of the previous antibodies and a pharmaceutically acceptable carrier.

[0249] In one aspect of the above embodiments, the present invention provides any of the previous antibodies for use as a drug.

[0250] In one aspect of the above embodiments, the present invention provides the use of any of the previous antibodies in the preparation of a drug for treating a neurological disorder. In one embodiment, the neurological disorder is selected from neuropathy, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, and CNS inflammation.

[0251] In another aspect of the above embodiments, the present invention provides the use of any of the previous antibodies for treating a neurological disorder. In one embodiment, the neurological disorder is selected from neuropathy disorder, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, and CNS inflammation.

[0252] In another aspect of the above embodiments, the present invention provides the use of any one of the previous antibodies for the transport of one or more compounds across the BBB.

[0253] In another aspect of the above embodiments, there is provided the use of any one of the previous antibodies in the preparation of a medicament for the transport of one or more compounds across the BBB.

[0254] In one aspect of the above embodiments, there is provided a method for transporting a compound across the BBB in a subject, comprising exposing any one of the previous antibodies to the BBB such that the antibody can transport the compound conjugated thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody conjugated to the compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is a TfR-saturating dose. In another embodiment, the antibody is administered at a dose and / or dose frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0255] In another aspect of the above embodiments, there is provided a method for increasing the exposure of the CNS of a subject to a compound, comprising exposing any one of the previous antibodies to the BBB such that the antibody can transport the compound conjugated thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody conjugated to the compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is TfR-saturated. In another embodiment, the antibody is administered at a dose and / or dose frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0256] In one aspect of the above embodiments, there is provided a method for increasing the residence of a compound administered to a subject in the CNS, comprising exposing any one of the previous antibodies to the BBB, thereby increasing the residence of the compound in the CNS. In one embodiment, the antibody conjugated to the compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is TfR-saturated. In another embodiment, the antibody is administered at a dose and / or dose frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0257] In one aspect of the above embodiments, a method of treating a neurological disorder in a mammal is provided, comprising treating the mammal with any one of the antibodies described previously. In one embodiment, the neurological disorder is selected from neuropathy, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, and CNS inflammation. In one embodiment, the neurological disorder is in a human subject. In one embodiment, the antibody conjugated to a compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is TfR-saturated. In another embodiment, the antibody is administered at a dose and / or dose frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0258] In one embodiment, the antibody is modified in one or more properties selected from the effector function of the antibody Fc region, the complement activation function of the antibody, and the affinity of the antibody for TfR.

[0259] In one embodiment, the property is the effector function of the antibody Fc region.

[0260] In one embodiment, the property is the complement activation function of the antibody.

[0261] In one embodiment, the property is the affinity of the antibody for TfR.

[0262] In one embodiment, the effector function or complement activation function has been reduced or eliminated relative to a wild-type antibody of the same isotype. In one embodiment, the effector function is reduced or eliminated by a method selected from reducing the glycosylation of the antibody, modifying the antibody isotype to an isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0263] In one embodiment, the effector function is reduced or eliminated by reducing the glycosylation of the antibody. In one embodiment, the glycosylation of the antibody is reduced by a method selected from producing the antibody in an environment that does not permit wild-type glycosylation; removing carbohydrate groups already present on the antibody; and modifying the antibody such that wild-type glycosylation does not occur.

[0264] In one embodiment, the glycosylation of the antibody is reduced by producing the antibody in an environment that does not permit wild-type glycosylation, such as producing the antibody in a non-mammalian cell production system or by synthetic production. In one embodiment, the antibody is produced in a non-mammalian cell production system. In another embodiment, the antibody is produced synthetically.

[0265] In one embodiment, the glycosylation of an antibody is reduced by modifying the antibody such that wild-type glycosylation does not occur, such as by including a mutation at position 297 in the Fc region of the antibody such that the wild-type asparagine residue at that position is replaced by another amino acid that interferes with glycosylation at that position.

[0266] In one embodiment, effector function is reduced or eliminated by at least one Fc region modification. In one embodiment, effector function or complement activation function is reduced or eliminated by deleting all or a portion of the Fc region, or by engineering an antibody such that it does not include an Fc region or a non-Fc region that is capable of effector function or complement activation function. In one embodiment, at least one Fc region modification is selected from: Fc region point mutations at the following positions that impair binding to one or more Fc receptors: 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, and 439; Fc region point mutations at the following positions that impair C1q binding: 270, 322, 329, and 321; deleting some or all of the Fc region; and a point mutation at position 132 of the CH1 domain. In one embodiment, the modification is an Fc region point mutation at the following positions that impairs C1q binding: 270, 322, 329, and 321. In another embodiment, the modification is deleting some or all of the Fc region. In another embodiment, complement initiation function is reduced or eliminated by deleting all or a portion of the Fc region, or by engineering an antibody such that it does not include an Fc region that participates in the complement pathway. In one embodiment, the antibody is selected from Fab or single-chain antibody. In another embodiment, the non-Fc region of the antibody is modified to reduce or eliminate activation of the antibody for the complement pathway. In one embodiment, the modification is a point mutation in the CH1 region that impairs binding to C3. In one embodiment, the point mutation is at position 132 (see, e.g., Vidarte et al., J. Biol. Chem. 276 (2001) 38217-38223).

[0267] In one aspect of the above embodiments, the affinity of the antibody for the TfR is reduced, as measured relative to a wild-type antibody of the same isotype that does not have reduced TfR affinity. In one such aspect, the antibody has a K D or IC 50 .

[0268] In one embodiment, the antibodies reported herein are effector function silent. In one embodiment, the antibody does not have effector function. In one embodiment, the antibody is of the human IgG1 subclass and has mutations L234A, L235A, and P329G in both heavy chains (numbering according to Kabat's EU index).

[0269] In one embodiment, the antibody is

[0270] a) a full-length antibody of the human IgG1 subclass, or

[0271] b) a full-length antibody of the human IgG4 subclass, or

[0272] c) a full-length antibody of the human IgG1 subclass having mutations L234A, L235A, and P329G,

[0273] d) a full-length antibody of the human IgG4 subclass having mutations S228P, L235E, and optionally P329G,

[0274] e) a full-length antibody of the human IgG1 subclass having mutations L234A, L235A, and P329G in both heavy chains, and having mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the other opposite heavy chain, or

[0275] f) a full-length antibody of the human IgG4 subclass having mutations S228P and optionally P329G in both heavy chains, and having mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the other opposite heavy chain.

[0276] In one aspect of the above embodiments, the present invention provides a pharmaceutical formulation comprising any of the previous antibodies and a pharmaceutically acceptable carrier.

[0277] In one aspect of the above embodiments, the present invention provides any of the previous antibodies for use as a medicament.

[0278] In another aspect of the above embodiments, the present invention provides the use of any of the previous antibodies in the preparation of a medicament for the treatment of a neurological disorder. In one embodiment, the neurological disorder is selected from neuropathy, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, and CNS inflammation.

[0279] In another aspect of the above embodiments, the present invention provides the use of any prior antibody in the treatment of neurological disorders. In one embodiment, the neurological disorder is selected from neuropathy, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, and CNS inflammation.

[0280] In another aspect of the above embodiments, the present invention provides the use of any prior antibody for the transport of one or more compounds across the BBB.

[0281] In another aspect of the above embodiments, there is provided the use of any prior antibody in the preparation of a medicament for the transport of one or more compounds across the BBB.

[0282] In one aspect of the above embodiments, there is provided a method for transporting a compound across the BBB in a subject, comprising exposing any prior antibody to the BBB such that the antibody can transport the compound conjugated thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody conjugated to the compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is a TfR-saturating dose. In another embodiment, the antibody is administered at a dose and / or dose frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0283] In another aspect of the above embodiments, there is provided a method for increasing the exposure of a subject's CNS to a compound, comprising exposing any prior antibody to the BBB such that the antibody can transport the compound conjugated thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody conjugated to the compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is TfR-saturating. In another embodiment, the antibody is administered at a dose and / or dose frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0284] In one aspect of the above embodiments, there is provided a method for increasing the residence of a compound administered to a subject in the CNS, comprising exposing any prior antibody to the BBB to increase the residence of the compound in the CNS. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody conjugated to the compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is TfR-saturating. In another embodiment, the antibody is administered at a dose and / or dose frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0285] In one aspect of the above embodiments, a method of treating a neurological disorder in a mammal is provided, including treating the mammal with any one of the antibodies described previously. In one embodiment, the neurological disorder is selected from neuropathy, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, and CNS inflammation. In another such aspect, the neurological disorder is in a human subject. In one embodiment, the antibody of the conjugate compound is administered at a therapeutic dose. In one embodiment, the therapeutic dose is TfR-saturated. In another embodiment, the antibody is administered at a dose and / or dosing frequency calibrated to minimize the acute clinical symptoms of antibody administration.

[0286] In another embodiment, a method of reducing the clearance of a compound administered to a subject is provided, wherein the compound is conjugated to an antibody that binds to TfR with low affinity, thereby reducing the clearance of the compound.

[0287] In another embodiment, a method of optimizing the pharmacokinetics and / or pharmacodynamics of a compound that is effective in the CNS of a subject is provided, wherein the compound is conjugated to an antibody that binds to TfR with low affinity, and the antibody is selected such that its affinity for TfR after conjugation to the compound results in a transport amount of the compound-conjugated antibody across the BBB that can optimize the pharmacokinetics and / or pharmacodynamics of the compound in the CNS.

[0288] In yet another aspect, the anti-transferrin receptor antibodies according to any one of the above aspects and embodiments can incorporate any single property or combination thereof described in Sections 1-5 below.

[0289] 1. Antibody affinity

[0290] In one embodiment, the Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed with the Fab form of the target antibody and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). To establish the conditions for testing, plates are coated with 5 μg / mL anti-Fab capture antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) The porous plate (Thermo Scientific) was incubated overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 °C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM 125 I]-antigen was mixed with serially diluted target Fab (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12, see Presta, L.G. et al., Cancer Res. 57 (1997) 4593-4599). Subsequently, the target Fab was incubated overnight; however, the incubation could be extended for a longer period (e.g., about 65 hours) to ensure equilibrium was reached. Thereafter, the mixture was transferred to the capture plate and incubated at room temperature (e.g., for one hour). Subsequently, the solution was removed and the plate was washed eight times with 0.1% polysorbate 20 in PBS . After the plate was dried, 150 μL / well of scintillant (MICROSCINT-20 TM ; Packard) was added, and then the plate was counted on a TOPCOUNT TM γ counter (Packard) for ten minutes. The concentration of each Fab that produced less than or equal to 20% of the maximum binding was selected for use in the competitive binding assay.

[0291] According to another embodiment, Kd was measured using surface plasmon resonance testing. For example, using or (BIAcore, Inc., Piscataway, NJ), the test was performed at 25 °C with immobilized antigen CM5 chip at ~10 response units (RU). In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg / mL (~0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of the coupled protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25 °C, serially diluted Fab (0.78 nM to 500 nM) was injected in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant at a flow rate of approximately 25 μL / min. By simultaneously fitting the binding and dissociation sensorgrams using a simple one-to-one Langmuir binding model ( evaluation software version 3.2), the association rate (kon ) and dissociation rate (k off ). According to the k off / k on ratio, the equilibrium dissociation constant (KD) is calculated (see, for example, Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). If the binding rate measured by the above surface plasmon resonance test exceeds 10 6 M -1 s -1 , then the binding rate can be determined by using fluorescence quenching technology, which measures the increase or decrease in the fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, at 25°C in the presence of increasing concentrations of antigen (excitation = 295 nm; emission = 340 nm, 16 nm bandpass), for example, it can be measured in a spectrometer such as a stopped-flow spectrometer (Aviv Instruments) or an 8000-series SLM-AMINCO TM spectrometer (ThermoSpectronic).

[0292] 2. Antibody fragments

[0293] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO 93 / 16185; US 5,571,894 and US 5,587,458. For a discussion of Fab and F(ab’)2 fragments that contain salvage receptor binding epitope residues and have an increased in vivo half-life, see US 5,869,046.

[0294] Bispecific antibodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific. See, e.g., EP 0 404 097; WO 1993 / 01161; Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Trispecific and tetravalent antibodies are also described in Hudson, P.J. et al., Nat. Med. 9 (20039 129-134).

[0295] Single-domain antibodies are antibody fragments that contain all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516).

[0296] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0297] 3. Chimeric and Humanized Antibodies

[0298] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, e.g., in US 4,816,567; and Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, a chimeric antibody contains a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0299] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody contains one or more variable domains in which the HVRs (e.g., CDRs) (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody optionally may also contain at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody may be replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0300] Reviews of humanized antibodies and methods of making them are found, for example, in Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and are further described, for example, in Riechmann, L. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321 and US 7,087,409; Kashmiri, S.V. et al., Methods 36 (2005) 25-34 (describing specific determining region (SDR) grafting); Padlan, E.A., Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing"); Dall'Acqua, W.F. et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing "directed selection" method of FR shuffling).

[0301] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims, M.J. et al., J. Immunol. 151 (1993) 2296-2308); framework regions from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L.G. et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening of FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, M.J. et al., J. Biol. Chem. 271 (1996) 22611-22618).

[0302] 4. Multispecific antibodies

[0303] In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different epitopes. In certain embodiments, one of the binding specificities is directed against the transferrin receptor, and the other is directed against any other antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express the transferrin receptor. Bispecific antibodies can be prepared in the form of full-length antibodies or antibody fragments.

[0304] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "stuffer" engineering (see, e.g., US 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553); using the "diabody" technique for preparing bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fc (scFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al. J. Immunol. 147 (1991) 60-69).

[0305] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0306] The antibodies or fragments herein also include "dual action Fab" or "DAF" which contain antigen-binding sites that bind the transferrin receptor as well as another different antigen (see, e.g., US 2008 / 0069820).

[0307] The antibodies or fragments herein also include multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 and WO 2010 / 145793.

[0308] In one embodiment of all aspects reported herein, the anti-transferrin receptor antibody is a bispecific antibody.

[0309] One aspect reported herein is a bivalent, bispecific antibody that comprises

[0310] a) a first light chain and a first heavy chain of an antibody that specifically binds a first antigen, and

[0311] b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are interchanged,

[0312] wherein the first antigen or the second antigen is the human transferrin receptor.

[0313] The antibody of a) does not contain the modification described in b), and the heavy chain and the light chain of a) are separate chains.

[0314] In the antibody of b),

[0315] In the light chain

[0316] the variable light chain domain VL is replaced by the variable heavy chain domain VH of the antibody,

[0317] and

[0318] In the heavy chain

[0319] the variable heavy chain domain VH is replaced by the variable light chain domain VL of the antibody.

[0320] In one embodiment

[0321] i) In the constant domain CL of the first light chain of a), the amino acid at position 124 (numbered according to Kabat) is replaced by a positively charged amino acid, and in the constant domain CH1 of the first heavy chain of a), the amino acid at position 147 or the amino acid at position 213 (numbered according to Kabat EU index) is replaced by a negatively charged amino acid,

[0322] or

[0323] ii) In the constant domain CL of the second light chain in b), the amino acid at position 124 (numbered according to Kabat) is replaced by a positively charged amino acid, and in the constant domain CH1 of the second heavy chain in b), the amino acid at position 147 or the amino acid at position 213 (numbered according to Kabat EU index) is replaced by a negatively charged amino acid.

[0324] In a preferred embodiment

[0325] i) In the constant domain CL of the first light chain in a), the amino acid at position 124 (numbered according to Kabat) is independently replaced by lysine (K), arginine (R), or histidine (H) (independently replaced by lysine (K) or arginine (R) in a preferred embodiment), and in the constant domain CH1 of the first heavy chain in a), the amino acid at position 147 or the amino acid at position 213 (numbered according to Kabat EU index) is independently replaced by glutamic acid (E) or aspartic acid (D).

[0326] or

[0327] ii) In the constant domain CL of the second light chain in b), the amino acid at position 124 (numbered according to Kabat) is independently replaced by lysine (K), arginine (R), or histidine (H) (independently replaced by lysine (K) or arginine (R) in a preferred embodiment), and wherein in the constant domain CH1 of the second heavy chain in b), the amino acid at position 147 or the amino acid at position 213 (numbered according to Kabat EU index) is independently replaced by glutamic acid (E) or aspartic acid (D).

[0328] In one embodiment, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are replaced by K (numbered according to Kabat EU index).

[0329] In one embodiment, in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are replaced by E (numbered according to Kabat EU index).

[0330] In a preferred embodiment, in the constant domain CL of the first light chain, the amino acids at positions 124 and 123 are replaced by K, and in the constant domain CH1 of the first heavy chain, the amino acids at positions 147 and 213 are replaced by E (numbered according to Kabat EU index).

[0331] In one embodiment, the amino acids at positions 124 and 123 in the constant domain CL of the second heavy chain are replaced with K, and the amino acids at positions 147 and 213 in the constant domain CH1 of the second light chain are replaced with E, and the amino acid at position 38 in the variable domain VL of the first light chain is replaced with K, the amino acid at position 39 in the variable domain VH of the first heavy chain is replaced with E, the amino acid at position 38 in the variable domain VL of the second heavy chain is replaced with K, and the amino acid at position 39 in the variable domain VH of the second light chain is replaced with E (numbering according to the Kabat EU index).

[0332] One aspect reported herein is a bivalent, bispecific antibody that comprises

[0333] a) a first light chain and a first heavy chain of an antibody that specifically binds a first antigen, and

[0334] b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are substituted with each other, and wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are substituted with each other,

[0335] wherein the first antigen or the second antigen is the human transferrin receptor.

[0336] The antibody of a) does not contain the modifications described in b), and the heavy and light chains of a) are separate chains.

[0337] In the antibody of b),

[0338] Within the light chain

[0339] the variable light chain domain VL is replaced with the variable heavy chain domain VH of the antibody, and the constant light chain domain CL is replaced with the constant heavy chain domain CH1 of the antibody;

[0340] and

[0341] Within the heavy chain

[0342] the variable heavy chain domain VH is replaced with the variable light chain domain VL of the antibody, and the constant heavy chain domain CH1 is replaced with the constant light chain domain CL of the antibody.

[0343] One aspect reported herein is a bivalent, bispecific antibody that comprises

[0344] a) a first light chain and a first heavy chain of an antibody that specifically binds a first antigen, and

[0345] b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are substituted with each other,

[0346] Wherein the first antigen or the second antigen is human transferrin receptor.

[0347] The antibody of a) does not contain the modifications reported in b), and the heavy and light chains of a) are separate chains.

[0348] In the antibody of b),

[0349] Within the light chain

[0350] The constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody;

[0351] And

[0352] Within the heavy chain

[0353] The constant heavy chain domain CH1 is replaced by the constant light chain domain CL of the antibody.

[0354] One aspect reported herein is a multispecific antibody that comprises

[0355] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0356] b) one, two, three, or four single-chain Fab fragments that specifically bind to one to four other antigens (i.e., the second and / or third and / or fourth and / or fifth antigens, preferably specifically bind to one other antigen, i.e., the second antigen),

[0357] Wherein the single-chain Fab fragment(s) described in b) is / are fused via a peptide linker to the full-length antibody of a) at the C- or N-terminus of the heavy or light chain of the full-length antibody,

[0358] Wherein the first antigen or one of the other antigens is human transferrin receptor.

[0359] In one embodiment, one or two identical single-chain Fab fragments that bind to the second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of the heavy or light chain of the full-length antibody.

[0360] In one embodiment, one or two identical single-chain Fab fragments that bind to the second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of the heavy chain of the full-length antibody.

[0361] In one embodiment, one or two identical single-chain Fab fragments that bind to the second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of the light chain of the full-length antibody.

[0362] In one embodiment, two identical single-chain Fab fragments that bind a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each heavy or light chain of the full-length antibody.

[0363] In one embodiment, two identical single-chain Fab fragments that bind a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each heavy chain of the full-length antibody.

[0364] In one embodiment, two identical single-chain Fab fragments that bind a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each light chain of the full-length antibody.

[0365] One aspect reported herein is a trivalent, bispecific antibody that comprises

[0366] a) a full-length antibody that specifically binds a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0367] b) a first polypeptide that consists of:

[0368] ba) an antibody heavy chain variable domain (VH),

[0369] or

[0370] bb) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1),

[0371] wherein the first polypeptide is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker at the N-terminus of its VH domain,

[0372] c) a second polypeptide that consists of

[0373] ca) an antibody light chain variable domain (VL),

[0374] or

[0375] cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL),

[0376] wherein the second polypeptide is fused to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker at the N-terminus of its VL domain,

[0377] and

[0378] wherein the antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding site that specifically binds a second antigen,

[0379] and

[0380] Wherein the first antigen or the second antigen is human transferrin receptor.

[0381] In one embodiment, the heavy chain variable domain (VH) of the polypeptide of b) and the light chain variable domain (VL) of the polypeptide of c) are linked and stabilized via an interchain disulfide bridge formed by introducing a disulfide bond between the following positions:

[0382] i) position 44 of the heavy chain variable domain and position 100 of the light chain variable domain, or

[0383] ii) position 105 of the heavy chain variable domain and position 43 of the light chain variable domain, or

[0384] iii) position 101 of the heavy chain variable domain and position 100 of the light chain variable domain (always according to the Kabat EU index number).

[0385] Techniques for introducing non-native disulfide bridges for stabilization are described, for example, in WO 94 / 029350, Rajagopal, V. et al., Prot. Eng. (1997) 1453-59; Kobayashi, H. et al., Nuclear Medicine & Biology, Vol. 25, (1998) 387-393; or Schmidt, M. et al., Oncogene (1999) 18 1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides of b) and c) is located between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides of b) and c) is located between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (always according to Kabat numbering). In one embodiment, a trivalent, bispecific antibody without said optional disulfide stabilization between the variable domains VH and VL of a single-chain Fab fragment is preferred.

[0386] One aspect reported herein is a trispecific or tetravalent antibody that comprises

[0387] a) a first light chain and a first heavy chain of a full-length antibody that specifically binds a first antigen, and

[0388] b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds a second antigen, wherein the variable domains VL and VH are substituted for each other, and / or wherein the constant domains CL and CH1 are substituted for each other, and

[0389] c) one to four antigen-binding peptides that specifically bind one or two other antigens (i.e., bind a third and / or fourth antigen) are fused via a peptide linker to the C-terminus or N-terminus of the light chain or heavy chain of a) and / or b),

[0390] wherein one of the first antigen, the second antigen, or one of the other antigens is the human transferrin receptor.

[0391] The antibody of a) does not contain the modification reported in b), and the heavy chain and light chain of a) are separate chains.

[0392] In one embodiment, the trispecific or tetra-specific antibody comprises, under c), one or two antigen-binding peptides that specifically bind one or two other antigens.

[0393] In one embodiment, the antigen-binding peptide is selected from scFv fragments and scFab fragments.

[0394] In one embodiment, the antigen-binding peptide is an scFv fragment.

[0395] In one embodiment, the antigen-binding peptide is an scFab fragment.

[0396] In one embodiment, the antigen-binding peptide is fused to the C-terminus of the heavy chain of a) and / or b).

[0397] In one embodiment, the trispecific or tetra-specific antibody comprises, under c), one or two antigen-binding peptides that specifically bind one other antigen.

[0398] In one embodiment, the trispecific or tetra-specific antibody comprises two identical antigen-binding peptides that specifically bind a third antigen under c). In a preferred embodiment, such two identical antigen-binding peptides are fused via the same peptide linker to the C-terminus of the heavy chains of a) and b). In a preferred embodiment, the two identical antigen-binding peptides are scFv fragments or scFab fragments.

[0399] In one embodiment, the trispecific or tetra-specific antibody comprises two antigen-binding peptides that specifically bind a third and a fourth antigen under c). In one embodiment, the two antigen-binding peptides are fused via the same peptide connector to the C-terminus of the heavy chains of a) and b). In a preferred embodiment, the two antigen-binding peptides are scFv fragments or scFab fragments.

[0400] One aspect reported herein is a bispecific, tetravalent antibody that comprises

[0401] a) The two light chains and two heavy chains of an antibody that specifically binds to a first antigen (and comprises two Fab fragments),

[0402] b) Two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein said additional Fab fragments are each fused via a peptide linker to the C- or N-terminus of the heavy chain of a),

[0403] and

[0404] wherein in the Fab fragments, the following modifications have been made

[0405] i) In two of the Fab fragments of a), or in two of the Fab fragments of b), the variable domains VL and VH are substituted for each other, and / or the constant domains CL and CH1 are substituted for each other,

[0406] or

[0407] ii) In two of the Fab fragments of a), the variable domains VL and VH are substituted for each other, and the constant domains CL and CH1 are substituted for each other,

[0408] and

[0409] in two of the Fab fragments of b), the variable domains VL and VH are substituted for each other, or the constant domains CL and CH1 are substituted for each other,

[0410] or

[0411] iii) In two of the Fab fragments of a), the variable domains VL and VH are substituted for each other, or the constant domains CL and CH1 are substituted for each other,

[0412] and

[0413] in two of the Fab fragments of b), the variable domains VL and VH are substituted for each other, and the constant domains CL and CH1 are substituted for each other,

[0414] or

[0415] iv) In two of the Fab fragments of a), the variable domains VL and VH are substituted for each other, and in two of the Fab fragments of b), the constant domains CL and CH1 are substituted for each other,

[0416] or

[0417] v) In two of the Fab fragments of a), the constant domains CL and CH1 are substituted for each other, and in two of the Fab fragments of b), the variable domains VL and VH are substituted for each other,

[0418] wherein the first antigen or the second antigen is the human transferrin receptor.

[0419] In one embodiment, said additional Fab fragment is fused via a peptide linker to the C-terminus of the heavy chain of a) or the N-terminus of the heavy chain of a).

[0420] In one embodiment, said additional Fab fragment is fused via a peptide linker to the C-terminus of the heavy chain of a).

[0421] In one embodiment, said additional Fab fragment is fused via a peptide linker to the N-terminus of the heavy chain of a).

[0422] In one embodiment, the following modifications are made in the Fab fragment:

[0423] i) In the two Fab fragments of a) or in the two Fab fragments of b), the variable domains VL and VH are substituted for each other,

[0424] and / or

[0425] the constant domains CL and CH1 are substituted for each other.

[0426] In one embodiment, the following modifications are made in the Fab fragment:

[0427] i) In the two Fab fragments of a), the variable domains VL and VH are substituted for each other,

[0428] and / or

[0429] the constant domains CL and CH1 are substituted for each other.

[0430] In one embodiment, the following modifications are made in the Fab fragment:

[0431] i) In the two Fab fragments of a), the constant domains CL and CH1 are substituted for each other.

[0432] In one embodiment, the following modifications are made in the Fab fragment:

[0433] i) In the two Fab fragments of b), the variable domains VL and VH are substituted for each other,

[0434] and / or

[0435] the constant domains CL and CH1 are substituted for each other.

[0436] In one embodiment, the following modifications are made in the Fab fragment:

[0437] i) In the two Fab fragments of b), the constant domains CL and CH1 are substituted for each other.

[0438] One aspect reported herein is a bispecific, trivalent antibody that comprises

[0439] a) The (modified) heavy chain of a first antibody that specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein the N-terminus of the second VH-CH1 domain pair of said first antibody is fused to the C-terminus of said heavy chain via a peptide linker,

[0440] b) Two light chains of said first antibody of a),

[0441] c) The (modified) heavy chain of a second antibody that specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein the N-terminus of the second VH-CL domain pair of said second antibody is fused to the C-terminus of said heavy chain via a peptide linker,

[0442] d) Two (modified) light chains of said second antibody of c), each comprising a CL-CH1 domain pair,

[0443] wherein the first antigen or the second antigen is the human transferrin receptor.

[0444] One aspect reported herein is a bispecific antibody that comprises

[0445] a) The heavy and light chains of a first full-length antibody that specifically binds to a first antigen, and

[0446] b) The heavy and light chains of a second full-length antibody that specifically binds to a second antigen, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker,

[0447] wherein the first antigen or the second antigen is the human transferrin receptor.

[0448] The antibody of a) does not contain the modification reported in b), and the heavy and light chains are separate chains.

[0449] One aspect reported herein is a bispecific antibody that comprises

[0450] a) A full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0451] b) An Fv fragment that specifically binds to a second antigen and comprises a VH 2 domain and a VL 2 domain, wherein the two domains are linked to each other via a disulfide bridge,

[0452] wherein only the VH 2 domain or the VL 2 domain is fused to the heavy or light chain of the full-length antibody that specifically binds to the first antigen via a peptide linker,

[0453] wherein the first antigen or the second antigen is the human transferrin receptor.

[0454] In this bispecific, the heavy and light chains of a) are separate chains.

[0455] In one embodiment, VH 2 domain or VL 2 domain of the other is not fused via a peptide linker to the heavy or light chain of a full-length antibody that specifically binds a first antigen.

[0456] In all aspects reported herein, the first light chain comprises a VL domain and a CL domain, while the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

[0457] One aspect reported herein is a bispecific trivalent antibody that comprises

[0458] a) two Fab fragments that specifically bind a first antigen,

[0459] b) one CrossFab fragment that specifically binds a second antigen, wherein the CH1 and CL domains are exchanged with each other,

[0460] c) an Fc region that comprises a first Fc-region heavy chain and a second Fc-region heavy chain,

[0461] wherein the C-terminus of the CH1 domain of the two Fab fragments is linked to the N-terminus of the heavy chain Fc-region polypeptide, and

[0462] wherein the C-terminus of the CL domain of the CrossFab fragment is linked to the N-terminus of the VH domain of one of the Fab fragments, and

[0463] wherein the first antigen or the second antigen is the human transferrin receptor.

[0464] One aspect reported herein is a bispecific trivalent antibody that comprises

[0465] a) two Fab fragments that specifically bind a first antigen,

[0466] b) one CrossFab fragment that specifically binds a second antigen, wherein the CH1 and CL domains are exchanged with each other,

[0467] c) an Fc region that comprises a first Fc-region heavy chain and a second Fc-region heavy chain,

[0468] wherein the C-terminus of the CH1 domain of the first Fab fragment is linked to the N-terminus of one of the heavy chain Fc-region polypeptides and the C-terminus of the CL-domain of the CrossFab fragment is linked to the N-terminus of the other heavy chain Fc-region polypeptide, and

[0469] wherein the C-terminus of the CH1 domain of the second Fab fragment is linked to the N-terminus of the VH domain of the first Fab fragment or to the N-terminus of the VH domain of the CrossFab fragment, and

[0470] wherein the first antigen or the second antigen is human transferrin receptor.

[0471] One aspect reported herein is a bispecific antibody, which comprises

[0472] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0473] b) a Fab fragment that specifically binds to a second antigen and comprises a VH 2 domain and a VL 2 domain, which comprises a heavy chain fragment and a light chain fragment, wherein

[0474] within the light chain fragment

[0475] the variable light chain domain VL 2 is replaced by the variable heavy chain domain VH 2 of the antibody,

[0476] and

[0477] within the heavy chain fragment

[0478] the variable heavy chain domain VH 2 is replaced by the variable light chain domain VL 2 of the antibody,

[0479] wherein the heavy chain Fab fragment is inserted between the CH1 domain of one heavy chain of the full-length antibody and the Fc-region of the corresponding full-length antibody, and the N-terminus of the light chain Fab fragment is conjugated to the C-terminus of the light chain of the full-length antibody, wherein the light chain of the full-length antibody pairs with the heavy chain of the full-length antibody into which the heavy chain Fab fragment is inserted, and

[0480] wherein the first antigen or the second antigen is human transferrin receptor.

[0481] One aspect reported herein is a bispecific antibody, which comprises

[0482] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0483] b) a Fab fragment that specifically binds to a second antigen and comprises a VH 2 domain and a VL 2 domain, which comprises a heavy chain fragment and a light chain fragment, wherein

[0484] within the light chain fragment

[0485] Variable light chain domain VL 2 is replaced by the variable heavy chain domain VH of the antibody 2 and

[0486] and

[0487] within the heavy chain fragment

[0488] Variable heavy chain domain VH 2 is replaced by the variable light chain domain VL of the antibody 2 and

[0489] wherein the C-terminus of the heavy chain fragment of the Fab fragment is conjugated to the N-terminus of one of the heavy chains of the full-length antibody, and the C-terminus of the light chain fragment of the Fab fragment is conjugated to the N-terminus of the light chain of the full-length antibody, wherein the light chain of the full-length antibody pairs with the full-length antibody heavy chain of the heavy chain fragment conjugated with the Fab fragment, and

[0490] wherein the first antigen or the second antigen is human transferrin receptor.

[0491] In one embodiment in all respects, the antibodies reported herein are multispecific antibodies that require heterodimerization of at least two heavy chain polypeptides, and wherein the antibody specifically binds to human transferrin receptor and a second non-human transferrin receptor antigen.

[0492] To support heterodimerization, several methods for CH3-modification have been described, see for example WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954, WO2013 / 096291, which are incorporated herein by reference. Generally, in methods known in the art, both the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner such that a heavy chain containing one engineered CH3 domain no longer homodimerizes with another heavy chain of the same structure (e.g., the first CH3-engineered heavy chain no longer homodimerizes with another CH3-engineered first heavy chain; and the second CH3-engineered heavy chain no longer homodimerizes with another CH3-engineered second heavy chain). Thereby, forcing a heavy chain containing one engineered CH3 domain to heterodimerize with another heavy chain containing a CH3 domain engineered in a complementary manner. For this embodiment of the invention, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitution to force the first and second heavy chains to heterodimerize, while the first and second heavy chains no longer homodimerize (e.g., for steric reasons).

[0493] The different methods described above and incorporated, known in the art for supporting heavy chain heterodimerization, can be considered as different alternative schemes for use in a multispecific antibody according to the invention, which comprises a "non-exchangeable Fab region" derived from a first antibody that specifically binds a first antigen, and a "crossed Fab region" derived from a second antibody that specifically binds a second antigen, and combines the specific amino acid substitutions described above for the present invention.

[0494] The CH3 domains of the multispecific antibodies reported herein can be modified by the "knob-into-hole" technology, which is described in detail in several examples such as WO 96 / 027011, Ridgway, J.B. et al., Protein Eng. 9 (1996) 617-621; and Merchant, A.M. et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of two CH3 domains are altered to enhance the heterodimerization of the two heavy chains containing these two CH3 domains. One of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole". The introduction of disulfide bridges can further stabilize the heterodimer (Merchant, A.M. et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increase the yield.

[0495] In a preferred embodiment, the multispecific antibodies reported herein contain the T366W mutation in the CH3 domain of the "knob chain" and the T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index). Other inter-chain disulfide bridges between the CH3 domains can also be used (Merchant, A.M. et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing the Y349C mutation into the CH3 domain of the "knob chain" and the E356C or S354C mutation into the CH3 domain of the "hole chain". Thus, in another preferred embodiment, the multispecific antibodies reported herein contain the Y349C and T366W mutations in one of the two CH3 domains and the E356C, T366S, L368A, and Y407V mutations in the other of the two CH3 domains, or the multispecific antibodies contained herein contain the Y349C and T366W in one of the two CH3 domains and the S354C, T366S, L368A, and Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain form an inter-chain disulfide bridge) (numbering according to the Kabat EU index).

[0496] Other knob-into-hole technologies as described in EP1870459A1 can be used in place of or additionally. In one embodiment, the multispecific antibodies reported herein comprise the R409D and K370E mutations in the CH3 domain of the "knob chain" and the D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

[0497] In one embodiment, the multispecific antibodies reported herein comprise the T366W mutation in the CH3 domain of the "knob chain" and the T366S, L368A and Y407V mutations in the CH3 domain of the "hole chain", and additionally the R409D and K370E mutations in the CH3 domain of the "knob chain" and the D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

[0498] In one embodiment, the multispecific antibodies reported herein comprise the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, or the multispecific antibodies reported herein comprise the Y349C and T366W mutations in one of the two CH3 domains and the S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, and additionally the R409D and K370E mutations in the CH3 domain of the "knob chain" and the D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

[0499] In addition to the "knob-into-hole technology", other techniques known in the art for modifying the CH3 domain of the heavy chain of a multispecific antibody to enhance heterodimerization. These techniques, especially those described in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 and WO2013 / 096291, can be considered herein as alternatives to the "knob-into-hole technology" for the multispecific antibodies reported herein.

[0500] In one embodiment of the multispecific antibodies reported herein, the method described in EP1870459 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody. This method is based on introducing charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3-domain-interface between both the first and second heavy chains.

[0501] Accordingly, this embodiment relates to multispecific antibodies reported herein, wherein in the tertiary structure of the antibody, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface located between the respective antibody CH3 domains, wherein the amino acid sequences of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain each comprise a set of amino acids located within said interface in the tertiary structure of the antibody, wherein the first amino acid of the set of amino acids located at the interface in the CH3 domain from one heavy chain is replaced with a positively charged amino acid, and the second amino acid of the set of amino acids located at the interface in the CH3 domain from the other heavy chain is replaced with a negatively charged amino acid. The multispecific antibody according to this embodiment is also referred to herein as a "CH3(+ / -) engineered multispecific antibody" (wherein the abbreviation "+ / -" indicates the oppositely charged amino acids introduced in the respective CH3 domains).

[0502] In one embodiment of the CH3(+ / -) engineered multispecific antibody reported herein, the positively charged amino acid is selected from K, R, and H, and the negatively charged amino acid is selected from E or D.

[0503] In one embodiment of the CH3(+ / -) engineered multispecific antibody reported herein, the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D.

[0504] In one embodiment of the CH3(+ / -) engineered multispecific antibody reported herein, the positively charged amino acid is K, and the negatively charged amino acid is E.

[0505] In one embodiment of the CH3(+ / -) engineered multispecific antibody reported herein, in the CH3 domain of one heavy chain, the amino acid R at position 409 is replaced with D and the amino acid K at position is replaced with E, and in the CH3 domain of the other heavy chain, the amino acid D at position 399 is replaced with K and the amino acid E at position 357 is replaced with K (numbering according to the Kabat EU index).

[0506] In one embodiment of the multispecific antibodies reported herein, the method described in WO 2013 / 157953 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody. In one embodiment of the multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is replaced by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is replaced by D (numbering according to Kabat EU index). In another embodiment of the multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is replaced by K, the amino acid L at position 351 is replaced by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is replaced by D (numbering according to Kabat EU index).

[0507] In another embodiment of the multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is replaced by K, and the amino acid L at position 351 is replaced by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is replaced by D (numbering according to Kabat EU index). Additionally, at least one of the following replacements is included in the CH3 domain of the other heavy chain: the amino acid Y at position 349 is replaced by E, the amino acid Y at position 349 is replaced by D, and the amino acid L at position 368 is replaced by E (numbering according to Kabat EU index). In one embodiment, the amino acid L at position 368 is replaced by E (numbering according to Kabat EU index).

[0508] In one embodiment of the multispecific antibodies reported herein, the method described in WO 2012 / 058768 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody. In one embodiment of the multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid L at position 351 is replaced by Y and the amino acid Y at position 407 is replaced by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is replaced by A and the amino acid K at position 409 is replaced by F (numbering according to Kabat EU index). In another embodiment, in addition to the above replacements, at least one of the amino acids at positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N), and 392 (originally K) in the CH3 domain of the other heavy chain is replaced (numbering according to Kabat EU index). Preferred replacements are:

[0509] - the amino acid T at position 411 is replaced by an amino acid selected from N, R, Q, K, D, E, and W (numbering according to Kabat EU index),

[0510] - The amino acid D at position 399 is replaced with an amino acid selected from R, W, Y, and K (numbering according to the Kabat EU index),

[0511] - The amino acid S at position 400 is replaced with an amino acid selected from E, D, R, and K (numbering according to the Kabat EU index),

[0512] - The amino acid F at position 405 is replaced with an amino acid selected from I, M, T, S, V, and W (numbering according to the Kabat EU index),

[0513] - The amino acid N at position 390 is replaced with an amino acid selected from R, K, and D (numbering according to the Kabat EU index); and

[0514] - The amino acid K at position 392 is replaced with an amino acid selected from V, M, R, L, F, and E (numbering according to the Kabat EU index).

[0515] In another embodiment of the multispecific antibody reported herein (engineered according to WO 2012 / 058768), in the CH3 domain of one heavy chain, the amino acid L at position 351 is replaced with Y and the amino acid Y at position 407 is replaced with A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is replaced with V and the amino acid K at position 409 is replaced with F (numbering according to the Kabat EU index). In another embodiment of the multispecific antibody reported herein, in the CH3 domain of one heavy chain, the amino acid Y at position 407 is replaced with A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is replaced with A and the amino acid K at position 409 is replaced with F (numbering according to the Kabat EU index). In the last-mentioned embodiment above, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is replaced with E, the amino acid T at position 411 is replaced with E, the amino acid D at position 399 is replaced with R, and the amino acid S at position 400 is replaced with R (numbering according to the Kabat EU index).

[0516] In one embodiment of the multispecific antibody reported herein, the method described in WO 2011 / 143545 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody. In one embodiment of the multispecific antibody reported herein, amino acid modifications are introduced at positions 368 and / or 409 in the CH3 domains of the two heavy chains (numbering according to the Kabat EU index).

[0517] In one embodiment of the multispecific antibodies reported herein, the method described in WO 2011 / 090762 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody. WO 2011 / 090762 relates to amino acid modifications according to the "knobs-into-holes" technology. In one embodiment of the CH3(KiH)-engineered multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is replaced by W, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is replaced by A (numbering according to the Kabat EU index). In another embodiment of the CH3(KiH)-engineered multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid T at position 366 is replaced by Y, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is replaced by T (numbering according to the Kabat EU index).

[0518] In one embodiment of the multispecific antibodies reported herein, which is of the IgG2 isotype, the method described in WO 2011 / 090762 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody.

[0519] In one embodiment of the multispecific antibodies reported herein, the method described in WO 2009 / 089004 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody. In one embodiment of the multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid K or N at position 392 is replaced with a negatively charged amino acid (in a preferred embodiment, replaced with E or D, and in a preferred embodiment, replaced with D), and in the CH3 domain of the other heavy chain, the amino acid D at position 399, the amino acid E or D at position 356, or the amino acid E at position 357 is replaced with a positively charged amino acid (in a preferred embodiment, replaced with K or R, and in a preferred embodiment, replaced with K, and in a preferred embodiment, the amino acid at position 399 or 356 is replaced with K) (numbering according to the KabatEU index). In a further embodiment, in addition to the above replacements, in the CH3 domain of one heavy chain, the amino acid K or R at position 409 is replaced with a negatively charged amino acid (in a preferred embodiment, replaced with E or D, and in a preferred embodiment, replaced with D) (numbering according to the Kabat EU index). In a still further embodiment, in addition to or in place of the above replacements, in the CH3 domain of one heavy chain, the amino acid K at position 439 and / or the amino acid K at position 370 are independently replaced with negatively charged amino acids (in a preferred embodiment, replaced with E or D, and in a preferred embodiment, replaced with D) (numbering according to the Kabat EU index).

[0520] In one embodiment of the multispecific antibodies reported herein, the method described in WO 2007 / 147901 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody. In one embodiment of the multispecific antibodies reported herein, in the CH3 domain of one heavy chain, the amino acid K at position 253 is replaced with E, the amino acid D at position 282 is replaced with K, and the amino acid K at position 322 is replaced with D, and in the CH3 domain of the other heavy chain, the amino acid D at position 239 is replaced with K, the amino acid E at position 240 is replaced with K, and the amino acid K at position 292 is replaced with D (numbering according to the Kabat EU index).

[0521] In one embodiment of the multispecific antibodies reported herein, the method described in WO 2007 / 110205 is used to support the heterodimerization of the first and second heavy chains of the multispecific antibody.

[0522] In one embodiment of all aspects and embodiments reported herein, the multispecific antibody is a bispecific antibody or a trispecific antibody. In a preferred embodiment of the invention, the multispecific antibody is a bispecific antibody.

[0523] In one embodiment of all aspects reported herein, the antibody is a bivalent or trivalent antibody. In one embodiment, the antibody is a bivalent antibody.

[0524] In one embodiment of all aspects reported herein, the multispecific antibody has a constant domain structure of an IgG-type antibody. In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG1 subclass, or of the human IgG1 subclass with mutations L234A and L235A. In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG2 subclass. In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG3 subclass. In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG4 subclass, or of the human IgG4 subclass with an additional mutation S228P. In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG1 subclass or the human IgG4 subclass. In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG1 subclass, having mutations L234A and L235A (numbering according to Kabat EU index). In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG1 subclass, having mutations L234A, L235A and P329G (numbering according to Kabat EU index). In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG4 subclass, having mutations S228P and L235E (numbering according to Kabat EU index). In yet another embodiment of all aspects reported herein, the multispecific antibody is characterized in that the multispecific antibody is of the human IgG4 subclass, having mutations S228P, L235E and P329G (numbering according to Kabat EU index).

[0525] In one embodiment of all aspects reported herein, an antibody comprising a heavy chain with a CH3 domain as described in detail herein contains an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of all aspects reported herein, an antibody comprising a heavy chain with a CH3 domain as described in detail herein contains an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index).

[0526] 5. Antibody variants

[0527] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics (e.g., antigen binding).

[0528] a) Substitution, insertion, and deletion variants

[0529] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Conservative substitutions". More substantial changes are provided in Table 1 under the heading "Exemplary substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the target antibody and the product screened for the desired activity, such as retained / enhanced antigen binding, reduced immunogenicity, or enhanced ADCC or CDC.

[0530] Table 1

[0531] Initial residue Exemplary substitution Conservative substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

[0532] Amino acids can be grouped according to common side chain properties:

[0533] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0534] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0535] (3) Acidic: Asp, Glu;

[0536] (4) Basic: His, Lys, Arg;

[0537] (5) Residues affecting the chain direction: Gly, Pro;

[0538] (6) Aromatic: Trp, Tyr, Phe.

[0539] Non-conservative substitutions require changing a member of one of these categories to another.

[0540] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study has an alteration (e.g., improvement) (e.g., increased affinity, reduced immunogenicity) in certain biological properties relative to the parental antibody and / or substantially retains certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which can be readily produced, e.g., using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, the variant antibody is displayed on a phage, and screened for a particular biological activity (e.g., binding affinity).

[0541] Alterations (e.g., substitutions) can be made in the HVRs, e.g., to increase antibody affinity. Such alterations can be made in HVR "hot spots", i.e., residues encoded by codons that undergo high frequency mutation during the process of somatic hypermutation (see, e.g., Chowdhury, P.S., Methods Mol. Biol. 207 (2008) 179-196), and / or residues that contact the antigen, and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by construction of a second library and re-selection has been described, e.g., in Hoogenboom, H.R. et al., Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity mutagenesis, diversity is introduced into the variable gene selected for mutation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A second library is then formed. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves HVR-directed methods, where several HVR residues (e.g., 4-6 residues at a time) are randomized. For example, using alanine scanning mutagenesis or modeling, the HVR residues involved in antigen binding can be specifically identified. CDR-H3 and CDR-L3 are particularly often targeted.

[0542] In certain embodiments, substitutions, insertions or deletions can occur within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the HVRs. For example, such changes can be made, for example, outside of antigen contact residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR can be unaltered, or can contain no more than one, two or three amino acid substitutions, respectively.

[0543] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described by Cunningham, B.C. and Wells, J.A., Science 244 (1989) 1081-1085. In this method, residues or a set of target residues (e.g., charged residues such as Arg, Asp, His, Lys and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Additional substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex can be used to identify the points of contact between the antibody and the antigen. Such contact residues and adjacent residues can be targeted for substitution or elimination as candidates. Variants can be screened to determine whether they possess the desired properties.

[0544] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing hundreds or more residues, as well as insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or with a polypeptide that increases the serum half-life of the antibody.

[0545] b) Glycosylation variants

[0546] In certain embodiments, the antibodies provided herein are altered to have an increased or decreased degree of antibody glycosylation. Addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0547] If the antibody comprises an Fc region, the carbohydrates linked thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, generally linked via N-linkage to Asn297 in the CH2 domain of the Fc region (see, e.g., Wright, A. and Morrison, S. L., TIBTECH 15: (1997) 26-32). The oligosaccharides can include a variety of sugars such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants having certain improved properties.

[0548] In one embodiment, antibody variants are provided that have a glycan structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the amount of fucose in the antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. It can be measured by MALDI-TOF mass spectrometry, and the average amount of fucose within the sugar chain at Asn297 is calculated relative to the sum of all glycan structures (such as complex, hybrid, and high-mannose structures) linked to Asn 297, thereby determining the amount of fucose, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acid positions upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylation variants can have improved ADCC function. See, for example, US2003 / 0157108; US2004 / 0093621. Published examples related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87:614 (2004) 614-622.Examples of cell lines capable of producing afucosylated antibodies include the protein fucosylation-deficient Lec13 CHO cells (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986): 533-545; US 2003 / 0157108; and WO 2004 / 056312, especially Example 11); and gene knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87: 614 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003 / 085107).

[0549] Antibody variants with bisected oligosaccharides are also provided, e.g., where the biantennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. These antibody variants can have reduced fucosylation and / or enhanced ADCC function. Examples of these antibody variants are described in, e.g., WO 2003 / 011878; US6,602,684; and US 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. These antibody variants can have enhanced CDC function. These antibody variants are described in, e.g., WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0550] c) Fc-region variants

[0551] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc-region variants. The Fc-region variants can comprise a human Fc-region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0552] In certain embodiments, the invention contemplates antibody variants that have some but not all effector functions, making them ideal candidates for certain applications in which the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains the ability to bind FcRn. The major cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assay procedures for evaluating the ADCC activity of a target molecule are described in US 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA) and Non-radioactive cytotoxicity assays (Promega, Madison, WI)). Effector cells suitable for these assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be evaluated in vivo, for example, in an animal model as disclosed in Clynes, R. et al., Proc. Nat’l Acad. Sci. USA 95 (1998) 652-656. A C1q binding assay can also be performed to demonstrate that the antibody is unable to bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, M.S. et al., Blood 101 (2003) 1045-1052; and Cragg, M.S. and M.J. Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, S.B. et al., Int’l. Immunol. 18 (2006) 1759-1769).

[0553] Antibodies with reduced effector function include those in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are replaced (US 6,737,056). These Fc mutants include Fc mutants having replacements at two or more positions among amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are replaced with alanine (US 7,332,581).

[0554] Certain antibody variants with enhanced or reduced FcR binding are described (see, for example, US 6,737,056; WO 2004 / 056312 and Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0555] In certain embodiments, the antibody variant comprises an Fc-region having one or more amino acid replacements that enhance ADCC, for example, replacements at positions 298, 333, and / or 334 of the Fc-region (EU numbering of residues).

[0556] In some embodiments, alterations are made in the Fc-region to result in altered (i.e., increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in US 6,194,551, WO 99 / 51642, and Idusogie, E.E. et al., J. Immunol. 164 (2000) 4178-4184.

[0557] Antibodies having an increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117 (1976) 587-593 and Kim, J.K. et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005 / 0014934. These antibodies comprise an Fc-region having one or more substitutions that increase Fc-region binding to FcRn. Such Fc variants include Fc variants having substitutions at one or more of the following Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, such as a substitution at Fc-region residue 434 (US 7,371,826).

[0558] For other examples of Fc-region variants, see also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94 / 29351.

[0559] d) Cysteine-engineered antibody variants

[0560] In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, such as "thioMAbs", in which one or more residues of the antibody are replaced with cysteine residues. In some embodiments, the residues to be replaced are at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy-chain Fc-region. Cysteine-engineered antibodies can be generated as described, for example, in US 7,521,541.

[0561] e) Antibody derivatives

[0562] In certain embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known and readily available in the art. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propanediol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in preparation due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, and the like.

[0563] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided, wherein the non-protein moiety can be selectively heated upon exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but can heat the non-protein moiety to a temperature capable of killing cells located in the vicinity of the antibody-non-protein moiety.

[0564] B. Blood-brain barrier shuttle module

[0565] In one embodiment in all aspects, the antibody is a multispecific antibody having at least one binding specificity for the transferrin receptor and at least one binding specificity for a therapeutic agent. In one embodiment, the antibody comprises a first antigen-binding site that binds the transferrin receptor and a second antigen-binding site that binds a brain antigen. In a further embodiment, the brain antigen is selected from Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), α-synuclein, CD20, human glucocerebrosidase, and amyloid precursor protein (APP). In a preferred embodiment, the multispecific antibody binds:

[0566] i) Transferrin receptor and Aβ, or

[0567] ii) Transferrin receptor and CD20, or

[0568] iii) Transferrin receptor and α-synuclein, or

[0569] iv) Transferrin receptor and phospho-tau protein, or

[0570] v) Transferrin receptor and HER2, or

[0571] vi) Transferrin receptor and glucocerebrosidase.

[0572] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO: 24 and light chain variable domains of SEQ ID NO: 37 that form a transferrin receptor binding site, and at least one pair of heavy chain variable domains of SEQ ID NO: 81 and light chain variable domains of SEQ ID NO: 82 that form an Aβ binding site.

[0573] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO: 24 and light chain variable domains of SEQ ID NO: 37 that form a transferrin receptor binding site, and at least one pair of heavy chain variable domains of SEQ ID NO: 79 and light chain variable domains of SEQ ID NO: 80 that form a human CD20 binding site. In one embodiment, the heavy chain variable region comprises substitution of the amino acid residue at Kabat position 11 with any amino acid other than leucine. In one embodiment, the substitution comprises substitution of the amino acid residue at Kabat position 11 with a non-polar amino acid. In a preferred embodiment, the substitution comprises substitution of the amino acid residue at Kabat position 11 in the heavy chain variable domain of SEQ ID NO: 79 with an amino acid residue selected from valine, leucine, isoleucine, serine, and phenylalanine.

[0574] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO: 24 and light chain variable domains of SEQ ID NO: 37 that form a transferrin receptor binding site, and at least one pair of heavy chain variable domains of SEQ ID NO: 83 and light chain variable domains of SEQ ID NO: 84 that form a human α-synuclein binding site.

[0575] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:85 and humanized light chain variable domains derived from SEQ ID NO:86 that form a human α-synuclein binding site.

[0576] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:87 and humanized light chain variable domains derived from SEQ ID NO:88 that form a human α-synuclein binding site.

[0577] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:89 and humanized light chain variable domains derived from SEQ ID NO:90 that form a human α-synuclein binding site.

[0578] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:91 and humanized light chain variable domains derived from SEQ ID NO:92 that form a human α-synuclein binding site.

[0579] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:93 and humanized light chain variable domains derived from SEQ ID NO:94 that form a human α-synuclein binding site.

[0580] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:24 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site, and a human glucocerebrosidase binding site.

[0581] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:7 that form a transferrin receptor binding site and light chain variable domains of SEQ ID NO:34, and at least one pair of heavy chain variable domains of SEQ ID NO:83 and light chain variable domains of SEQ ID NO:84 that form a human α-synuclein binding site.

[0582] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:7 that form a transferrin receptor binding site and light chain variable domains of SEQ ID NO:34, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:85 and humanized light chain variable domains derived from SEQ ID NO:86 that form a human α-synuclein binding site.

[0583] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:7 that form a transferrin receptor binding site and light chain variable domains of SEQ ID NO:34, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:87 and humanized light chain variable domains derived from SEQ ID NO:88 that form a human α-synuclein binding site.

[0584] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:7 that form a transferrin receptor binding site and light chain variable domains of SEQ ID NO:34, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:89 and humanized light chain variable domains derived from SEQ ID NO:90 that form a human α-synuclein binding site.

[0585] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:7 that form a transferrin receptor binding site and light chain variable domains of SEQ ID NO:34, and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:91 and humanized light chain variable domains derived from SEQ ID NO:92 that form a human α-synuclein binding site.

[0586] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:7 and light chain variable domains of SEQ ID NO:34 that form a transferrin receptor binding site and at least one pair of humanized heavy chain variable domains derived from SEQ ID NO:93 and humanized light chain variable domains derived from SEQ ID NO:94 that form a human α-synuclein binding site.

[0587] In one embodiment, the antibody is a bispecific antibody comprising at least one pair of heavy chain variable domains of SEQ ID NO:7 and light chain variable domains of SEQ ID NO:37 that form a transferrin receptor binding site and a human glucocerebrosidase binding site.

[0588] Monovalent binding entities that specifically bind to blood-brain barrier receptors can be characterized in terms of their binding and transcytosis properties as follows:

[0589] - As a monovalent binding entity, effective cell binding to BBBR-expressing cells,

[0590] - As a monovalent binding entity, effective in vivo transcytosis,

[0591] - Human-macaque cross-reactivity (e.g., in BIAcore and FACS assays).

[0592] Transcytosis screening can be performed in an hCMEC / D3-based assay. Testing can be performed in a pulse-chase mode. Incubate hCMEC / D3 brain endothelial cells with the monovalent binding entity for 1 hour, then wash and measure the following parameters at 0 and 4 hours after washing:

[0593] i) The amount of monovalent binding entity absorbed into the cells during the loading period,

[0594] ii) The basolateral amount of the monovalent binding entity 4 hours after loading and washing;

[0595] iii) The apical amount of the monovalent binding entity 4 hours after loading and washing;

[0596] iv) The amount of the monovalent binding entity in the cells (by cell lysis) at 0 and 4 hours after loading and washing;

[0597] v) The total amount of the monovalent binding entity at 0 and 4 hours after loading and washing.

[0598] To be capable of serving as a monovalent binding entity in the blood-brain barrier shuttle module reported herein, an anti-transferrin receptor antibody (e.g., as a monovalent binding entity) must i) be taken up (endocytosed) by hCMEC / D3 cells, ii) be transported to the exterior of hCMEC / D3 cells (exocytosed), and iii) be stable inside hCMEC / D3 cells (no or low trafficking to endosomes for degradation).

[0599] Thus, in one embodiment, in hCMEC / D3-based assays, a monovalent binding entity is characterized by i) (substantially) being taken up into hCMEC / D3 cells during a one-hour loading period, ii) being released to the apical and / or basolateral compartments after the loading period and wash steps and within 4 hours after washing, and iii) a low (intracellular) degradation rate.

[0600] In one embodiment, the loading is carried out for one hour at a concentration of about 2.67 μg / mL of the monovalent binding entity.

[0601] It has been found that to be capable of serving as a monovalent binding entity of the blood-brain barrier shuttle module reported herein, the monovalent binding entity must exhibit the following thresholds in the above-described hCMEC / D3-based assays:

[0602] i) During the loading period, the amount of the monovalent binding entity taken up into the cells is 400 pg or higher.

[0603] ii) Four hours after loading and washing, the amount of the monovalent binding entity in the basolateral side is 100 pg or higher, and

[0604] iii) Four hours after loading and washing, the amount of the monovalent binding entity in the apical side is 150 pg or higher.

[0605] Murine anti-human transferrin receptor antibody 128.1 (for the variable region sequences, see WO93 / 10819 and SEQ ID NO: 64 and 65) can be used as a reference. In this case, to be capable of serving as a monovalent binding entity of the blood-brain barrier shuttle module reported herein, the monovalent binding entity must exhibit the following thresholds in the above-described hCMEC / D3-based assays:

[0606] i) During the loading period, the amount of the monovalent binding entity taken up into the cells is 60% or higher of the loading amount of antibody 128.1.

[0607] ii) Four hours after loading and washing, the basolateral content of the monovalent binding entity is 60% or higher of the basolateral content of antibody 128.1, and

[0608] iii) The apical content of the univalent binding entity is 60% or higher of the apical content of antibody 128.1, 4 hours after loading and washing.

[0609] The hCMEC / D3-based assay (an embodiment for all aspects reported herein) was conducted as follows.

[0610] The medium and supplements for hCMEC / D3 (see WO 2006 / 056879 and Weksler, B.B. et al., FASEBJ. 19 (2005) 1872-1874) can be obtained from Lonza. hCMEC / D3 cells (passages 26-29) were cultured / grown to confluence on collagen-coated coverslips (microscopy) or in flasks in EBM2 medium containing 2.5% FBS, one-quarter of the supplied growth factors, and fully supplemented with the supplied hydrocortisone, gentamicin, and ascorbic acid.

[0611] For all transcytosis assays, high-density well (1×10 8 well / cm 2 ) PET membrane filter inserts (0.4 μm pore size, 12 mm diameter) were used / can be used in 12-well cell culture plates. For the apical and basolateral chambers, the medium volumes were calculated to be 400 μL and 1600 μL, respectively. The apical chambers of the filter inserts were coated with rat tail collagen I (7.5 μg / cm 2 ) followed by fibronectin (5 μg / mL), and each was incubated at RT for 1 h. hCMEC / D3 cells were grown / could be grown in EBM2 medium for 10-12 days to a confluent monolayer (~2×10 5 cells / cm 2 ). Before the assay, the empty filters were blocked in PBS containing 1% BSA for 1 hour or overnight (o / n), and then calibrated in EBM2 for at least 1 hour before the assay.

[0612] The assay was conducted in serum-free EBM2 medium (or reconstituted as described herein). (For the assay protocol, see Figure 1)。On the day of the experiment, the cells were serum-starved for 60 min to deplete the natural ligands of the target blood-brain barrier receptor. The filter inserts with or without cells (but blocked overnight in complete medium) were incubated with the monoclonal antibody (univalent binding entity) under discussion at 37 °C for 1 h on the apical side. The monolayer was washed three times at room temperature (RT) for 3 - 5 min each time in serum-free medium on the apical side (400 μL) and the basolateral side (1600 μL). Pre-warmed medium was added to the apical chamber, and the filter was transferred to a new 12-well plate containing 1600 μL of pre-warmed medium (blocked overnight with PBS containing 1% BSA). At this point, the filter with or without cells was lysed in 500 μL of RIPA buffer to determine the uptake of the specific antibody (univalent binding entity). The remaining filters were incubated at 37 °C or 4 °C, and samples were collected at different time points to determine the apical and / or basolateral release of the antibody (univalent binding entity). High-sensitivity IgG ELISA can be used to quantify the antibody content in the samples (see Example 9). For each time point, data should be generated from two empty filters and three filter cell cultures.

[0613] C. Recombinant methods and compositions

[0614] Antibodies can be produced using recombinant methods and compositions, such as those described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-transferrin receptor antibody as described herein is provided. This nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising this nucleic acid are provided. In a further embodiment, a host cell comprising this nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for preparing an anti-transferrin receptor antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell medium).

[0615] For recombinant production of anti-transferrin receptor antibodies, the nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody).

[0616] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, when glycosylation and Fc effector functions are not required, antibodies can be produced in bacteria. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., in: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibodies can be separated from the bacterial cell paste in the soluble fraction and further purified.

[0617] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathways have been "humanized", resulting in the production of antibodies with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech (2006) 24:210-215.

[0618] Suitable host cells for glycosylated antibody expression can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0619] Plant cell cultures can also be used as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing the PLANTIBODIES TM technology for producing antibodies in transgenic plants).

[0620] Vertebrate cells can also be used as hosts. For example, a mammalian cell line adapted for suspension growth can be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (293 or 293 cells described, for example, in Graham, F. L. et al., J. Gen Virol. 36 (1997) 59); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, J. P., Biol. Reprod. 23 (1980) 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, such as those described in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo. B. K. C. (ed.), Humana Press, Totowa, NJ (2004) pp. 255-268.

[0621] D. Assays

[0622] The anti-transferrin receptor antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0623] 1. Binding assays

[0624] In one aspect, the antibodies of the invention can be tested for their antigen-binding activity, for example, by known methods such as ELISA, αLISA, Western blotting, antibody or reverse phase arrays, etc.

[0625] In an exemplary ELISA or αLISA assay, transferrin receptor in a solution (such as cell supernatant, cell or tissue lysate, body fluid, etc.) is bound by a capture antibody (the capture antibody specifically binds to a first epitope on the transferrin receptor or the transferrin receptor in a specific conformation) and a detection antibody conjugated to a detection entity (the detection antibody specifically binds to a second epitope or conformation of transferrin). A readout is obtained based on the detection entity (such as chemiluminescence, fluorescence, energy transfer-induced luminescence, etc.).

[0626] In the case of an antibody array, antibodies are spotted on a glass or nitrocellulose chip. The slide is blocked and incubated with a solution containing transferrin receptor, washed to remove unbound antibodies, and the bound antibodies are detected with a fluorescently labeled corresponding secondary antibody. The fluorescence signal is measured by a fluorescence slide scanner. Similarly, for a reverse-phase array, recombinant transferrin receptor, cell supernatant, cell or tissue lysate, body fluid, etc. are spotted on a glass or nitrocellulose chip. The slide is blocked and the array is incubated with an antibody directed against a specific epitope on the transferrin receptor. Unbound antibodies are washed away, and the bound antibodies are detected with a fluorescently labeled corresponding secondary antibody. The fluorescence signal is measured by a fluorescence slide scanner (Dernick, G. et al., J. Lipid Res., 52 (2011) 2323-2331).

[0627] E. Methods and Compositions for Diagnosis and Detection

[0628] In some embodiments, any anti-transferrin receptor antibody provided herein is used to detect the presence of human transferrin receptor in a biological sample. The term "detect" as used herein includes quantitative or qualitative detection. In some embodiments, the biological sample includes cells or tissues, such as brain tissue.

[0629] In one embodiment, anti-transferrin receptor antibodies are provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of transferrin receptor in a biological sample is provided. In some embodiments, the method includes contacting the biological sample with an anti-transferrin receptor antibody described herein under conditions that permit binding of the anti-transferrin receptor antibody to the transferrin receptor, and detecting whether a complex has formed between the anti-transferrin receptor antibody and the transferrin receptor. Such a method can be an in vitro or in vivo method. In one embodiment, anti-transferrin receptor antibodies are used to select a subject suitable for treatment with an anti-transferrin receptor antibody, for example, when transferrin receptor is a biomarker for patient selection.

[0630] Exemplary disorders that can be diagnosed using the antibodies of the present invention include neurodegeneration with brain iron accumulation type 1 (NBIA1), pure autonomic failure, Down syndrome, Guam complex, and severe Lewy body diseases such as diffuse Lewy body disease (DLBD), Lewy body variant of Alzheimer's disease (LBVAD), some forms of Gaucher's disease, and Parkinson's disease dementia (PDD).

[0631] In some embodiments, labeled anti-transferrin receptor antibodies are provided. Labels include, but are not limited to, labels or moieties that can be directly detected (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as moieties that can be indirectly detected (e.g., through enzymatic reactions or molecular interactions), such as enzymes or ligands. Exemplary labels include, but are not limited to, radioactive isotopes 32 P, 14 C, 125 I, 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (US 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, glucose oxidase, such as glucose oxidase, galactose oxidase, and 6-phosphoglucose dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that couple the oxidation of hydrogen peroxide to dye precursors such as HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, etc.

[0632] F. Pharmaceutical Preparations

[0633] Pharmaceutical formulations of anti-transferrin receptor antibodies as described herein can be prepared, for example, by admixing the antibody having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Science, 16th edition, Osol, A. (ed.) (1980)) and preparing in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; chlorhexidine digluconate; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as poly(vinylpyrrolidone); amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20( Baxter International, Inc.). Certain exemplary sHASEGP and methods of using the same, including rhuPH20, are described in US 2005 / 0260186 and US 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glucosaminoglycanases, such as chondroitinase).

[0634] Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0635] The formulations herein may also contain more than one active ingredient, preferably those having complementary activities and not adversely affecting each other, as required for the particular indication being treated. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0636] The active ingredient can be entrapped in, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in coarse emulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th Edition, Osol, A (ed.) (1980).

[0637] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices can be in the form of shaped articles such as films or microcapsules.

[0638] Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0639] G. Methods of Treatment and Compositions

[0640] Any anti-TfR antibody provided herein can be used in a therapeutic method. In one aspect, an anti-TfR antibody is provided for use as a medicament. For example, the present invention provides a method of transporting a therapeutic compound across the blood-brain barrier, the method comprising exposing an anti-TfR antibody conjugated to a therapeutic compound (e.g., a multispecific antibody that binds both TfR and a brain antigen) to the BBB such that the antibody transports the therapeutic compound conjugated thereto across the BBB. In another example, the present invention provides a method of transporting a neurological disorder drug across the blood-brain barrier, the method comprising exposing an anti-TfR antibody of the present invention conjugated to a brain disorder drug (e.g., a multispecific antibody that binds both TfR and a brain antigen) to the BBB such that the antibody transports the neurological disorder drug conjugated thereto across the BBB. In one embodiment, the BBB is in a mammal (e.g., a human), such as a mammal (e.g., a human) having a neurological disorder, the neurological disorder including, but not limited to: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, traumatic brain injury, etc. In one embodiment, the neurological disorder is selected from neuropathy, amyloidosis, cancer (e.g., involving the CNS or brain), ocular diseases or disorders, viral or microbial infections, inflammation (e.g., of the CNS or brain), ischemia, neurodegenerative diseases, epilepsy, behavioral disorders, lysosomal storage diseases, etc. The antibodies of the present invention are particularly suitable for treating these neurological disorders because they are capable of transporting one or more linked active ingredients / conjugated therapeutic compounds across the BBB into the CNS / brain, where there is a molecular, cellular, or viral / microbial basis for these disorders. Neuropathy is a disease or disorder of the nervous system characterized by inappropriate or uncontrolled nerve signal conduction or lack of signal conduction, and it includes, but is not limited to, chronic pain (including nociceptive pain), pain caused by physical tissue damage, including cancer-related pain, neuropathic pain (pain caused by abnormalities in the nerves, spinal cord, or brain), and psychogenic pain (entirely or mostly related to psychological disorders), headache, migraine, neuropathy, and the symptoms and syndromes that often accompany such neuropathy, such as dizziness or nausea.

[0641] For neuropathy, the selectable neurological drugs include analgesics, including but not limited to, narcotic / opioid analgesics (i.e., morphine, fentanyl, hydrocodone, meperidine, methadone, oxymorphone, pentazocine, propoxyphene, tramadol, codeine, and oxycodone), non-steroidal anti-inflammatory drugs (NSAIDs) (i.e., ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicam, nabumetone, oxaprozin, piroxicam, sulindac, and tolmetin), corticosteroids (i.e., cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone, and triamcinolone), anti-migraine agents (i.e., sumatriptin, almotriptan, frovatriptan, sumatriptan, rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan, and ergotamine), acetaminophen, salicylates (i.e., aspirin, choline salicylate, magnesium salicylate, diflunisal, and salsalate), anticonvulsants (i.e.,carbamazepine, clonazepam, gabapentin, lamotrigine, pregabalin, tiagabine, and topiramate), anesthetics (i.e., isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, propoxycaine, procaine, novocaine, proparacaine, tetracaine, articaine, bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, saxitoxin, and tetrodotoxin), and cox-2-inhibitors (i.e., celecoxib, rofecoxib, and valdecoxib). For neuropathies accompanied by dizziness, alternative neurological drugs include anti-vertigo agents, including but not limited to meclizine, diphenhydramine, promethazine, and diazepam. For neuropathies accompanied by nausea, alternative neurological drugs include anti-nausea agents, including but not limited to promethazine, chlorpromazine, prochlorperazine, trimethobenzamide, and metoclopramide.,

[0642] Amyloidosis is a group of diseases and conditions associated with extracellular proteinaceous deposits in the CNS, which includes, but is not limited to, secondary amyloidosis, age-related amyloidosis, Alzheimer's disease (AD), mild cognitive impairment (MCI), dementia with Lewy bodies, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guamanian Parkinson-dementia complex, cerebral amyloid angiopathy, Huntington's disease, progressive supranuclear palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, transmissible spongiform encephalopathy, HIV-associated dementia, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), and eye diseases involving β-amyloid protein deposition (i.e., macular degeneration, drusen-related optic neuropathy, and cataracts).

[0643] For amyloidosis, alternative neurological medications include, but are not limited to, antibodies or other binding molecules (including but not limited to small molecules, peptides, aptamers, or other protein conjugates) that specifically bind to a target selected from the following: beta-secretase, tau, presenilin, amyloid precursor protein or a portion thereof, amyloid-beta peptide or its oligomers or fibrils, death receptor 6 (DR6), receptor for advanced glycation end products (RAGE), parkin, and huntingtin; cholinesterase inhibitors (i.e., galantamine, donepezil, rivastigmine, and tacrine); NMDA receptor antagonists (i.e., memantine), monoamine depleting agents (i.e., tetrabenazine); ergoloid mesylate; anticholinergic antiparkinsonian drugs (i.e., procyclidine, diphenhydramine, trihexylphenidyl, benztropine, biperiden, and trihexyphenidyl); dopaminergic antiparkinsonian drugs (i.e., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa, pergolide, tolcapone, and amantadine); tetrabenazine; anti-inflammatory drugs (including but not limited to non-steroidal anti-inflammatory drugs (i.e., indomethicin and other compounds listed above); hormones (i.e., estrogen, progesterone, and leuprolide); vitamins (i.e., folic acid and niacinamide); dimebolin; homotaurine (i.e., 3-aminopropanesulfonic acid; 3APS); 5-hydroxytryptamine receptor activity modulators (i.e., xaliproden); interferons, and glucocorticoids.

[0644] CNS cancers are characterized by the abnormal proliferation of one or more CNS cells (i.e., nerve cells) and include, but are not limited to, glioma, glioblastoma multiforme, meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma, medulloblastomas, ganglioglioma, Schwannoma, neurofibroma, neuroblastoma, and epidural, intramedullary or intradural tumors, or CNS metastases of peripheral tumors such as CD20 or HER2 positive cancers.

[0645] For cancers, the selectable neurological agents are chemotherapeutic agents.

[0646] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); δ-9-tetrahydrocannabinol (dronabinol, );beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan) CPT-11 (irinotecan, ) Acetylcamptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183 - 186(1994)); enediyne anthracycline antibiotics (dynemicin), including dynemicin A; anesperamicin; and neocarzinostatin chromophore and related pigment - protein enediyne chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6 - diazo - 5 - oxo - L - norleucine,. Doxorubicin (including morpholino-doxorubicin, cyano-morpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone;aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; bestrabucil; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Cytarabine (“Ara-C”); Thiotepa; Taxoids, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Cremophor-free ABRAXANETM, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and Docetaxel ( Rorer, Antony, France); Chlorambucil; Gemcitabine 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, such as cisplatin and carboplatin; Vinblastine Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine Oxaliplatin; Leucovovin; Vinorelbine Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid; Capecitabine A pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisone; and FOLFOX, oxaliplatin in combination with 5-FU and leucovovin (ELOXATIN TM) Abbreviation of the treatment regimen.

[0647] This definition of chemotherapeutic agents also includes: antihormonal agents, which act to modulate, reduce, block or inhibit the effects of hormones that can promote cancer growth and are typically in the form of systemic or whole-body treatment. It can be the hormone itself. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene); anti-progesterones; estrogen receptor downregulators (ERD); agents that act to inhibit or shut down the ovaries, such as luteinizing hormone-releasing hormone (LHRH) agonists, such as and leuprolide acetate, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole ( vorozole), letrozole ( letrozole), and anastrozole. In addition, this definition of chemotherapeutic agents also includes bisphosphonates, such as clodronate (e.g., or etidronate, NE-58095, Zoledronic acid / zoledronate ( zoledronic acid / zoledronate), Alendronate ( alendronate), Pamidronate ( pamidronate), Tiludronate ( tiludronate) or Risedronate ( risedronate); and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as Vaccines and gene therapy vaccines, e.g. vaccine, Vaccines and vaccine; Topoisomerase 1 inhibitor; ABARELIX rmRH; lapatinib ditosylate (ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any one of the above.

[0648] Another group of compounds that can be selected as neurological drugs for cancer treatment or prevention are anti-cancer immunoglobulins (including but not limited to trastuzumab, pertuzumab, bevacizumab, alemtuzumab, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, panitumumab and rituximab). In some cases, antibodies conjugated to toxic markers or conjugates can be used to target and kill desired cells (i.e., cancer cells), including but not limited to tositumomab with 131I radiolabeling, or trastuzumab emtansine.

[0649] Eye diseases or disorders are diseases or disorders of the eye, which for the purposes of this article is considered a CNS organ isolated by the BBB. Eye diseases or disorders include, but are not limited to, sclera, cornea, iris, and ciliary body disorders (i.e., scleritis, keratitis, corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson superficial punctate keratopathy, corneal neovascularization, Fuchs dystrophy, keratoconus, keratoconjunctivitis sicca, iritis, and uveitis), lens disorders (i.e., cataract), choroid and retina disorders (i.e., retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, age-related macular degeneration, macular degeneration (wet or dry), epiretinal membrane, retinitis pigmentosa, and macular edema), glaucoma, floaters, optic nerve and visual pathway disorders (i.e., Leber hereditary optic neuropathy and drusen of the optic disc), extraocular muscle / binocular motility / accommodation / refraction disorders (i.e., strabismus, ophthalmoplegia, progressive external ophthalmoplegia, esotropia, exotropia, hyperopia, myopia, astigmatism, refractive error, presbyopia, and ophthalmoplegia), visual impairment and blindness (i.e., amblyopia, Leber congenital amaurosis, scotoma, color blindness, achromatopsia, night blindness, blindness, onchocerciasis, and microphthalmia / coloboma), red eye, Argyll Robertson pupil, fungal keratitis, dry eye, and aniridia.

[0650] For eye diseases or disorders, the select neurological medications are: anti-angiogenic ophthalmic agents (i.e., bevacizumab, ranibizumab, and pegaptanib), ophthalmic glaucoma agents (i.e., carbachol, epinephrine, demecarium bromide, apraclonidine, brimonidine, brinzolamide, levobunolol, timolol, betaxolol, dorzolamide, bimatoprost, carteolol, metipranolol, dipivefrin, travoprost, and latanoprost), carbonic anhydrase inhibitors (i.e., methazolamide and acetazolamide), ophthalmic antihistamines (i.e., naphazoline, phenylephrine, and tetrahydrozoline), ophthalmic lubricants, ophthalmic steroids (i.e., fluorometholone, prednisolone, loteprednol, dexamethasone, difluprednate, rimexolone, fluocinolone, medrysone, and triamcinolone), ophthalmic anesthetics (i.e., lidocaine, proparacaine, and tetracaine), ophthalmic anti-infectives (i.e.,Levofloxacin, gatifloxacin, ciprofloxacin, moxifloxacin, chloramphenicol, bacitracin / polymyxin B, sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin, sulfisoxazole, gentamicin, idoxuridine, erythromycin, natamycin, gramicidin, neomycin, ofloxacin, trifluridine, ganciclovir, vidarabine, ophthalmic anti-inflammatory agents (i.e., nepafenac, ketorolac, flurbiprofen, suprofen, cyclosporine, triamcinolone, diclofenac, and bromfenac), and ophthalmic antihistamines or decongestants (i.e., ketotifen, olopatadine, epinastine, naphazoline, cromolyn, tetrahydrozoline, pemirolast, bepotastine, naphazoline, phenylephrine, nedocromil, lodoxamide, phenylephrine, emedastine, and azelastine).

[0651] CNS viral or microbial infections include, but are not limited to, infections caused by viruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria spp., Streptococcus spp., Pseudomonas spp., Proteus spp., Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus spp., and Mycobacterium tuberculosis), and other microorganisms such as fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., Toxoplasma gondii), or amoeba, which result in CNS pathophysiology including, but not limited to, meningitis, encephalitis, myelitis, vasculitis, and abscesses, which may be acute or chronic.

[0652] For viral or microbial diseases, the selectable neurological drugs include, but are not limited to: antiviral compounds (including, but not limited to, adamantane antivirals (i.e., rimantadine and amantadine), antiviral interferons (i.e., pegylated interferon α-2b), chemokine receptor antagonists (i.e., maraviroc), integrase strand transfer inhibitors (i.e., raltegravir), neuraminidase inhibitors (i.e., oseltamivir and zanamivir), non-nucleoside reverse transcriptase inhibitors (i.e., efavirenz, etravirine, delavirdine and nevirapine), nucleoside reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir, zalcitabine, telbivudine and didanosine), protease inhibitors (i.e., darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir, amprenavir, indinavir and saquinavir), purine nucleosides (i.e., valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir and cidofovir) and miscellaneous antiviral agents (i.e., enfuvirtide, foscarnet, palivizumab and fomivirsen), antibiotics (including, but not limited to, aminopenicillins (i.e.,Amoxicillin, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucoxacillin, temocillin, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, and bacampicillin), cephalosporin (i.e., cefazolin, cephalexin, cephalothin, cefamandole, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cephradine, loracarbef, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin), carbapenem / penem (i.e., imipenem, meropenem, ertapenem, faropenem, and doripenem), monobactam (i.e., aztreonam, tigemonam, norcardicin A, and tabtoxinine-beta-lactam), beta-lactamase inhibitor in combination with another beta-lactam antibiotic (i.e., clavulanic acid, tazobactam, and sulbactam), aminoglycoside (i.e.,Amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromomycin), ansamycin (i.e., geldanamycin and herbimycin), carbacephem (i.e., loracarbef), glycopeptide (i.e., teicoplanin and vancomycin), macrolide (i.e., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spectinomycin), monobactam (i.e., aztreonam), quinolone (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamide (i.e., sulfonamidochrysoidine, mafenide, sulfacetamide, sulfadiazine, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim, and sulfamethoxazole), tetracyclines (i.e.,tetracycline, demeclocycline, doxycycline, minocycline, and oxytetracycline), antineoplastic or cytotoxic antibiotics (i.e., doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin), and miscellaneous antibacterial compounds (i.e., bacitracin, colistin, and polymyxin B), antifungals (i.e., metronidazole, nitazoxanide, tinidazole, chloroquine, iodoquinol, and paromomycin), and anti - parasites (including but not limited to quinine, chloroquine, amodiaquine, pyrimethamine, sulphadoxine, proguanil, mefloquine, atovaquone, primaquine, artemesinin, halofantrine, doxycycline, clindamycin, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin, amphotericin B, melarsoprol, efornithin, and albendazole).

[0653] CNS inflammation includes, but is not limited to, inflammation caused by CNS injury, which can be physical injury (i.e., due to accident, surgery, brain trauma, spinal cord injury, concussion) and injury caused by or associated with one or more other CNS diseases or disorders (i.e., abscess, cancer, viral or microbial infection).

[0654] For CNS inflammation, neurological drugs can be selected that address the inflammation itself (i.e., non-steroidal anti-inflammatory agents such as ibuprofen or naproxen), or neurological drugs that treat the underlying cause of the inflammation (i.e., antiviral or anti-cancer agents).

[0655] As used herein, CNS ischemia refers to a group of disorders or their etiologies related to abnormal blood flow or vascular behavior in the brain, and includes, but is not limited to, focal cerebral ischemia, global cerebral ischemia, stroke (i.e., subarachnoid hemorrhage and intracerebral hemorrhage), and aneurysm.

[0656] For ischemia, neurological drugs that can be selected include, but are not limited to, thrombolytics (i.e., urokinase, alteplase, reteplase, and tenecteplase), platelet aggregation inhibitors (i.e., aspirin, cilostazol, clopidogrel, prasugrel, and dipyridamole), statins (i.e., lovastatin, pravastatin, fluvastatin, rosuvastatin, atorvastatin, simvastatin, cerivastatin, and pitavastatin), and compounds that improve blood flow or vascular elasticity, including, for example, blood pressure drugs.

[0657] Neurodegenerative diseases are a group of diseases and disorders associated with the loss of function or death of nerve cells in the CNS, and include, but are not limited to, adrenoleukodystrophy, Alexander disease, Alpers disease, amyotrophic lateral sclerosis, ataxia-telangiectasia, Batten disease, Cockayne syndrome, corticobasal degeneration, degeneration caused by or associated with amyloidosis, Friedreich ataxia, frontotemporal degeneration, Kennedy disease, multiple system atrophy, multiple sclerosis, primary lateral sclerosis, progressive supranuclear palsy, spinal muscular atrophy, transverse myelitis, Refsum disease, and spinocerebellar ataxia.

[0658] For neurodegenerative diseases, the neurological drugs that can be selected are growth hormone or neurotrophic factors; examples include but are not limited to brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4 / 5, fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemisinin, persephin, interleukin, glial cell line-derived neurotrophic factor receptor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone morphogenetic protein (BMP), netrins, saposin, semaphorin, and stem cell factor (SCF).

[0659] Seizure disorders and conditions of the CNS involve inappropriate and / or abnormal electrical conductance in the CNS and include, but are not limited to, epilepsy (i.e., absence seizures, atonic seizures, benign Rolandic epilepsy, childhood absence, clonic seizures, complex partial seizures, frontal lobe epilepsy, febrile seizures, infantile spasms, juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner Syndrome, Dravet’s syndrome, Otahara syndrome, West syndrome, myoclonic seizures, mitochondrial disorders, progressive myoclonic epilepsies, psychogenic seizures, reflex epilepsy, Rasmussen's Syndrome, simple partial seizures, secondarily generalized seizures, temporal lobe epilepsy, tonic-clonic seizures, tonic seizures, psychomotor seizures, limbic epilepsy, partial-onsetseizures), generalized-onset seizures, status epilepticus, abdominal epilepsy, akinetic seizures, autonomic seizures, massive bilateral myoclonus, catamenial epilepsy, drop seizures, emotional seizures, focal seizures, gelastic seizures, Jacksonian March, Lafora Disease, motor seizures, multifocal seizures, nocturnal seizures, photosensitive seizure, pseudo seizures, sensory seizures, subtle seizures, sylvan seizures, withdrawal seizures, and visual reflex seizures. For seizure disorders, the neurological medications of choice are anticonvulsants or antiepileptics, including but not limited to barbiturate anticonvulsants (i.e., primidone, metharbital, mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal, barbital, brallobarbital, and phenobarbital), benzodiazepine anticonvulsantsanticonvulsants) (i.e., diazepam, clonazepam, and lorazepam), carbamate anticonvulsants (i.e., felbamate), carbonic anhydrase inhibitor anticonvulsants (i.e., acetazolamide, topiramate, and zonisamide), dibenzazepine anticonvulsants (i.e., rufinamide, carbamazepine, and oxcarbazepine), fatty acid derivative anticonvulsants (i.e., divalproex and valproic acid), γ-aminobutyric acid analogs (i.e., pregabalin, gabapentin, and vigabatrin), γ-aminobutyric acid reuptake inhibitors (i.e., tiagabine), γ-aminobutyric acid transaminase inhibitors (i.e., vigabatrin), hydantoin anticonvulsants (i.e., phenytoin, ethotoin, fosphenytoin, and mephenytoin), miscellaneous anticonvulsants (i.e., lacosamide and magnesium sulfate), progestins (i.e., progesterone), oxazolidinedione anticonvulsants (i.e., paramethadione and trimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam), succinimide anticonvulsants (i.e., ethosuximid and methsuximide), triazine anticonvulsants (triazineanticonvulsants) (i.e., lamotrigine) and urea anticonvulsants (i.e., phenacemide and pheneturide).

[0660] Behavioral disorders are CNS disorders characterized by abnormal behaviors exhibited by a patient and include, but are not limited to, sleep disorders (i.e., insomnia, parasomnias, night terrors, circadian rhythm sleep disorders, and narcolepsy), mood disorders (i.e., depression, suicidal depression, anxiety, chronic affective disorders, phobias, panic attacks, obsessive-compulsive disorder, attention deficit hyperactivity disorder (ADHD), attention deficit disorder (ADD), chronic fatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolar disorder), eating disorders (i.e., anorexia or bulimia), psychoses, developmental behavioral disorders (i.e., autism, Rett’s syndrome, Aspberger’s syndrome), personality disorder, and psychotic disorders (i.e., schizophrenia, delusional disorder, etc.).

[0661] For behavioral disorders, the neurological drugs can be selected from behavior-modifying compounds, including but not limited to atypical antipsychotics (i.e., risperidone, olanzapine, aripiprazole, quetiapine, paliperidone, asenapine, clozapine, iloperidone, and ziprasidone), phenothiazine antipsychotics (i.e., prochlorperazine, chlorpromazine, fluphenazine, perphenazine, trifluoperazine, thioridazine, and mesoridazine), thioxanthene (i.e., thiothixene), miscellaneous antipsychotics (i.e., pimozide, lithium, molindone, haloperidol, and loxapine), selective serotonin reuptake inhibitors (i.e., citalopram, escitalopram, paroxetine, fluoxetine, and sertraline), serotonin-norepinephrine reuptake inhibitors (i.e., duloxetine, venlafaxine, desvenlafaxine), tricyclic antidepressants (i.e., doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine, imipramine, protriptyline, and desipramine), tetracyclic antidepressants (i.e., mirtazapine and maprotiline), phenylpiperazine antidepressants (i.e.,Trazodone and nefazodone, monoamine oxidase inhibitors (i.e., isocarboxazid, phenelzine, selegiline, and tranylcypromine), benzodiazepines (i.e., alprazolam, estazolam, flurazeptam, clonazepam, lorazepam, and diazepam), norepinephrine-dopamine reuptake inhibitors (i.e., bupropion), CNS stimulants (i.e., phentermine, diethylpropion, methamphetamine, dextroamphetamine, amphetamine, methylphenidate, dexmethylphenidate, lisdexamfetamine, modafinil, pemoline, phendimetrazine, benzphetamine, phendimetrazine, armodafinil, diethylpropion, caffeine, atomoxetine, doxapram, and mazindol), anxiolytics / sedatives / hypnotics (including but not limited to barbiturates (i.e.,Secobarbital, phenobarbital, and mephobarbital, benzodiazepine (as described above), and miscellaneous anxiolytics / sedatives / hypnotics (i.e., diphenhydramine, sodium oxybate, zaleplon, hydroxyzine, chloral hydrate, aolpidem, buspirone, doxepin, eszopiclone, ramelteon, meprobamate, and ethclorvynol), secretin (see, e.g., Ratliff-Schaub et al., Autism 9 (2005): 256-265), opioid peptides (see, e.g., Cowen et al., J. Neurochem. 8 (2004) 273-285), and neuropeptides (see, e.g., Hethwa et al., Am. J. Physiol. 289 (2005) E301-305).

[0662] Lysosomal storage diseases are metabolic disorders that are associated with or have CNS-specific symptoms in some cases; such disorders include, but are not limited to: Tay-Sachs disease, Gaucher disease, Fabry disease, mucopolysaccharidoses (type I, II, III, IV, V, VI, and VII), glycogen storage diseases, GM1 gangliosidosis, metachromatic leukodystrophy, Farber disease, Canavan disease, and neuronal ceroid lipofuscinosis types 1 and 2, Niemann-Pick disease, Pompe disease, and Krabbe disease.

[0663] For lysosomal storage diseases, alternative neurological drugs are the enzyme itself that is impaired in the disease, or a drug that mimics the activity of the enzyme that is impaired in the disease. Exemplary recombinant enzymes for treating lysosomal storage diseases include, but are not limited to, those described, for example, in U.S. Patent Application Publication No. 2005 / 0142141 (i.e., α-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, α-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, β-galactosidase, arylsulfatase B, β-glucuronidase, acid α-glucosidase, glucocerebrosidase, α-galactosidase A, hexosaminidase A, acid sphingomyelinase, β-galactocerebrosidase, β-galactosidase, arylsulfatase A, acid ceramidase, aspartylglucosaminidase, palmitoyl-protein thioesterase 1, and tripeptidyl peptidase 1).

[0664] In one aspect, the antibodies of the invention are used to detect neurological disorders and / or to evaluate the severity or duration of a disease or disorder prior to the onset of symptoms. In some aspects, the antibodies permit detection and / or imaging of neurological disorders, including imaging by radiography, tomography, or magnetic resonance imaging (MRI).

[0665] In one aspect, there is provided the use of a low-affinity anti-TfR antibody of the invention as a medicament. In other aspects, there is provided the use of a low-affinity anti-TfR antibody for treating a neurological disease or disorder (e.g., Alzheimer's disease) without depletion of red blood cells (i.e., reticulocytes). In certain embodiments, there is provided a modified low-affinity anti-TfR antibody for use in a treatment method as described herein. In certain embodiments, the invention provides a method for treating an individual suffering from a neurological disease or disorder with a low-affinity anti-TfR antibody modified to have improved safety, the method comprising administering to the individual an effective amount of the anti-TfR antibody (optionally conjugated to a neurological disorder drug). In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent. In other embodiments, the invention provides an anti-TfR antibody modified to improve its safety for reducing or inhibiting amyloid plaque formation in a patient at risk of or suffering from a neurological disease or disorder (e.g., Alzheimer's disease). According to any of the above embodiments, the "individual" is optionally a human. In certain aspects, the anti-TfR antibodies of the invention for use in the methods of the invention can enhance the uptake of a neurological disorder drug conjugated thereto.

[0666] In another aspect, the invention provides the use of a low-affinity anti-TfR antibody of the invention in the preparation or manufacture of a medicament. In some embodiments, the medicament is for treating a neurological disease or disorder. In another embodiment, the medicament is for use in a method of treating a neurological disease or disorder, the method comprising administering to an individual suffering from a neurological disease or disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.

[0667] In another aspect, the invention provides a method of treating Alzheimer's disease. In one embodiment, the method comprises administering to an individual suffering from Alzheimer's disease an effective amount of a bispecific antibody of the invention that binds both BACE1 and TfR or both Aβ and TfR. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. According to any of the above embodiments, the "individual" can be a human.

[0668] The anti-TfR antibodies of the present invention can be used alone or in combination with other agents for treatment. For example, the anti-TfR antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the other therapeutic agent is a therapeutic agent that is effective in treating a neurological disorder that is the same as or different from the neurological disorder being treated with the anti-TfR / antibody. Exemplary other therapeutic agents include, but are not limited to: the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rivastigmine, and tacrine), NMDA receptor antagonists (such as memantine), amyloid β peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid β peptide immunization, phosphodiesterase inhibitors, 5-hydroxytryptamine receptor antagonists, and anti-amyloid β peptide antibodies. In certain embodiments, the at least one other therapeutic agent is selected because of its ability to mitigate one or more side effects of a neurological drug.

[0669] In certain other such embodiments, the at least one other therapeutic agent is selected because of its ability to inhibit or prevent complement pathway activation upon administration of the anti-TfR antibody. Examples of such therapeutic agents include, but are not limited to, active agents that have the ability to interfere with anti-TfR antibody binding or activation of the complement pathway and active agents that inhibit one or more molecular interactions within the complement pathway, and are generally described in Mollnes and Kirschfink (Molec. Immunol. 43 (2006) 107-121), the content of which is hereby expressly incorporated by reference.

[0670] These combination therapies, as noted above and herein, encompass combination administration (wherein two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case the administration of the antibody of the present invention can occur before, simultaneously with, and / or after the administration of the other therapeutic agent and / or adjuvant. In some embodiments, the administration of the anti-TfR antibody and the administration of the other therapeutic agent occur within about one month of each other, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days. The antibodies of the present invention can also be used in combination with other intervention therapies, such as, but not limited to, radiotherapy, behavioral therapy, or other therapies known in the art and appropriate for the neurological disorder to be treated or prevented.

[0671] The anti-TfR antibodies (and any other therapeutic agent) of the present invention can be administered by any suitable means, including parenterally, intraluminally, and intranasally, and, if local treatment is desired, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, depending in part on whether the administration is short-term or long-term, by injection, such as intravenous or subcutaneous injection. A variety of dosing regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulse infusion.

[0672] The antibodies of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered herein include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the active agent is to be delivered, the method of administration, the dosing regimen, and other factors known to the medical practitioner.

[0673] The antibodies need not, but may optionally, be formulated with one or more agents currently used to prevent or treat the disorder under discussion or to prevent, mitigate, or ameliorate one or more side effects of the antibody administration. The effective amount of the other agent depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These agents are generally used in the same dosage and by the route of administration described herein, or in about 1% to 99% of the dosage described herein, or in any dosage and by any route empirically / clinically determined to be appropriate.

[0674] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibodies may be suitably administered to the patient either as a single dose or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage of antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) may be administered to the patient, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until there is an optimal suppression of disease symptoms. An exemplary dosage of the antibody will range from about 0.05 mg / kg to about 40 mg / kg. Thus, one or more dosages (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, or 40 mg / kg may be administered to the patient. Such dosages may be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient receives about two to about twenty or, for example, about six dosages of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses. However, other treatment regimens may be useful. It should be understood that one way to mitigate the effect of anti-TfR antibody administration on the reticulocyte population is to vary the amount or schedule of the dosage such that there is an overall lower amount of circulating antibody in the bloodstream interacting with the reticulocytes. In a non-limiting example, a lower dosage of anti-TfR antibody may be administered at a higher frequency compared to a higher dosage. The dosage used may be balanced between the amount of antibody that must be delivered to the CNS (which is itself related to the affinity of the CNS antigen-specific portion of the antibody), the affinity of the antibody for TfR, and whether a compound that protects red blood cells (i.e., reticulocytes), stimulates their growth and development, or inhibits the complement pathway is co-administered with or continuously administered with the antibody. The progress of the treatment can be readily monitored by conventional techniques and assays as described herein and as known in the art.

[0675] It should be understood that any of the above formulations or treatment methods may be practiced using the immunoconjugates of the invention in place of or in addition to the anti-TfR antibodies.

[0676] III. Articles

[0677] In another aspect of the invention, there is provided an article of manufacture comprising a material useful for treating, preventing, and / or diagnosing the above-described disorders. The article of manufacture includes 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 container can be formed from a variety of materials such as glass or plastic. The container contains a composition which can be present alone or can be combined with another composition effective to treat, prevent, and / or diagnose the condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for treating the selected condition. Additionally, the article of manufacture can include (a) a first container containing a composition, wherein the composition includes an antibody of the invention; and (b) a second container containing a composition, wherein the composition includes another cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention can also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture can also include a second (or third) container including a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. From a commercial and user standpoint, it can also include other desirable materials including other buffers, diluents, filters, needles, and syringes.

[0678] It should be understood that the immunoconjugates of the invention can be included in any of the above articles of manufacture in place of or in addition to the bispecific antibodies of the invention.

[0679] IV. EXAMPLES

[0680] The following are examples of the methods and compositions of the invention. It should be understood that numerous other embodiments can be practiced in view of the general description provided above.

[0681] Materials and methods

[0682] Recombinant DNA technology

[0683] DNA was manipulated using standard methods as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

[0684] Gene and oligonucleotide synthesis

[0685] At Geneart GmbH (Regensburg, Germany), the desired gene fragments were prepared by chemical synthesis. The synthesized gene fragments were cloned into an Escherichia coli plasmid for propagation / amplification. The DNA sequence of the subcloned gene fragments was verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled via PCR or by annealing chemically synthesized oligonucleotides. The corresponding oligonucleotides were obtained from metabion GmbH (Planegg-Martinsried, Germany).

[0686] Reagents

[0687] Unless otherwise indicated, all commercial chemicals, antibodies, and kits were used according to the manufacturer's protocols.

[0688] Example 1

[0689] Immunization of rabbits and mice

[0690] Immunization of mice

[0691] NMRI mice were genetically immunized with a plasmid expression vector encoding full-length human or macaque TfR by intradermal administration of 100 μg of carrier DNA followed by electroporation (2 rectangular pulses of 1000 V / cm, duration 0.1 ms, interval 0.125 s; followed by 4 rectangular pulses of 287.5 V / cm, duration 10 ms, interval 0.125 s). Mice received 6 or 7 consecutive immunizations on days 0, 14, 28, 42, 56, 70, and 84. The fourth and sixth immunizations were performed with a vector encoding macaque TfR; all other immunizations were performed with a vector encoding human TfR. Blood was collected on days 36, 78, and 92, and sera were prepared and used for ELISA titration assays (see below). Animals with the highest titers were selected and boosted on day 96: intravenous injection of 10 6 human TF-1 cells or 50 μg of recombinant human soluble TfR lacking the helical domain (the extracellular domain of human TfR starting at Leu122 and ending at Asn608, expressed in HEK293F cells as an N-terminal fusion to the human Fc-region and purified by protein A affinity chromatography and size exclusion chromatography), and monoclonal antibodies were isolated by hybridoma technology based on the ability to bind to human and macaque transferrin receptors expressed on the surface of stably transfected CHO-K1 cells (see Example 3).

[0692] Immunization of rabbits

[0693] Genetic immunization was performed on New Zealand white rabbits or transgenic rabbits expressing a humanized antibody library by intradermal administration of 400 μg of vector DNA followed by electroporation (5 rectangular pulses of 750 V / cm, duration 10 ms, interval 1 s) using a plasmid expression vector encoding full-length human or macaque TfR. The rabbits received 6 consecutive immunizations on days 0, 14, 28, 56, 84, and 112. The fourth and sixth immunizations were performed using a vector encoding macaque TfR; a vector encoding human TfR was used for all other immunizations. Blood was collected (10% of the estimated total blood volume) on days 35, 63, 91, and 119. Serum was prepared and used for ELISA titration assays (see below), and peripheral mononuclear cells were isolated and used as a source of antigen-specific B cells in the B cell cloning procedure (see Example 2).

[0694] Determination of serum titer (ELISA)

[0695] Human recombinant soluble TfR (R&D Systems catalog no. 2474-TR) was immobilized at 3 μg / mL in PBS, 100 μL / well, on 96-well NUNC Maxisorb plates, followed by: blocking the plates with 2% Crotein C in PBS, 200 μL / well; applying serial dilutions of antiserum in 0.5% Crotein C in PBS, in duplicate, 100 μL / well; detecting with: (1) HRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch / Dianova 115-036-071; 1 / 16000) for all mouse sera, (2) HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch / Dianova 711-036-152; 1 / 16000) for all rabbit sera, (3) rabbit anti-human IgG antibody (Pierce / Thermo Scientific 31423; 1 / 5000) only for sera from transgenic rabbits, (4) biotinylated goat anti-human κ antibody (SouthernBiotech / Biozol 2063-08, 1 / 5000) and streptavidin-HRP only for sera from transgenic rabbits; diluted in 0.5% Crotein C in PBS, 100 μL / well. For all steps, the plates were incubated at 37 °C for 1 h. Between all steps, the plates were washed 3 times with 0.05% Tween 20 in PBS. Signal was generated by adding BM Blue POD Substrate soluble (Roche), 100 μL / well; and stopped by adding 1 M HCl, 100 μL / well. Absorbance was read at 450 nm relative to 690 nm as reference. The titer was defined as the antiserum dilution resulting in half-maximal signal.

[0696] Example 2

[0697] B-cell clones from rabbit

[0698] Isolation of rabbit peripheral blood mononuclear cells (PBMC)

[0699] Blood samples were obtained from a total of 6 animals (2 wild-type (wt) rabbits and 4 transgenic (tg) rabbits). These rabbits were derived from 2 different immunization series: The first immunization series used 2 wt and 2 tg rabbits, while the second immunization series used 2 tg rabbits (see also Example "Immunization of rabbits"). Whole blood containing EDTA was diluted two-fold with 1×PBS (PAA, Pasching, Austria), and then density centrifugation was performed using mammalian lympholyte (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the manufacturer's instructions. PBMCs were washed twice with 1×PBS.

[0700] EL-4B5 medium

[0701] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM glutamine, 1% penicillin / streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany), and 0.05 mM β-mercaptoethanol (Gibco, Paisley, Scotland).

[0702] Depletion of cells

[0703] First immunization series: Macrophages / monocytes were depleted using sterile 6-well plates (cell culture grade) coated with confluent monolayers of CHO cells by non-specific adhesion and non-specific binding of lymphocytes.

[0704] Second immunization series: The depletion step using CHO cell-coated wells was omitted because we could not rule out that those B-cells that produce antibodies cross-reactive with the hamster transferrin receptor antibody were depleted. Therefore, blank sterile 6-well plates (cell culture grade) were used to deplete macrophages and monocytes by non-specific adhesion, so that potential B-lymphocytes that produce hamster cross-reactive (and possibly mouse cross-reactive) surface antibodies could reach the next step of the workflow.

[0705] For each immunization series: Each well was filled with a maximum of 4 mL of medium and up to 6×106 PBMCs from immunized rabbits were taken and allowed to bind for 1 h at 37 °C in an incubator. The cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.

[0706] Enrichment of B-cells on human transferrin receptor

[0707] Up to 6×10 6 PBLs / 4 mL of medium were seeded into a 6-well tissue culture plate coated with a monolayer of human transferrin receptor-positive CHO cells and allowed to bind for 1 h at 37 °C in an incubator. The wells were carefully washed 1 - 2 times with 1×PBS to remove non-adherent cells. The remaining adherent cells were detached by treatment with trypsin for 10 min at 37 °C in an incubator. The trypsinization was stopped with EL-4B5 medium. The cells were kept on ice until immunofluorescence staining.

[0708] Immunofluorescence staining and flow cytometry

[0709] Anti-IgG FITC (AbD Serotec, Düsseldorf, Germany) was used for single cell sorting. For surface staining, cells from the depletion and enrichment steps were incubated with anti-IgG FITC antibody in PBS and incubated in the dark at 4 °C for 45 min. After staining, the PBMCs were washed twice with ice-cold PBS. Finally, the PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Before FACS analysis, propidium iodide at a concentration of 5 μg / mL (BD Pharmingen, San Diego, CA, USA) was added to distinguish dead cells from live cells.

[0710] A Becton Dickinson FACSAria equipped with a computer and FACSDiva software (BD Biosciences, USA) was used for single cell sorting.

[0711] B-cell culture

[0712] The culture of rabbit B-cells was prepared by a method similar to that described by Zubler et al. (1985). Briefly, sorted single rabbit B-cells were incubated in a 96-well plate containing 200 μL / well of EL-4B5 medium in an incubator at 37 °C in a 5% CO2 atmosphere for 7 days, where the medium contained Pansorbin Cell (1:100000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant (charge TSN-M13(10242), MicroCoat, Bernried, Germany) and γ-irradiated murine EL-4-B5 thymoma cells (2.5×10 4 / well). The supernatant of the B-cell culture was collected for screening, and the remaining cells were immediately collected and frozen at -80 °C in 100 μL of RLT buffer (Qiagen, Hilden, Germany).

[0713] Example 3

[0714] Identification of human and macaque TfR-binding antibodies by cell ELISA

[0715] To screen rabbit B-cell or mouse hybridoma supernatants for antibodies that recognize human and macaque TfR, a cell ELISA using stably transfected CHO-K1 cells was utilized. Stable transfectants were obtained by transfecting CHO-K1 cells with an expression plasmid containing an expression cassette for human or macaque TfR and neomycin-phosphotransferase. After transfection, the cells were diluted in growth medium containing 500 μg / mL G418 (Life Technologies). After the appearance of growing clones, the cells were isolated, stained with MEM-75 (Abcam) or 13E4 (Life Technologies) and a PE-labeled human or macaque TfR secondary antibody, and the highly fluorescent cells were sorted as single cells into the wells of a 96-well plate (FACS Aria). After 7 days of growth, the TfR expression of the clones was examined again, and the best-expressing clones were selected for the cell ELISA experiment.

[0716] Briefly, 15,000 cells were seeded into each well of a 384-well plate and incubated at 37 °C, 5% CO2 for 18 h. The supernatant was removed using an automated washer (BIOTEK), and 30 μL of the supernatant containing the antibody was added to each well, followed by 24 μL of growth medium. After 2 h of incubation, the wells were emptied and 30 μL of 0.05% glutaraldehyde in PBS was added for 45 min at RT. The wells were washed 3 times with PBS / 0.025% Tween20 (PBST), 30 μL of anti-rabbit-HRP or anti-mouse-HRP (Southern Biotech) diluted 1:5000 in blocking buffer was added and the plate was incubated for 1 h at RT. The wells were washed 6 times with PBST and a signal was generated using 30 μL of TMB / well, and the absorbance was measured at 450 nm.

[0717] Example 4

[0718] Cloning and expression of anti-TfR antibody

[0719] Recombinant DNA technology

[0720] Standard methods were used to manipulate DNA according to Sambrook, J et al., Molecular cloning: A Laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

[0721] Gene and oligonucleotide synthesis

[0722] The required gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthetic gene fragments were cloned into an E. coli plasmid for propagation / amplification. The DNA sequence of the subcloned gene fragments was verified by DNA sequencing. Alternatively, short DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The corresponding oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0723] PCR amplification of V-domains

[0724] Total RNA was prepared from B-cell lysates using the NucleoSpin 8 / 96 RNA kit (Macherey & Nagel; 740709.4, 740698) according to the manufacturer's protocol. The RNA was diluted with 60 μL of RNase-free water. cDNA was generated using 6 μL of RNA by reverse transcriptase reaction using Superscript III First-Strand Synthesis SuperMix (Invitrogen 18080-400) and oligo-dT primers according to the manufacturer's instructions. All steps were performed on a Hamilton ML Star system. Immunoglobulin heavy and light chain variable regions (VH and VL) were amplified using AccuPrime SuperMix (Invitrogen 12344-040) and 4 μL of cDNA in a final volume of 50 μL, using the primers rbHC.up and rbHC.do for the heavy chain, rbLC.up and rbLC.do for the light chain of wild-type rabbit B cells, and BcPCR_FHLC_leader.fw and BcPCR_huCkappa.rev for the light chain of transgenic rabbit B cells (see table below). All forward primers were signal peptide-specific (for VH or VL), while the reverse primers were constant region-specific (for VH or VL). The PCR conditions for RbVH+RbVL were as follows: hot start at 94 °C for 5 min; 94 °C for 20 sec, 70 °C for 20 sec, 68 °C for 45 sec, 35 cycles, and a final extension at 68 °C for 7 min. The PCR conditions for HuVL were as follows: hot start at 94 °C for 5 min; 94 °C for 20 sec, 52 °C for 20 sec, 68 °C for 45 sec, 40 cycles, and a final extension at 68 °C for 7 min.

[0725]

[0726] 8 μL of the 50 μL PCR solution was loaded onto a 2% 48E-gel (Invitrogen G8008-02). Positive PCR reactions were purified using the NucleoSpin Extract II kit (Macherey & Nagel; 740609250) according to the manufacturer's protocol and eluted in 50 μL of elution buffer. All purification steps were performed on a Hamilton ML Starlet system.

[0727] Recombinant expression of rabbit monoclonal bivalent antibodies

[0728] To recombinantly express rabbit monoclonal bivalent antibodies, PCR products encoding VH or VL were cloned as cDNAs into expression vectors by the blunt-end cloning method (RS Haun et al., BioTechniques (1992) 13, 515 - 518; MZ Li et al., Nature Methods (2007) 4, 251 - 256). The expression vectors contained an expression cassette consisting of a 5’ CMV promoter including intron A and a 3’ BGH polyadenylation sequence. In addition to the expression cassette, the plasmid also contained a pUC18-derived origin of replication and a β-lactamase gene conferring ampicillin resistance for plasmid amplification in Escherichia coli. Three variants of this basic plasmid were used: one plasmid contained a rabbit IgG constant region designed to accept the VH region, and the other two plasmids contained rabbit or human κ LC constant regions to accept the VL region.

[0729] Using overlapping primers, the linearized expression plasmids encoding κ or γ constant regions and the VL / VH inserts were amplified by PCR.

[0730] The purified PCR products were incubated with T4 DNA-polymerase to generate single-stranded overhangs. The reaction was stopped by adding dCTP.

[0731] In the next step, the plasmids and inserts were mixed and incubated with recA, which induces site-specific recombination. The recombinant plasmids were transformed into Escherichia coli. The following day, growing colonies were picked and the correct recombinant plasmids were examined by plasmid preparation, restriction analysis, and DNA-sequencing.

[0732] For antibody expression, the isolated HC and LC plasmids were transiently co-transfected into HEK293 cells and the supernatant was harvested after 1 week.

[0733] Generation of vectors for expression of rabbit monoclonal univalent antibodies

[0734] To recombinantly express the selected candidates as monoclonal monovalent antibodies, the rabbit constant regions of all VH chains were converted to human constant regions that included a club mutation in the CH3 segment. For the VL chains derived from rabbit wild-type B-cells, the rabbit Cκ constant region was converted to human. 4 μL of the cDNA of the selected candidates was used to amplify the immunoglobulin heavy and light chain variable regions in a 50 μL final volume, where AccuPrime SuperMix (Invitrogen 12344-040) was used, and signal peptide-specific forward primers and CDR3-J region-specific reverse primers with overlapping sequences (20 bp) homologous to the human constant region (of VH or VL) at the 3' end were used. The PCR conditions for VH and VL chain amplification were as follows: hot start at 94 °C for 5 min; 94 °C for 20 sec, 68 °C for 20 sec, 68 °C for 45 sec, 35 cycles, and a final extension at 68 °C for 7 min.

[0735] By the blunt-end cloning method (RS Haun et al., BioTechniques (1992) 13, 515-518; MZ Li et al., Nature Methods (2007) 4, 251-256), the PCR products encoding VH or VL were cloned as cDNA into the expression vector. The expression vector contained an expression cassette consisting of a 5' CMV promoter including intron A and a 3' BGH polyadenylation sequence. In addition to the expression cassette, the plasmid also contained a pUC18-derived origin of replication and a β-lactamase gene conferring ampicillin resistance for plasmid amplification in E. coli. Two variants of this basic plasmid were used: one plasmid contained the human IgG constant region designed to accept the newly amplified VH chains, and the second plasmid contained the human κLC constant region to accept the VL chains.

[0736] Using overlapping primers, the linearized expression plasmid encoding the κ or γ constant region and the VL / VH insert were amplified by PCR.

[0737] The purified PCR products were incubated with T4 DNA-polymerase to generate single-stranded overhangs. The reaction was stopped by adding dCTP.

[0738] In the next step, the plasmid and the insert were mixed and incubated with recA, which induces site-specific recombination. The recombinant plasmid was transformed into E. coli. The next day, the growing colonies were picked and the correct recombinant plasmids were checked by plasmid preparation, restriction analysis, and DNA-sequencing.

[0739] Example 5

[0740] Transient expression of monovalent anti-TfR antibodies

[0741] Antibodies are produced in vivo in transiently transfected HEK293 cells (derived from the human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). For transfection, the "293-Free" transfection reagent (Novagen) is used. Antibodies and antibody-based modified molecules as described above are expressed from individual expression plasmids. Transfection is performed as described in the manufacturer's instructions. Three to seven days after transfection, the cell culture supernatant containing the recombinant protein is collected. The supernatant is stored at a reduced temperature (e.g., -80 °C) until purification.

[0742] General information on the recombinant expression of human immunoglobulins in, for example, HEK293 cells is given in Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197 - 203.

[0743] Example 6

[0744] High-throughput purification of single-arm transferrin receptor antibodies

[0745] 50 mL of the clarified supernatant containing the single-arm antibody in a 96-deep well plate is loaded onto a 200 μL MabSelect SuRe column. After a washing step with PBS at pH 7.4, the protein is eluted with 2.5 mM HCl using a Tecan / Atoll-system to obtain 0.5 mL of eluate. The eluate is neutralized with 2 M Tris at pH 8. The purified protein is quantified using a Nanodrop spectrophotometer and analyzed by CE-SDS and analytical SEC under denaturing and reducing conditions. To obtain proteins with high purity (>95%), most antibodies have to be further purified on size exclusion chromatography to separate from half-antibodies, club-club antibodies, and larger aggregates. Hereinafter, using Dionex UltiMate 3000, 500 μL of the sample is injected onto a Superdex 200 10 / 300 GL in 20 mM histidine containing 140 mM NaCl at pH 6.0. This method allows the fractionation of 25 - 30 samples / day and thus enables the polishing of a large number of screening hits in single-arm mode. The fractions are combined and analyzed again as described above.

[0746] Example 7

[0747] hCMEC / D3 cell cultures for the transcytosis assay

[0748] The medium and supplements for hCMEC / D3 (Weksler, B.B. et al., FASEB J. 19 (2005), 1872 - 1874) were obtained from Lonza. hCMEC / D3 cells (passages 26 - 29) were cultured to confluence on collagen - coated coverslips (for microscopy) or in flasks in EBM2 medium containing 2.5% FBS, one - quarter of the provided growth factors, and fully supplemented with the provided hydrocortisone, gentamicin, and ascorbic acid.

[0749] For all transcytosis assays, high - density wells (1×10 8 wells / cm 2 ) PET membrane filter inserts (0.4 μm, 12 mm diameter) were used in 12 - well cell culture plates. For the apical and basolateral chambers, the calculated optimal medium volumes were 400 μL and 1600 μL, respectively. The apical chamber of the filter insert was coated with rat - tail collagen I (7.5 μg / cm 2 ), followed by fibronectin (5 μg / mL), with each incubation lasting 1 hour at RT. hCMEC / D3 cells were grown in EMB2 medium for 10 - 12 days to a confluent monolayer (approximately 2×10 5 cells / cm 2 ).

[0750] Example 8

[0751] Transcytosis assay of univalent antibodies

[0752] The entire assay was performed in serum - free EBM2 medium (otherwise reconstituted as described in Example 1). The filter insert with cells was incubated with univalent antibody (concentration: 2.67 μg / mL) in the apical side at 37 °C for 1 hour, after which the entire apical and basolateral media were collected. From these values, the paracellular flux was calculated. The monolayer was washed 3×3 - 5 min on the apical side (400 μL) and basolateral side (1600 μL) in serum - free medium at RT. All washings were collected to monitor the removal efficiency of unbound antibody. Pre - warmed medium was added to the apical chamber and the filter was transferred to a new 12 - well plate containing 1600 μL of pre - warmed medium (blocked overnight with PBS containing 1% BSA). At this point, the cells on the filter were lysed in 500 μL of RIPA buffer to determine specific antibody uptake. The remaining filter was incubated at 37 °C and samples were collected at different time points to determine the apical and / or basolateral release of the antibody. The amount of antibody in the samples was quantified using high - sensitivity IgG ELISA (see Example 3). For each time point, data were generated from three filter cell cultures.

[0753] Example 9

[0754] Sensitive IgG ELISA after transcytosis assay

[0755] The entire procedure was carried out under RT, and an automated washer was used for the washing steps. The 384-well plates were coated with 1 μg / mL anti-human / mouse-IgG (Fcγ-specific) in PBS at 30 μL / well for 2 h, followed by incubation in blocking buffer PBS containing 1% BSA or 1% CroteinC for 1 h (for human and mouse IgG assays, respectively). Serial dilutions of samples from the transcytosis assay and the standard concentrations of antibodies used in the transcytosis assay were added to the plates and incubated for 2 h. After four washes, 50 ng / mL anti-human / mouse-F(ab)2-biotin in blocking buffer was added at 30 μL / well and incubated for another 2 h. After six washes, 50 ng / mL (huIgG assay) or 100 ng / mL (mIgG assay) poly-HRP40-streptavidin (Fitzgerald; in PBS containing 1% BSA and 0.05% Tween-20) was added at 30 μL / well and incubated for 30 min. After four washes, the immune complexes were detected by adding BM chemiluminescence substrate (Roche) at 30 μL / well. The luminescence signals were measured using a luminescence plate reader, and the concentrations were calculated using a fitted standard curve. The sensitivity range of the assay was from 10 pg / mL to 10 ng / mL.

[0756] Example 10

[0757] Cell ELISA was performed with CHO cells transfected with hTfR mutants for epitope mapping

[0758] Based on: Although there is significant homology (77% identity) between human and mouse TfR, no antibody against the extracellular portion is known to show good cross-reactivity between the two orthologs. Therefore, to be able to define the epitope regions on the human transferrin receptor (hTfR), mutations were introduced into the hTfR sequence at positions where the surface-exposed amino acid clusters have different amino acids in the aligned mouse TfR sequence (see the table below). Cloning of the plasmids with the corresponding mutations was described above. To map the binding of the human TfR binder to the epitope, CHO-K1 cells were transiently transfected with the plasmids described, and antibody binding was measured in cell ELISA. Briefly, one day before the experiment, 10 4Cells were seeded in normal growth medium (RPMI / 10% FCS) in each well of a 96-well plate. On another day, the medium was changed to OPTI-MEM serum-reduced medium (Gibco), and after a 30-minute pre-incubation, 10 μL of a mixture of 1200 μL OPTI-MEM, 12 μg plasmid DNA, and 12 μL XtremeGENE transfection reagent (Roche) was added to the wells. The cells were incubated at 37 °C / 7.5% CO2 for 2 days, then the medium was removed and TfR antibody at a concentration of 1 nM to 100 nM in growth medium was added, followed by incubation at 4 °C for 2 h. Thereafter, 0.05% glutaraldehyde in PBS was substituted for the antibody solution, and the cells were fixed at RT for 15 min, then washed twice with PBS and incubated with an HRP-conjugated anti-human-Fc secondary antibody (BioRad; 1:2000 in ELISA blocking reagent (Roche)) at RT for 1.5 h. After washing 3 times with PBS, 50 μL TMB / well was used to generate a signal, and the absorbance was measured at 450 nm.

[0759]

[0760] Example 11

[0761] Surface Plasmon Resonance-based binding assay for human TfR antibody interaction

[0762] Binding experiments were performed on a BIAcore B 4000 (GE Healthcare) equipped with a C1 sensor chip (GE Healthcare, catalog number BR1005-35), where the chip was pre-treated with an anti-human Fab antibody (GE Healthcare catalog number 28-9583-25) using standard amine coupling chemistry procedures according to the supplier's manual.

[0763] For kinetic measurements, at 25 °C, in phosphate-buffered saline, pH 7.4, 0.05% Tween 20, the sample antibody was immobilized using a contact time of 60 s and a flow rate of 10 μL / min. Recombinant His6-tagged human transferrin receptor (R&D systems, catalog number 2474-TR-050) was applied at increasing concentrations, and the signal was monitored over time. The average time span of a 150-s binding time and a 600-s dissociation time at a flow rate of 30 μL / min was recorded. A 1:1 binding model (Langmuirisotherm) was used to fit the data.

[0764] Example 12

[0765] Humanization of the VH and VL domains of mouse and rabbit - anti-transferrin receptor antibodies

[0766] The non-human anti-transferrin receptor antibody was humanized as follows: Based on the characterization of the coding sequences and amino acid sequences of VH and VL domains of a non-human anti-transferrin receptor antibody of the IgG1 class with a κ light chain, and based on the human germline framework VH4_3 and VK1_10 combination used for cloning 299, the corresponding humanized anti-transferrin receptor antibody was generated by CDR grafting with reverse / forward mutations.

[0767] Example 13

[0768] Method for determining human / macaque TfR receptor affinity

[0769] In this example, a method for determining human transferrin receptor affinity for comparing dissociation behavior is listed.

[0770] For all assays, the Biotin CAPture kit from GE Healthcare (Instruction 28-9242-34AB) was used. First, the chip was rehydrated by docking it in a BIAcore T200 instrument. Thereafter, the chip was left overnight in the running buffer for standby. For surface preparation, the Biotin CAPture reagent was diluted 1:100 in the running buffer (1×PBS supplemented with 0.25 M NaCl). This solution was injected onto flow cells 1 to 4 for 360 sec at a flow rate of 2 μL / min. Next, the sensor surface was conditioned by injecting the regeneration solution provided in the Biotin CAPture kit three times for one minute each. This is mandatory for the docking procedure or for the first time or after storage. A 100 nM solution of human or macaque mono-biotinylated transferrin receptor should be injected onto flow cell 2 for 30 sec at a flow rate of 10 μL / min. For affinity determination, six concentrations (500, 250, 125, 62.5, 31.25, 15.625, and 0 nM) were injected. They were injected onto the "hu-TfR-flow cell" (e.g., Flow Cell 2 prepared as described above) with an injection time of 180 sec (binding) and a flow rate of 10 μL / min. After a 600-sec dissociation period, the surface was regenerated using the regeneration solution provided in the BiotinCAPture kit according to the manufacturer's instructions, and the next cycle was performed.

[0771] The kinetic data were evaluated using the BIAcore T200 evaluation software. In particular, after applying the 1:1 Langmuir binding model, the dissociation rate constants of different human transferrin receptor binders were considered.

[0772] Example 14

[0773] Relative Ranking of B4000 for Human / Macaque Transferrin Receptor Dissociation

[0774] According to the manufacturer's instructions, the CAP sensor chip (provided in the Biotin CAPture kit, series S#28-9202-34GE) was installed in the BIAcore B4000 system and normalized and addressed hydrodynamically. In the first cycle, the CAP reagent (provided in the kit) was addressed to spots 1, 2, 4, and 5, where a flow rate of 10 μL / min was used for 300 sec. Human transferrin receptor capture occurred at spots 1 (human transferrin receptor-biotinylated) and 5 (macaque transferrin receptor-biotinylated), where a flow rate of 10 μL / min and a contact time of 30 sec were used. These receptors were diluted to a concentration of 50 nM with running buffer (1×PBS #28995084, GE Healthcare, supplemented with 0.25 M NaCl). Antibodies were injected into all flow cells at a series of concentrations of 100 nM, 50 nM, 25 nM, and 0 nM, where a flow rate of 30 μL / min was used for 180 sec. The dissociation time was set to 300 sec. Using the regeneration solution provided in the Biotin CAPture kit, regeneration of the entire complex from the CAP chip was performed (using a flow rate of 10 μL / min for 120 sec). To control the active protein concentration, a second cycle was performed at spot 5 using biotinylated protein A (#P2165-2MG, Sigma) with a flow rate of 10 μL / min and a contact time of 30 sec. In this control cycle, spot 1 remained empty. Antibodies and regeneration were manipulated similarly to cycle 1. Relevant kinetic data were calculated using the BIAcore B4000 evaluation software. Applying a 1:1 dissociation fit, dissociation from the human transferrin receptor was determined.

[0775] Some embodiments of the present invention:

[0776] 1. A humanized antibody that specifically binds to the human transferrin receptor, wherein the antibody

[0777] in the heavy chain variable domain, contains the HVRs of SEQ ID NO: 66, 68, and 72, and

[0778] in the light chain variable domain, contains the HVRs of SEQ ID NO: 75, 76, and 78.

[0779] 2. The humanized antibody according to claim 1, comprising the heavy chain variable domain of SEQ ID NO: 24 and the light chain variable domain of SEQ ID NO: 37.

[0780] 3. The humanized antibody according to any one of claims 1 to 2, wherein the humanized antibody has effector function silenced.

[0781] 4. The humanized antibody according to any one of claims 1 to 3, wherein the humanized antibody specifically binds to the human transferrin receptor and the cynomolgus transferrin receptor.

[0782] 5. The humanized antibody according to any one of claims 1 to 4, wherein the humanized antibody is a multispecific antibody having at least one binding specificity for the human transferrin receptor and at least one binding specificity for a therapeutic target.

[0783] 6. The humanized antibody according to claim 5, wherein the humanized antibody comprises a first antigen-binding site that binds to the human transferrin receptor and a second antigen-binding site that binds to a brain antigen.

[0784] 7. The humanized antibody according to claim 6, wherein the brain antigen is selected from Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), α-synuclein, CD20, amyloid precursor protein (APP), and glucocerebrosidase.

[0785] 8. The humanized antibody according to any one of claims 5 to 7, wherein the multispecific antibody binds to:

[0786] i) the human transferrin receptor and Aβ, or

[0787] ii) the human transferrin receptor and CD20, or

[0788] iii) the human transferrin receptor and α-synuclein, or

[0789] iv) the human transferrin receptor and phospho-tau protein, or

[0790] v) the human transferrin receptor and glucocerebrosidase.

[0791] 9. The humanized antibody according to any one of claims 1 to 8, wherein the humanized antibody is a bispecific antibody that comprises

[0792] i) a first binding site comprising a heavy chain variable domain of SEQ ID NO: 24 and a light chain variable domain of SEQ ID NO: 37,

[0793] and

[0794] ii) a second binding site selected from the following

[0795] a) The heavy chain variable domain of SEQ ID NO:81 and the light chain variable domain of SEQ ID NO:82, or

[0796] b) The heavy chain variable domain of SEQ ID NO:83 and the light chain variable domain of SEQ ID NO:84, or

[0797] c) The heavy chain variable domain of SEQ ID NO:85 and the light chain variable domain of SEQ ID NO:86, or

[0798] d) The heavy chain variable domain of SEQ ID NO:87 and the light chain variable domain of SEQ ID NO:88, or

[0799] e) The heavy chain variable domain of SEQ ID NO:91 and the light chain variable domain of SEQ ID NO:92, or

[0800] f) The heavy chain variable domain of SEQ ID NO:89 and the light chain variable domain of SEQ ID NO:90, or

[0801] g) The heavy chain variable domain of SEQ ID NO:93 and the light chain variable domain of SEQ ID NO:94, or

[0802] h) The heavy chain variable domain of SEQ ID NO:79 and the light chain variable domain of SEQ...

Claims

1. A humanized antibody that specifically binds to the human transferrin receptor, wherein the antibody comprises: - A heavy chain variable domain of SEQ ID NO: 23 and a light chain variable domain of SEQ ID NO:

37.

2. The humanized antibody according to claim 1, wherein the humanized antibody is a bispecific antibody.

3. The humanized antibody according to any one of claims 1-2, wherein the humanized antibody is a) A full-length antibody of the human IgG1 subclass, or b) A full-length antibody of the human IgG4 subclass, or c) A full-length antibody of the human IgG1 subclass having mutations L234A, L235A, and P329G, or d) A full-length antibody of the human IgG4 subclass having mutations S228P, L235E, and optionally P329G, or e) A full-length antibody of the human IgG1 subclass having mutations L234A, L235A, and P329G in both heavy chains, having mutations T366W and S354C in one heavy chain, and having mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain, or f) A full-length antibody of the human IgG4 subclass having mutations S228P, L235E, and optionally P329G in both heavy chains, having mutations T366W and S354C in one heavy chain, and having mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

4. The humanized antibody according to any one of claims 1-2, wherein the humanized antibody comprises i) A homodimeric Fc-region of the human IgG1 subclass optionally having mutations P329G, L234A, and L235A, or ii) A homodimeric Fc-region of the human IgG4 subclass optionally having mutations P329G, S228P, and L235E, or iii) A heterodimeric Fc-region, wherein a) One Fc-region polypeptide comprises the mutation T366W, while the other Fc-region polypeptide comprises the mutations T366S, L368A, and Y407V, or b) One Fc-region polypeptide comprises the mutations T366W and Y349C, while the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or c) One Fc-region polypeptide comprises the mutations T366W and S354C, while the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C, or iv) A heterodimeric Fc-region of the human IgG4 subclass, wherein both Fc-region polypeptides comprise the mutations P329G, L234A, and L235A, and a) One Fc-region polypeptide comprises the mutation T366W, while the other Fc-region polypeptide comprises the mutations T366S, L368A, and Y407V, or b) One Fc-region polypeptide comprises the mutations T366W and Y349C, while the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or c) One Fc-region polypeptide contains the mutations T366W and S354C, while the other Fc-region polypeptide contains the mutations T366S, L368A, Y407V and Y349C, or v) A heterodimeric Fc-region of the human IgG4 subclass, wherein both Fc-region polypeptides contain the mutations P329G, S228P and L235E, and a) One Fc-region polypeptide contains the mutation T366W, while the other Fc-region polypeptide contains the mutations T366S, L368A and Y407V, or b) One Fc-region polypeptide contains the mutations T366W and Y349C, while the other Fc-region polypeptide contains the mutations T366S, L368A, Y407V and S354C, or c) One Fc-region polypeptide contains the mutations T366W and S354C, while the other Fc-region polypeptide contains the mutations T366S, L368A, Y407V and Y349C.

5. A pharmaceutical preparation comprising a humanized antibody according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. Use of an antibody according to any one of claims 1 to 4 in the manufacture of a medicament for transporting one or more compounds across the BBB.

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