Anti-transferrin receptor antibodies with tailored affinity

Abstract

Herein is reported an anti-transferrin receptor antibody that specifically binds to a human transferrin receptor and a macaque 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 antibody has a dissociation rate for the human transferrin receptor equal to or less than (i.e., at most) the dissociation rate of the anti-transferrin receptor antibody 128.1 for the macaque transferrin receptor wherein the dissociation rate is determined by surface plasmon resonance, and the 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. 202111224085.8, filed on June 22, 2016, with the invention name “Anti-transferrin receptor antibodies with customized affinity”. Field of the Invention

[0002] The present invention relates to an anti-transferrin receptor antibody with a designed dissociation rate against human transferrin receptor and use thereof as a blood-brain barrier shuttling module. Background Art

[0003] Brain penetration of drugs for neurological disorders (e.g., large biotherapeutics or small molecule drugs with low brain permeability) is severely limited by the extensive and impermeable blood-brain barrier (BBB) ​​and other cellular components in the neurovascular unit (NVU). Many strategies to overcome this obstacle have been tested, and one of them is to utilize the transcytosis pathway mediated by endogenous receptors expressed on the brain capillary endothelium (blood-brain barrier receptors). Recombinant proteins, such as monoclonal antibodies or peptides, have been designed to antagonize these receptors, enabling receptor-mediated delivery of biotherapeutics to the brain. 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 that lead to degradation of biotherapeutics) remain undeveloped.

[0004] Monoclonal antibodies and other biotherapeutics have enormous 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 (approximately 0.1%) of injected IgG in the bloodstream is able to penetrate the CNS compartment (Felgenhauer, Klin. Wschr. 52 (1974) 1158-1164). Due to the low concentration of antibodies in the CNS, this will certainly limit any pharmacological effect.

[0005] It was previously discovered that the percentage of antibodies partitioning into the CNS can be increased by utilizing BBB receptors (ie, transferrin receptor, insulin receptor, etc.) (see, eg, WO 95 / 02421).

[0006] Therefore, there is a need for neurological disorder drug delivery systems that cross the BBB to effectively shuttle drugs into the brain.

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

[0008] In WO 2014 / 189973, anti-transferrin receptor antibodies and methods of use are reported. It is further reported that targeting BBB receptors with traditional specific high-affinity antibodies generally results in a limited increase in BBB transport. It was later found that in the anti-BBB antibodies studied, the antibodies were absorbed into the CNS and the number of levels distributed in the CNS was negatively correlated with their binding affinity to BBB receptors. For example, low-affinity antibodies to transferrin receptor (TfR) greatly improved the BBB transport and CNS retention of anti-TfR antibodies when administered at therapeutic dose levels relative to higher affinity anti-TfR antibodies, which makes it easier to obtain therapeutic concentrations in the CNS (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43). Using bispecific antibodies that bind both TfR and amyloid precursor protein (APP) splitting enzyme (β-secretase (BACE1)), evidence of the BBB transport was obtained. Compared with monospecific anti-BACE1 alone, a single systemic administration of a bispecific anti-TfR / BACE1 antibody engineered with a low-affinity antibody not only resulted in significant antibody uptake in the brain, but also significantly reduced brain Aβ1-40 levels, 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 increasing complexity of antibody engineering and the variability caused by the diversity of recombinant production cell systems for antibody production, it is very important to conduct further thorough non-clinical safety evaluations for monoclonal antibodies (mAbs) intended for therapeutic applications. In addition, in addition to the concerns caused by 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 safety considerations (Lynch, CM et al., mAbs 1 (2009) 2-11; Kim, SJ 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 being studied and provide information for product development. The main goals of nonclinical evaluation are (1) to identify the target organs of toxicity and determine whether the toxicity is reversible after treatment, (2) to identify a safe starting dose for use in 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 product labels. To achieve these goals, in vitro and in vivo nonclinical studies aimed at defining and understanding the pharmacological properties of antibodies are performed (Lynch, CM et al., mAbs 1 (2009) 2-11; Cavagnaro, JA, in: Cavagnaro, JA (ed.) "Preclinical safety evaluation of biopharmaceuticals"; Hoboken, NJ: Wiley 2008; 45-65).

[0011] For successful nonclinical safety evaluation of mAbs, the most relevant animal species should be selected for toxicity testing (Lynch, CM et al., mAbs 1 (2009) 2-11; Chapman, K. et al., Nat. Rev. Drug Discov. 6 (2007) 120-126). The relevant species is one in which the antibody has pharmacological activity and in which the target antigen should be present or expressed, and the tissue cross-reactivity profile should be similar to that of humans (Lynch, CM 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 JA (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205; Hall, WC et al., In: Cavagnaro, JA (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240). Using immunochemical or functional assays, relevant animal species that express the desired epitope and exhibit tissue cross-reactivity characteristics similar to those of human tissues can be identified (Lynch, CM et al., mAbs 1 (2009) 2-11; Hall, WC et al., In: Cavagnaro, JA (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 multispecies tissue microarrays (Lynch, CM et al., mAbs 1 (2009) 2-11; Hall, WC et al., In: Cavagnaro, JA (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240).Alternatively, antibody binding to cells from these animals can be assessed by flow-activated cell sorting (FACS), which is generally more sensitive than immunohistochemical analysis of tissue sections (Lynch, CM et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., In: Cavagnaro JA (eds); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205). DNA and amino acid sequences of the target antigen should be compared across species; homology between species should be determined (Lynch, CM et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., In: Cavagnaro JA (eds); 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 evaluation of toxicity caused by antibody binding to the target antigen, which is called on-target toxicity (Lynch, CM et al., mAbs 1 (2009) 2-11; 19, 20). In addition, the strong similarity in the tissue distribution of the target antigen between animal species and humans makes it more likely that the target organs of toxicity identified in animals will predict potential toxicity in humans. The lack of similarity in the tissue distribution of the antigen between animal species and humans does not completely exclude the use of the animal species for toxicity studies, but these differences must be taken into account for human risk assessment. As for antigen density or affinity, absolute equivalence between animal models and humans is similarly not required. A rationale for the relevance of the species chosen for toxicity testing should be included in the regulatory submission. If only one species is used for safety evaluation, the experimental summary will need to state that no other relevant species are present (Lynch, CM et al., mAbs 1 (2009) 2-11)

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

[0014] Therefore, in order to identify an animal model for a specific mAb, the above considerations must be made. However, the mAb in question must have cross-reactivity with the target antigen in the test species. Otherwise, even the most suitable test species cannot be used. Therefore, there is a need for mAbs that do not have intraspecies cross-reactivity but have interspecies cross-reactivity for their targets in humans and the species intended for nonclinical trials.

[0015] In EP 2 708 560, antibodies that specifically recognize transferrin receptor are reported. In FR 2 953 841, antibodies against 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 blood-brain barrier shuttling modules to deliver brain effector entities across the blood-brain barrier to the brain. In certain embodiments, the blood-brain barrier shuttling module is a monovalent binding entity that specifically binds to the transferrin receptor. When used as a blood-brain barrier shuttling 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] Reported herein are anti-transferrin receptor antibodies that specifically bind to human transferrin receptor (huTfR) and macaque transferrin receptor (cyTfR). In certain embodiments, the anti-transferrin receptor antibodies

[0018] Binds to human transferrin receptor (huTfR) and macaque transferrin receptor (cyTfR);

[0019] has an off-rate from human transferrin receptor that is equal to or less than (i.e., is at most) the off-rate of anti-transferrin receptor antibody 128.1 from macaque transferrin receptor, where the off-rate is determined by surface plasmon resonance and the 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] • Binds to the human transferrin receptor with an off-rate of 0.1 1 / s to 0.005 1 / s, inclusive.

[0021] One aspect as reported herein is an anti-transferrin receptor antibody specifically binding to human transferrin receptor and macaque transferrin receptor comprising

[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 antibody has an off-rate for human transferrin receptor that is equal to or less than (i.e., at most) the off-rate of anti-transferrin receptor antibody 128.1 for macaque transferrin receptor,

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

[0026] The 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 off-rate for human transferrin receptor is between 0.1 1 / s and 0.005 1 / s, inclusive.

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

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

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

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

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

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

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

[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 (numbering according to Kabat).

[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 (numbering according to Kabat).

[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 as reported herein is an anti-transferrin receptor antibody specifically binding to human transferrin receptor (huTfR) comprising

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

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

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

[0043] In one embodiment, the off-rate for human transferrin receptor is between 0.1 1 / s and 0.005 1 / s, inclusive.

[0044] One preferred aspect as reported herein is an anti-transferrin receptor antibody specifically binding to human transferrin receptor (huTfR) comprising

[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 of all aspects, the antibody is a multispecific antibody having at least one binding specificity for 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 to transferrin receptor and a second antigen binding site that binds to 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 to:

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

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

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

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

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

[0053] vi) Both transferrin receptor and glucocerebrosidase.

[0054] 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 a human Aβ binding site.

[0055] 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 a replacement of the amino acid residue at Kabat position 11 with any amino acid other than leucine. In one embodiment, the replacement comprises a replacement of the amino acid residue at Kabat position 11 with a non-polar amino acid. In a preferred embodiment, the replacement comprises a replacement 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 the group consisting of valine, leucine, isoleucine, serine, and phenylalanine.

[0056] 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.

[0057] 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.

[0058] 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.

[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 SEQ ID 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 a binding site 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 a deletion of at least one amino acid residue 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 of all aspects the antibody comprises

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

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

[0066] iii) a 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) a heterodimeric Fc-region of 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) a heterodimeric Fc-region of 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 comprises the mutations T366W and S354C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

[0080] In one embodiment of all aspects the antibody is a CrossMab.

[0081] One aspect as reported herein is an anti-transferrin receptor antibody comprising

[0082] i) a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57 and 58 and a light chain variable domain selected from the group consisting of SEQ ID NOs: 60, 61, 62 and 63, or

[0083] ii) a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 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 the group consisting of SEQ ID NOs: 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 as reported herein is a pharmaceutical formulation comprising an antibody as reported herein and a pharmaceutically acceptable carrier.

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

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

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

[0088] One aspect as reported herein is the use of an antibody as 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 to anti-pTau antibody mAb86; 3: 567; 4: 932; 5: 567 fused to 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 embodiments of the invention

[0093] Herein we report a humanized variant of the rabbit antibody 299 which shows a high transcytosis activity in a transcytosis assay according to Example 8, is cross-reactive with the human and macaque transferrin receptor, i.e. binds specifically to both orthologues of transferrin, shows good cell staining and has a similar half-life (reflected by the dissociation rate) as the murine antibody 128.1 for both the macaque transferrin receptor and the human transferrin receptor.

[0094] One aspect as reported herein is a humanized antibody specifically binding to human transferrin receptor, wherein the antibody comprises HVRs of SEQ ID NOs: 66, 68 and 72 in the heavy chain variable domain and HVRs of SEQ ID NOs: 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 SEQ ID NO:37.

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

[0097] In one embodiment, the humanized antibody specifically binds to human transferrin receptor and 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 the mutations L234A, L235A and P329G,

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

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

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

[0105] One aspect as reported herein is a bispecific antibody comprising

[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 the group consisting of

[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 as reported herein is a pharmaceutical formulation comprising an antibody as reported herein and optionally a pharmaceutically acceptable carrier.

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

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

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

[0120] One aspect as reported herein is a method of treatment comprising administering an antibody as reported herein for the treatment of a neurological disorder.

[0121] I. Definition

[0122] For the purposes of the present invention, an "acceptor 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 derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or may contain amino acid sequence changes. 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 acceptor human framework sequence is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0123] "Affinity" refers to the total 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 a 1:1 interaction between a binding pair member (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a 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 hereinafter.

[0124] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alterations.

[0125] The term "antibody" 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] "Antibody fragments" are molecules other than intact antibodies that comprise a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; 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 possessed by its heavy chain. There are five major antibody classes: 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 called α, δ, ε, γ, and μ, respectively.

[0129] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the 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 (e.g., B cell receptor); and B-cell activation.

[0130] An effective amount of a pharmaceutical agent (eg, a pharmaceutical formulation) is an amount effective, at dosages and for durations necessary, to achieve the desired therapeutic or prophylactic result.

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

[0132] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in VH (or VL): 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 to refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having heavy chains that comprise an Fc-region as defined herein.

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

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

[0136] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. 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) represents an antibody that has undergone humanization.

[0137] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are highly variable in sequence ("complementarity determining regions" or "CDRs") and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0138] The HVRs in this article 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, AM, J. Mol. Biol. 196 (1987) 901-917);

[0140] (b) 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, EA 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) antigenic contact 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) A combination of (a), (b) and / or (c), comprising 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 indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to the Kabat EU index numbering system (Kabat et al., supra).

[0144] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating 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 the components of its natural environment. An isolated nucleic acid includes a 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 that is different from its natural chromosomal location.

[0147] The term "monoclonal antibody" as used herein means the antibody derived from a substantially homogeneous antibody colony, that is, except for the possible variant antibodies (for example, variant antibodies containing natural mutations or produced in the production process of monoclonal antibody products) that are usually present in a very small amount, each antibody constituting the colony is identical and / or in conjunction with identical epi-positions. Different from the polyclonal antibody products that usually include different antibodies for different determinants (epi-positions), each monoclonal antibody of the monoclonal antibody products is directed to a determinant on the antigen. Thus, the modifier "monoclonal" represents that the antibody derives from the feature of a substantially homogeneous antibody colony, and should not be construed as needing to produce the antibody by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by various techniques, including, but not limited to, hybridoma method, recombinant DNA method, phage display method, and the method using all or part of the transgenic animals comprising human immunoglobulin locus, and these methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0148] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N- to C-, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-, each light chain has a variable region (VL), also referred to as a variable light domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain.

[0149] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or cautions concerning the use of the therapeutic product.

[0150] "Percentage (%) 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, if necessary, gaps are introduced to achieve maximum sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in a variety of ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. 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, DC, 20559, along with user documentation, where it is registered 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 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 do not change.

[0151] When ALIGN-2 is used for amino acid sequence comparison, 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 be alternatively expressed as a given amino acid sequence A having or comprising a particular % 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 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 will be understood that 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 to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0154] The term "pharmaceutical formulation" means a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no other components that are unacceptably toxic to a subject to which the formulation is to be administered.

[0155] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is 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 grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of the disease or slow the progression of the disease.

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

[0158] As used herein, the term "vector" 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 a host cell into which they are 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 a physiological barrier between peripheral circulation and the brain and spinal cord, which is formed by the tight junctions in the cerebrospinal membrane of the brain capillary endothelial cells, forming a tight barrier that limits molecules, or even very small molecules (such as urea (60 daltons)), to be transported to 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 in the CNS, and are collectively referred to herein as blood-brain barrier or BBB. The BBB also encompasses blood-CSF barrier (choroid plexus), wherein the barrier is composed of ependymal cells rather than capillary endothelial cells.

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

[0161] The term "blood-brain barrier receptor" (BBBR) refers to a receptor protein connected to the extracellular membrane of cells expressed on brain endothelial cells, which can transport molecules across the BBB, or can be used to transport molecules applied exogenously. Examples of BBBR include, but are not limited to, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptor (LDL-R), 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). Exemplary BBBR is transferrin receptor (TfR).

[0162] The term "brain effector entity" refers to a molecule that is transported across the BBB. Effector entities typically possess a desired characteristic therapeutic activity to be delivered to the brain. Effector entities include neurological disorder drugs 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 that can bind to the BBBR specifically and in a monovalent binding mode. The blood-brain shuttle module and / or conjugate as reported herein is characterized in that there is a single unit of a monovalent binding entity, that is, the blood-brain shuttle module and / or conjugate of the present invention contains only one unit of a 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, for example, single-chain Fab, scFv. The monovalent binding entity can, for example, be a scaffold protein engineered using existing technologies (such as phage display or immunization). The monovalent binding entity can also be a polypeptide. 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 coupled 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 BBBR, wherein the interaction between the monovalent binding entity and BBBR is through a single epitope. This monovalent binding mode can prevent any dimerization / multimerization of BBBR due to the single epitope interaction point. The monovalent binding mode prevents alteration of the intracellular sorting of BBBR.

[0165] The term "epitope" refers to any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitopic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen to which an antibody binds.

[0166] "Transferrin receptor" (TfR) is a transmembrane glycoprotein (having a molecular weight of about 180,000 Da) composed of two subunits bound by a disulfide bond (each subunit has an apparent molecular weight of about 90,000 Da) and is involved in iron absorption in vertebrates. In one embodiment, the TfR herein is a 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 its presence and / or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds that have associated labeling entities that allow detection.

[0168] The terms "CNS antigens" and "brain targets" 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 neurotrophic factor receptor (p75NTR), glucocerebrosidase, and caspase 6.

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

[0170] As used herein, the term "CH2-CH3 Ig entity" refers to a protein entity derived from the CH2 or CH3 domain of an immunoglobulin. A "CH2-CH3 Ig entity" comprises two "CH2-CH3" polypeptides that form a dimer. The immunoglobulin may 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 native sequence and variant CH2-CH3 domains of the CH2-CH3 domain. In one embodiment, the "CH2-CH3 Ig entity" is derived from a human heavy chain CH2-CH3 IgG domain that extends from Cys226 or 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, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, 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 links the different entities of the blood-brain barrier shuttle module and / or fusion polypeptide and / or conjugate reported herein. The linker can, for example, link the brain effector entity to a monovalent binding entity. For example, if the monovalent binding entity comprises a CH2-CH3 Ig entity and a scFab for a blood-brain barrier receptor, 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 connected by peptide bonds can be used. In certain embodiments, the amino acids are selected from 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 of at least 25 amino acid residues in length, and in a preferred embodiment, has a length of 32 to 50 amino acid residues. In one embodiment, the peptide linker is a (GxS)n linker, wherein 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, 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] Various chemical linkers can be used for conjugation. For example, various bifunctional protein coupling agents can be used to conjugate monovalent binding entities or fusion polypeptides and brain effector entities, 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), diazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). The linker can be a "cleavable linker" that facilitates release of the effector entity after delivery to the brain. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52 (1992) 127-131; US ​​5,208,020) can be used.

[0175] Covalent conjugation can be direct or via a linker. In certain embodiments, direct conjugation is to construct a polypeptide fusion (for example, two genes encoding a monovalent binding entity and an effector entity for BBBR and expressed as a single polypeptide (chain)). In certain embodiments, direct conjugation is to form a covalent bond between a reactive group on one of the two parts of the monovalent binding entity for BBBR and a corresponding group or receptor on the brain effector entity. In certain embodiments, direct conjugation is to modify (i.e., genetically modify) one of the two molecules to be conjugated to include a reactive group (as a non-limiting example, a sulfhydryl group or a carboxyl group), which forms a covalent bond with another molecule to be conjugated under suitable conditions. As a non-limiting example, a molecule (i.e., amino acid) with a desired reactive group (i.e., a cysteine ​​residue) can be introduced into a monovalent binding entity for example for a BBBR antibody and form a disulfide bond with a neuropharmaceutical. Methods for covalently conjugating nucleic acids to proteins are also known in the art (ie, photocrosslinking, see, for example, Zatsepin et al., Russ. Chem. Rev. 74 (2005) 77-95). Conjugation can also be performed using a variety of linkers. For example, various bifunctional protein coupling agents can be used to conjugate monovalent binding entities and effector entities, 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), diazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active 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 release of the effector entity after delivery to the brain. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker 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) is often used to describe molecular interactions. It is used as a measure of the strength (e.g., affinity) of the interaction between two molecules. Thus, K D The value is a measure of the strength of biomolecular interactions.

[0178] But K D The value does not describe the dynamics of the molecular interactions, i.e., on the one hand, the K D The value cannot be used to infer how quickly two molecules bind to each other (the binding rate constant or "on rate"), and on the other hand, the K D The K value does not allow for inference about how quickly the molecule dissociates (the dissociation rate constant or "off-rate"). D The use of K values ​​to characterize biomolecular interactions will ignore the fact that the same K D The value can be composed of very different (orders of magnitude) association and dissociation rates because K D The value is the ratio of the association rate to the dissociation rate.

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

[0180] In order to have a long-lasting (i.e., low-frequency dosing requirements) or customized (e.g., based on ambient conditions) interaction, the off-rate must be determined experimentally. This is even more important since it is almost impossible to predict the off-rate. In addition, as mentioned above, the correlation between off-rate and binding affinity is poor. For example, since K D The value is the ratio of the on-rate to the off-rate; even weak binders can remain bound to their target for extended periods, whereas strong binders dissociate rapidly.

[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 performed without prior antigen labeling and using crude bacterial lysates. Ylera, F. et al. (Anal. Biochem. 441 (2013) 208-213) reported an off-rate screening method for selecting high-affinity anti-drug antibodies in crude E. coli lysates containing monovalent Fab fragments. They selected the off-rate as the sorting parameter because the off-rate is concentration-independent. They chose a monovalent format to avoid the influence of affinity (avidity) during the off-rate sorting and affinity determination process (affinity influence will be observed when using complete IgG). Ylera et al. have found that clones with the best koff-rate were identified by the koff sorting step, while the clone would not be identified using only ELISA signal intensity as the selection criterion.

[0182] Murray, JB 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 (dissociation rate screening (ORS) by surface plasmon resonance is an effective method to kinetically sample hit to lead chemical space from unpurified reaction products). It summarizes that the dissociation rate constant kd (dissociation rate) is the most significant factor in ligand-protein binding that may enhance the efficacy of a compound. The authors point out that kinetic measurements of affinity provide more information than steady-state affinity balance measurements throughout a 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 balance 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, and similarly, data measured with an older BIAcore T100 instrument had a 15% difference. When comparing between instruments and time, the authors observed an average difference of only 30% in kd. This demonstrates that carryover, long-term storage, and different equipment have a moderate effect 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 of 14% to 40% has been reported (Murray, JB et al., J. Med. Chem. 57 (2014) 2845-2850; Katsamba, PS et al., Anal. Biochem. 352 (2006) 208-221).

[0183] In WO 2014 / 189973, anti-transferrin receptor antibodies and methods of use are reported. It is further reported that targeting BBB receptors with traditional specific high-affinity antibodies generally results in a limited increase in BBB transport. It was later found that, in the anti-BBB antibodies studied, the number of levels of antibody absorption into the CNS and distribution in the CNS is negatively correlated with its binding affinity to BBB receptors. For example, low-affinity antibodies to transferrin receptor (TfR) administered at therapeutic dose levels, relative to higher affinity anti-TfR antibodies, greatly improve the BBB transport and CNS retention of anti-TfR antibodies, and make it easier to reach therapeutic concentrations in the CNS (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43). Using bispecific antibodies that combine both TfR and amyloid precursor protein (APP) splitting enzyme (β-secretase (BACE1)), proof of such BBB transport was obtained. A single systemic dose of a bispecific anti-TfR / BACE1 antibody engineered with a low-affinity antibody not only resulted in significant antibody uptake in the brain, but also significantly reduced brain Aβ1-40 levels compared to monospecific anti-BACE1 alone, suggesting 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).

[0184] Available data and experiments indicate several causal mechanisms behind the improved antibody uptake into the CNS using a low-affinity antibody approach.

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

[0186] Second, it was proposed that the lower affinity of the antibody for the BBB-R would impair 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 the antibody on the CNS side of the BBB is nonsaturating due to the rapid diffusion of the antibody into the CNS compartment.

[0187] Third, in vivo and as observed for the TfR system, antibodies with lower affinity for the BBB-R will not be cleared from the system as effectively as antibodies with higher affinity for the BBB-R, and therefore will retain higher circulating concentrations than their higher affinity counterparts. This is advantageous because circulating antibody levels of lower affinity antibodies will be maintained at therapeutic levels for a longer period of time than higher affinity antibodies, thereby improving the absorption of antibodies in the brain for a longer period of time. Further, this improvement in both plasma and brain exposure can reduce 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 therapeutic compounds bound thereto.

[0188] These previous studies utilized mouse antibodies that specifically bind to mouse TfR but do not specifically recognize primate or human TfR. Therefore, 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 of antibody engineering and the variability caused by the diversity of recombinant production cell systems used for antibody production, a thorough nonclinical safety evaluation of monoclonal antibodies (mAbs) intended for therapeutic applications is very important. In addition, in addition to the problems caused by the long-term clinical use of mAbs to treat chronic diseases, the complex structure, unique biological functions and longer half-life of mAbs compared to traditional small molecule drugs also increase safety considerations (Lynch, CM et al., mAbs 1 (2009) 2-11; Kim, SJ et al., Mol. Cells 20 (2005) 17-29).

[0190] The overall goal of nonclinical studies for mAbs is to determine the toxicological properties of the mAb being studied and provide information for product development. The main goals of nonclinical evaluation are (1) to identify the target organs of toxicity and determine whether the toxicity is reversible after treatment, (2) to identify a safe starting dose for use in 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 product labels. To achieve these goals, in vitro and in vivo nonclinical studies aimed at defining and understanding the pharmacological properties of antibodies are performed (Lynch, CM et al., mAbs 1 (2009) 2-11; Cavagnaro, JA, in: Cavagnaro, JA (ed.) "Preclinical safety evaluation of biopharmaceuticals"; Hoboken, NJ: Wiley 2008; 45-65).

[0191] For successful nonclinical safety evaluation of mAbs, the most relevant animal species should be selected for toxicity testing (Lynch, CM et al., mAbs 1 (2009) 2-11; Chapman, K. et al., Nat. Rev. Drug Discov. 6 (2007) 120-126). The relevant species is one in which the antibody has pharmacological activity and in which the target antigen should be present or expressed, and the tissue cross-reactivity profile should be similar to that of humans (Lynch, CM 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 JA (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205; Hall, WC et al., In: Cavagnaro, JA (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240). Using immunochemical or functional assays, relevant animal species that express the desired epitope and exhibit tissue cross-reactivity characteristics similar to those of human tissues can be identified (Lynch, CM et al., mAbs 1 (2009) 2-11; Hall, WC et al., In: Cavagnaro, JA (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 multispecies tissue microarrays (Lynch, CM et al., mAbs 1 (2009) 2-11; Hall, WC et al., In: Cavagnaro, JA (ed.); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 207-240).Alternatively, antibody binding to cells from these animals is assessed by flow-activated cell sorting (FACS), which is generally more sensitive than immunohistochemical analysis of tissue sections (Lynch, CM et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., In: Cavagnaro JA (eds); Preclinical safety evaluation of biopharmaceuticals. Hoboken, NJ: Wiley 2008; 181-205). DNA and amino acid sequences of the target antigen should be compared across species; homology between species should be determined (Lynch, CM et al., mAbs 1 (2009) 2-11; Subramanyam, M. and Mertsching, E., In: Cavagnaro JA (eds); 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 called on-target toxicity (Lynch, CM et al., mAbs 1 (2009) 2-11; 19, 20). In addition, the strong similarity in the tissue distribution of the target antigen between animal species and humans makes it more likely that the target organ of toxicity identified in animals will predict potential toxicity in humans. The lack of similarity in the tissue distribution of the antigen between animal species and humans does not completely exclude the use of the animal species in toxicity studies, but these differences must be considered when conducting human risk assessments. As for antigen density or affinity, absolute equivalence between animal models and humans is similarly not required. A rationale for the relevance of the species chosen for toxicity testing should be included in the regulatory submission. If only one species is used for safety evaluation, the experimental summary will need to state that no other relevant species are present (Lynch, CM et al., mAbs 1 (2009) 2-11).

[0193] If the monoclonal antibody that is intended to be used for therapeutic purposes does not have species cross reactivity, then surrogate antibodies or different species must be used for model.Therefore, surrogate antibodies are a potential solution to limited safety test (safety test is limited and is possible when using the humanized monoclonal antibody with species cross reactivity of restriction).But, there is not yet at present the clear and definite standard (Regulatory Toxicology and Pharmacology Volume 40, Issue 3, December 2004, 219-226 page) that should be used for judging this potential surrogate antibody before the safety problem of potential surrogate antibody is used to determine clinical drugs.

[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 in question must have cross-reactivity with the target antigen in the test species. Otherwise, even the most suitable test species cannot be used. Therefore, there is a need for mAbs that do not have intra-species cross-reactivity but have inter-species cross-reactivity for their targets in humans and the species intended for non-clinical trials.

[0195] A. Exemplary Anti-Transferrin Antibodies

[0196] Reported herein is anti-transferrin receptor antibody, this antibody has the dissociation rate in a certain range for binding to human transferrin receptor, to ensure suitable BBB transport.It has been found that one end of this range is limited by the dissociation rate of mouse anti-transferrin receptor antibody 128.1 (the variable domain amino acid sequence provided in SEQ ID NO:64 and 65) measured for macaque transferrin receptor by surface plasmon resonance, and the other end is limited by 5% of this dissociation rate (that is, 20 times of dissociation slowly).In one embodiment, the dissociation rate for human transferrin receptor is between 0.1 1 / s to 0.005 1 / s (including 0.1 1 / s and 0.005 1 / s).

[0197] By using standard humanization technology, the humanized antibody of clone 229 reported herein can not be obtained.It is necessary to introduce non-standard sudden change in amino acid sequence, to obtain the humanized antibody with transferrin receptor binding dissociation rate (comprising 0.1 1 / s and 0.005 1 / s) in the expected range.This is particularly important because the antibody reported herein is to develop for the treatment of payload transported to the brain across the human blood-brain barrier.

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

[0199] It was further found to be advantageous to change the 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 showed comparable properties to anti-transferrin receptor antibody 128.1. This can be seen in the table below.

[0202]

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

[0204]

[0205]

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

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

[0208]

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

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

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

[0212] In the table below, the off-rates of a humanized variant of the murine light chain variable domain of clone 494 in combination with a humanized variant of the murine heavy chain variable domain of clone 494 are shown. The binding partner is the human transferrin receptor.

[0213]

[0214] In more detail, in one aspect, the present invention is based in part on the discovery that the anti-transferrin receptor antibodies reported herein can be used as blood-brain barrier shuttling modules to deliver brain effector entities across the blood-brain barrier into the brain. In certain embodiments, the blood-brain barrier shuttling 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 shuttling modules, for example, for the diagnosis or treatment of neurological disorders such as Alzheimer's disease, Parkinson's disease, and co-morbidities of Alzheimer's disease and Parkinson's disease.

[0215] It was found that an antibody comprising the heavy chain variable domain of SEQ ID NO: 24 and the light chain variable domain of SEQ ID NO: 37 mirrored the binding properties of murine antibody 128.1 relative to macaque transferrin receptor with respect to association and dissociation rates relative to human transferrin receptor.

[0216] Thus, one aspect as reported herein is an isolated antibody that binds to human transferrin receptor (huTfR) and macaque transferrin receptor (cyTfR), wherein said antibody has an off-rate towards human transferrin receptor of 0.1 to 0.005 1 / s as determined by surface plasmon resonance.

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

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

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

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

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

[0222] In one embodiment, the off-rate is from 0.08 1 / s to 0.008 1 / s.

[0223] In one embodiment of all aspects the off-rate is determined at 25°C.

[0224] In one embodiment of all aspects the off-rate is the off-rate measured at 25°C.

[0225] One aspect as reported herein is an anti-transferrin receptor antibody specifically binding to human transferrin receptor (huTfR) and macaque 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 (numbering according to Kabat).

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

[0227] In one embodiment, the antibody further has a serine residue (S) at position 65 in the heavy chain variable domain (numbering 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 as reported herein is an anti-transferrin receptor antibody that specifically binds to human transferrin receptor (huTfR) and macaque 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 an off-rate for human transferrin receptor in units of 1 / s that is equal to or less than (i.e. is at most) the off-rate of anti-transferrin receptor antibody 128.1 towards macaque transferrin receptor in units of 1 / s, wherein the off-rate is determined by surface plasmon resonance, and wherein the 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 an off-rate for human transferrin receptor in 1 / s that is i) equal to or less than (i.e., at most) the off-rate for anti-transferrin receptor antibody 128.1 for macaque transferrin receptor in 1 / s, and ii) equal to or greater than (i.e., at least 5% of the off-rate for anti-transferrin receptor antibody 128.1 for macaque transferrin receptor in 1 / s.

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

[0232] Binds to human transferrin receptor (huTfR) and macaque transferrin receptor (cyTfR);

[0233] has an off-rate in 1 / s for human transferrin receptor that is equal to or less than (i.e., at most) the off-rate in 1 / s of anti-transferrin receptor antibody 128.1 for macaque transferrin receptor, wherein the off-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 an off-rate of 0.1 1 / s to 0.005 1 / s, inclusive.

[0235] In one aspect, provided herein is an anti-transferrin receptor antibody comprising 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 VH HVR sequences selected from the group consisting of: (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 group consisting of: (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 group consisting of: (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 comprising the amino acid sequence of SEQ ID NO: 71, 72, or 73. In another embodiment, the antibody comprises HVR-H3 and HVR-L3, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 71, 72, or 73, and HVR-L3 comprising 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 comprising the amino acid sequence of SEQ ID NO: 71, 72, or 73, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78, and HVR-H2 comprising 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 the group consisting of: (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 75; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78. In one embodiment, the antibody comprises: (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 75; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78.

[0238] In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (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 the group consisting of: (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 present 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 an acceptor human framework, eg, 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 has a dissociation rate that is approximately 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 to the transferrin receptor with 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 substitution, insertion or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antibody transferrin receptor antibody comprises the VH sequence of SEQ ID NO: 24, including post-translational modifications of the sequence. In some embodiments, VH comprises one, two or three HVRs 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.

[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 that is approximately 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 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 with 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 HVR (i.e., in the FR). Optionally, the antibody 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 the following: (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 the VH of any one of the embodiments provided above and the VL of any one 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 another aspect of the present invention, the anti-transferrin receptor antibody according to any one of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-transferrin receptor antibody is an antibody fragment, for example, Fv, Fab, Fab', scFv, diabody or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, for example, complete IgG1 antibody or other antibody classes or isotypes, 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 label.

[0247] In another embodiment, the antibody is a multispecific antibody and the therapeutic compound optionally forms a 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 neurotrophic factor receptor (p75NTR), glucocerebrosidase and caspase 6. In another embodiment, the multispecific antibody binds to both TfR and BACE1. In another embodiment, the multispecific antibody binds to both TfR and Aβ. In another embodiment, the multispecific antibody binds to both TfR and α-synuclein. In another embodiment, the multispecific antibody binds to both TfR and CD20. In another embodiment, the multispecific antibody binds to both TfR and glucocerebrosidase. In another embodiment, the therapeutic compound is a neurological disorder drug.

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

[0249] In an aspect of the above embodiments, the invention provides any one of the antibodies described above for use as a medicament.

[0250] In one aspect of the above embodiments, the invention provides the use of any of the above antibodies in the preparation of a medicament 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 disorders, and CNS inflammation.

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

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

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

[0254] In one aspect of the above embodiment, a method for transporting a compound across the BBB in a subject is provided, comprising exposing any one of the antibodies to the BBB so that the antibody can transport the compound coupled thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody coupled 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 that is calibrated to minimize acute clinical symptoms of antibody administration.

[0255] In another aspect of the above embodiment, there is provided a method of increasing the exposure of the CNS of a subject to a compound, comprising exposing any of the previous antibodies to the BBB so that the antibody can transport the compound coupled thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody coupled 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 that is calibrated to minimize acute clinical symptoms of antibody administration.

[0256] In one aspect of the above embodiment, there is provided a method for increasing the retention of a compound administered to a subject in the CNS, comprising exposing any of the previous antibodies to the BBB, thereby increasing the retention of the compound in the CNS. In one embodiment, the antibody coupled 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 that is calibrated to minimize acute clinical symptoms of antibody administration.

[0257] In one aspect of the above embodiments, there is provided a method for treating a neurological disorder in a mammal, comprising treating a mammal with any of the previous antibodies. 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 disorders and CNS inflammation. In one embodiment, the neurological disorder is in a human subject. In one embodiment, the antibody coupled 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 that is 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 group consisting of: effector function of the antibody Fc region, complement activation function of the antibody, and 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 the group consisting of reducing 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 the group consisting of: producing the antibody in an environment that does not allow wild-type glycosylation; removing carbohydrate groups already present on the antibody; and modifying the antibody so that wild-type glycosylation does not occur.

[0264] In one embodiment, glycosylation of the antibody is reduced by producing the antibody in an environment that does not allow wild-type glycosylation, such as producing the antibody in a non-mammalian cell production system or producing the antibody synthetically. 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 the antibody is reduced by modifying the antibody so that wild-type glycosylation does not occur, such as by including a mutation at position 297 of 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, the effector function is reduced or eliminated by modifying at least one Fc region. In one embodiment, the effector function or complement activation function is reduced or eliminated by deleting all or part of the Fc region, or by engineering the antibody so that it does not include an Fc region or a non-Fc region that can cause effector function or complement activation function. In one embodiment, at least one Fc region modification is selected from: an Fc region point mutation that impairs binding to one or more Fc receptors selected from the following positions: 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, 30 335, 338, 340, 373, 376, 382, ​​388, 389, 414, 416, 419, 434, 435, 437, 438, and 439; an Fc region point mutation that impairs C1q binding selected from the group consisting of: 270, 322, 329, and 321; elimination of 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 that impairs C1q binding selected from the group consisting of: 270, 322, 329, and 321. In another embodiment, the modification is elimination of some or all of the Fc region. In another embodiment, complement priming function is reduced or eliminated by deleting all or a portion of the Fc region, or by engineering the antibody so that it does not include an Fc region involved in the complement pathway. In one embodiment, the antibody is selected from a Fab or a single-chain antibody. In another embodiment, the non-Fc region of the antibody is modified to reduce or eliminate activation of the complement pathway by the antibody. In one embodiment, the modification is a point mutation that impairs the CH1 region that binds 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 embodiment, the affinity of the antibody for TfR is reduced, as measured relative to a wild-type antibody of the same isotype that does not have reduced affinity for TfR. In one such aspect, the antibody has a K of about 1 pM to about 100 μM for TfR. D or IC 50 .

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

[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 human IgG1 subclass having the mutations L234A, L235A and P329G,

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

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

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

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

[0277] In an aspect of the above embodiment, the invention provides any antibody hereinbefore for use as a medicament.

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

[0279] In another aspect of the above embodiment, the present invention provides the use of any of the above antibodies for treating a neurological disorder. In one embodiment, the neurological disorder is selected from the group consisting of neuropathy, neurodegenerative disease, cancer, eye disease, epilepsy, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorders and CNS inflammation.

[0280] In another aspect of the above embodiment, the invention provides any of the antibodies hereinbefore described for use in transporting one or more compounds across the BBB.

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

[0282] In one aspect of the above embodiment, a method for transporting a compound across the BBB in a subject is provided, comprising exposing any one of the antibodies to the BBB so that the antibody can transport the compound coupled thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody coupled 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 that is calibrated to minimize acute clinical symptoms of antibody administration.

[0283] In another aspect of the above embodiment, there is provided a method of increasing the exposure of the CNS of a subject to a compound, comprising exposing any of the previous antibodies to the BBB so that the antibody can transport the compound coupled thereto across the BBB. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody coupled 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 that is calibrated to minimize acute clinical symptoms of antibody administration.

[0284] In one aspect of the above embodiment, there is provided a method for increasing the retention of a compound administered to a subject in the CNS, comprising exposing any of the previous antibodies to the BBB, thereby increasing the retention of the compound in the CNS. In one embodiment, the BBB is in a human subject. In one embodiment, the antibody coupled 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 that is calibrated to minimize acute clinical symptoms of antibody administration.

[0285] In one aspect of the above embodiments, there is provided a method for treating a neurological disorder in a mammal, comprising treating a mammal with any of the previous antibodies. 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 disorders and CNS inflammation. In another such aspect, the neurological disorder is in a human subject. In one embodiment, the antibody of the conjugated 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 that is 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 for optimizing the pharmacokinetics and / or pharmacodynamics of a compound effective in the CNS of a subject is provided, wherein the compound is conjugated to an antibody that binds to TfR with low affinity, wherein the antibody is selected such that its affinity for TfR after conjugation to the compound results in an amount of transport of the compound-conjugated antibody across the BBB that optimizes the pharmacokinetics and / or pharmacodynamics of the compound in the CNS.

[0288] In yet another aspect, the anti-transferrin receptor antibody according to any of the above aspects and embodiments may incorporate any of the individual properties or combinations thereof described in Sections 1-5 below.

[0289] 1. Antibody affinity

[0290] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed using a Fab form of the antibody of interest and its antigen. For example, by titrating the antibody against a minimal concentration of ( 125 The solution binding affinity of Fab for antigen was measured by equilibrating Fab with I)-labeled antigen and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). To establish the conditions for the assay, 5 μg / mL anti-Fab capture antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) was used to coat the plate. Multiwell plates (Thermo Scientific) were blocked overnight and then 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 The antigen is mixed with serially diluted Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12, see Presta, LG et al., Cancer Res. 57 (1997) 4593-4599). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 in PBS. Wash the plate eight times. After the plate is dry, add 150 μL / well scintillant (MICROSCINT-20 TM ; Packard), and then place the plate on the TOPCOUNT TM The concentrations of each Fab that produced less than or equal to 20% of maximal binding were selected for use in the competition binding assay.

[0291] According to another embodiment, using Surface plasmon resonance assay to measure Kd. For example, using -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) with immobilized antigen CM5 chip, tested at 25 ° C with ~ 10 response units (RU). In one embodiment, 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. 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 coupled protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, 24 h at 25 ° C with a flow rate of approximately 25 μL / min in a solution containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant-free PBS (PBST), and two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected. By simultaneously fitting the association and dissociation sensorgrams, a simple one-to-one Langmuir binding model ( Evaluation software version 3.2), and the binding rate (k on ) and dissociation rate (k off ). According to k off / k on The equilibrium dissociation constant (KD) was calculated from the ratio (see, for example, Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). 6 M -1 s -1 , the binding rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in the presence of increasing concentrations of antigen in PBS, pH 7.2 at 25° C., as can be measured, for example, in a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an 8000-series SLM-AMINCO with a stirred cuvette. TM Spectrophotometer (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, as well as other fragments described below. For a review of certain antibody fragments, see Hudson, PJ 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, Rosenburg 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 comprising salvage receptor binding epitope residues and having increased in vivo half-life, see US 5,869,046.

[0294] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 0 404 097; WO 1993 / 01161; Hudson, PJ et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat. Med. 9 (2003) 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, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516).

[0296] As described herein, 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 (eg, E. coli or phage).

[0297] 3. Chimeric and humanized antibodies

[0298] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US 4,816,567; and Morrison, SL et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, a chimeric antibody comprises 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 another example, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0299] In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while maintaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR (e.g., CDR) (or part thereof) is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies optionally can also include at least a portion of human constant region. In some embodiments, some FR residues in humanized antibodies can be replaced with corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0300] Humanized antibodies and methods for their preparation are reviewed in, for example, Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and further described in, for example, Riechmann, I. 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, SV et al., Methods 36 (2005) 25-34 (describing specificity determining region (SDR) grafting); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, WF 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 the “guided selection” method of FR shuffling).

[0301] The 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, MJ et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived 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, LG et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (19969 22611-22618).

[0302] 4. Multispecific Antibodies

[0303] In certain embodiments, the antibody provided herein is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In certain embodiments, one of the binding specificities is for transferrin receptor, and the other is for any other antigen. Bispecific antibodies can also be used for cytotoxic agents to be positioned in cells expressing transferrin receptor. Bispecific antibodies can be prepared as full-length antibodies or antibody fragment forms.

[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, AC, Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" engineering (see, e.g., US 5,731,168). Electrostatic steering effects can also be engineered to prepare antibody Fc-heterodimers to form multispecific antibodies (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., US 4,676,980 and Brennan, M. et al., Science 10 (1991) 3655-3659); and the like. 229 (1985) 81-83); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology for making 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 making 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-acting Fabs" or "DAFs," which contain an antigen binding site that binds to transferrin receptor as well as another, different antigen (see, eg, US 2008 / 0069820).

[0307] The antibodies or fragments herein also include the 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 as reported herein the anti-transferrin receptor antibody is a bispecific antibody.

[0309] One aspect as reported herein is a bivalent, bispecific antibody comprising

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

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

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

[0313] The antibody of a) does not contain the modification described in b), and the heavy and light chains of a) are separated 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 said 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 said 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 (numbering according to Kabat) is substituted 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 (numbering according to the Kabat EU index) is substituted by a negatively charged amino acid,

[0322] or

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

[0324] In a preferred embodiment

[0325] i) in the constant domain CL of the first light chain of a), the amino acid at position 124 (numbering according to Kabat) is independently substituted by lysine (K), arginine (R) or histidine (H) (in a preferred embodiment independently substituted by lysine (K) or arginine (R)), 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 (numbering according to the Kabat EU index) is independently substituted by glutamic acid (E) or aspartic acid (D),

[0326] or

[0327] ii) in the constant domain CL of the second light chain of b), the amino acid at position 124 (numbering according to Kabat) is independently substituted by lysine (K), arginine (R) or histidine (H) (in a preferred embodiment independently substituted by lysine (K) or arginine (R)), and wherein in the constant domain CH1 of the second heavy chain of b), the amino acid at position 147 or the amino acid at position 213 (numbering according to the Kabat EU index) is independently substituted 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 substituted by K (numbering according to the 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 substituted by E (numbering according to the 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 substituted by K, and in the constant domain CH1 of the first heavy chain, the amino acids at positions 147 and 213 are substituted by E (numbering according to the 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 substituted by K and in the constant domain CH1 of the second light chain the amino acids at positions 147 and 213 are substituted by E, and in the variable domain VL of the first light chain the amino acid at position 38 is substituted by K, the amino acid at position 39 in the variable domain VH of the first heavy chain is substituted by E, the amino acid at position 38 in the variable domain VL of the second heavy chain is substituted by K, and the amino acid at position 39 in the variable domain VH of the second light chain is substituted by E (numbering according to the Kabat EU index).

[0332] One aspect as reported herein is a bivalent, bispecific antibody comprising

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

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

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

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

[0337] In the antibody of b),

[0338] In the light chain

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

[0340] and

[0341] In the heavy chain

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

[0343] One aspect as reported herein is a bivalent, bispecific antibody comprising

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

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

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

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

[0348] In the antibody of b),

[0349] In 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] In the heavy chain

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

[0354] One aspect as reported herein is a multispecific antibody comprising

[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 further antigens (i.e. the second and / or third and / or fourth and / or fifth antigen, preferably specifically bind to one further antigen, i.e. the second antigen),

[0357] wherein the single-chain Fab fragment in b) is fused to the C- or N-terminus of the heavy chain or light chain of the full-length antibody in a) via a peptide linker,

[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 binding to a second antigen are fused to the full length antibody at the C-terminus of the heavy or light chain of the full length antibody via a peptide linker.

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

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

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

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

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

[0365] One aspect as reported herein is a trivalent, bispecific antibody comprising

[0366] 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

[0367] b) a first polypeptide consisting of:

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

[0369] or

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

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

[0372] c) a second polypeptide consisting of

[0373] ca) 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 at the N-terminus of the VL domain via a peptide linker,

[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 to the second antigen,

[0379] and

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

[0381] In one embodiment, the antibody heavy chain variable domain (VH) of the polypeptide of b) and the antibody 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:

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

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

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

[0385] Techniques for introducing non-natural 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, the trivalent, bispecific antibody preferably lacks the optional disulfide-stabilized domains VH and VL of the single-chain Fab fragment.

[0386] One aspect as reported herein is a trispecific or tetraspecific antibody comprising

[0387] a) a first light chain and a first heavy chain of a full-length antibody that specifically binds to 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 to 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) wherein one to four antigen-binding peptides that specifically bind to one or two other antigens (i.e., bind to a third and / or fourth antigen) are fused to the C-terminus or N-terminus of the light or heavy chain of a) and / or b) via a peptide linker,

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

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

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

[0393] In one embodiment, the antigen binding peptide is selected from the group consisting of a scFv fragment and a scFab fragment.

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

[0395] In one embodiment, the antigen binding peptide is a 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 tetraspecific antibody comprises under c) one or two antigen-binding peptides that specifically bind to one further antigen.

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

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

[0400] One aspect as reported herein is a bispecific, tetravalent antibody comprising

[0401] a) 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 further Fab fragments of an antibody that specifically binds to a second antigen, wherein said further Fab fragments are both fused to the C- or N-terminus of the heavy chain of a) via a peptide linker,

[0403] and

[0404] In the Fab fragment, the following modifications have been made

[0405] i) in the two Fab fragments of a), or in the two Fab fragments of b), the variable domains VL and VH are replaced by one another, and / or the constant domains CL and CH1 are replaced by one another,

[0406] or

[0407] ii) in the two Fab fragments of a), the variable domains VL and VH are replaced by one another, and the constant domains CL and CH1 are replaced by one another,

[0408] and

[0409] In the two 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 the two 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 the two 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 the two Fab fragments of a), the variable domains VL and VH are substituted for one another, and in the two Fab fragments of b), the constant domains CL and CH1 are substituted for one another,

[0416] or

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

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

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

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

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

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

[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 one another,

[0424] and / or

[0425] The constant domains CL and CH1 replace each other.

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

[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 replace each other.

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

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

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

[0433] i) in the two Fab fragments of b), the variable domains VL and VH are substituted for one another,

[0434] and / or

[0435] The constant domains CL and CH1 replace each other.

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

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

[0438] One aspect as reported herein is a bispecific, trivalent antibody comprising

[0439] a) a (modified) heavy chain of a first antibody, which specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein the N-terminus of a 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 the first antibody of a),

[0441] c) a (modified) heavy chain of a second antibody, which 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 the second antibody of c), each comprising a CL-CH1 domain pair,

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

[0444] One aspect as reported herein is a bispecific antibody comprising

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

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

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

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

[0449] One aspect as reported herein is a bispecific antibody comprising

[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) specifically binds to a second antigen and comprises VH 2 Domain and VL 2 domains, wherein the two domains are linked to each other via a disulfide bridge,

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

[0453] The first antigen or the second antigen is 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 The other of the domains is not fused via a peptide linker to the heavy or light chain of the full-length antibody that specifically binds to the first antigen.

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

[0457] One aspect as reported herein is a bispecific trivalent antibody comprising

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

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

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

[0461] wherein the C-termini of the CH1 domains of the two Fab fragments are connected to the N-termini 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] The first antigen or the second antigen is human transferrin receptor.

[0464] One aspect as reported herein is a bispecific trivalent antibody comprising

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

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

[0467] c) an Fc-region comprising 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 connected 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] The first antigen or the second antigen is human transferrin receptor.

[0471] One aspect as reported herein is a bispecific antibody comprising

[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) specifically binds to a second antigen and comprises VH 2 Domain and VL 2 A Fab fragment of a structural domain, comprising a heavy chain fragment and a light chain fragment, wherein

[0474] In the light chain fragment

[0475] Variable light chain domain VL 2 The variable heavy chain domain VH of the antibody 2 Alternative,

[0476] and

[0477] Within the heavy chain fragment

[0478] Variable heavy chain domain VH 2 The variable light chain domain VL of the antibody 2 Alternative,

[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 is paired with the heavy chain of the full-length antibody into which the heavy chain Fab fragment is inserted, and

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

[0481] One aspect as reported herein is a bispecific antibody comprising

[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) specifically binds to a second antigen and comprises VH 2 Domain and VL 2 A Fab fragment of a structural domain, comprising a heavy chain fragment and a light chain fragment, wherein

[0484] In the light chain fragment

[0485] Variable light chain domain VL 2 The variable heavy chain domain VH of the antibody 2 Alternative,

[0486] and

[0487] Within the heavy chain fragment

[0488] Variable heavy chain domain VH 2 The variable light chain domain VL of the antibody 2 Alternative,

[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 is paired with the heavy chain of the full-length antibody to which the heavy chain fragment of the Fab fragment is conjugated, and

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

[0491] In one embodiment of all aspects the antibody as reported herein is a multispecific antibody which requires heterodimerization of at least two heavy chain polypeptides and wherein the antibody specifically binds 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. Typically, in methods known in the art, the CH3 domain of a first heavy chain and the CH3 domain of a second heavy chain are engineered in a complementary manner such that a heavy chain comprising one engineered CH3 domain no longer homodimerizes with another heavy chain of the same structure (e.g., a CH3-engineered first heavy chain no longer homodimerizes with another CH3-engineered first heavy chain; and a CH3-engineered second heavy chain no longer homodimerizes with another CH3-engineered second heavy chain). Thus, a heavy chain comprising one engineered CH3 domain is forced to heterodimerize with another heavy chain comprising a complementary engineered CH3 domain. 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 for supporting heavy chain heterodimerization cited and included above, which are known in the art, can be considered as different alternatives for use in the multispecific antibodies according to the present invention, which comprise a "non-exchanged Fab region" derived from a first antibody that specifically binds to a first antigen, and a "crossed Fab region" derived from a second antibody that specifically binds to a second antigen, and are combined with the specific amino acid replacements described above for the present invention.

[0494] The CH3 domains of the multispecific antibodies reported herein can be modified using the "knob-into-hole" technique, described in detail with several examples in, for example, WO 96 / 027011, Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681. In this approach, the interaction surfaces of the two CH3 domains are modified to enhance heterodimerization of the two heavy chains containing them. One of the two CH3 domains (of the two heavy chains) can be a "knob" and the other a "hole." The introduction of disulfide bridges can further stabilize the heterodimer (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and improve the yield.

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

[0496] Other knob-in-hole technologies described in EP1870459A1 may be used instead or in addition. In one embodiment, the multispecific antibody as reported herein comprises R409D and K370E mutations in the CH3 domain of the "knob chain" and 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 antibody as reported herein comprises a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A and Y407V mutations in the CH3 domain of the "hole chain", and additionally R409D and K370E mutations in the CH3 domain of the "knob chain" and 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 antibody as reported herein comprises Y349C and T366W mutations in one of the two CH3 domains and S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains, or the multispecific antibody as reported herein comprises Y349C and T366W mutations in one of the two CH3 domains and S354C, T366S, L368A and Y407V mutations in the other of the two CH3 domains and additionally R409D and K370E mutations in the CH3 domain of the “knob chain” and 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-in-hole technique," other techniques for modifying the heavy chain CH3 domain of multispecific antibodies to enhance heterodimerization are known in the art. These techniques, particularly 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, are contemplated herein as alternatives to the "knob-in-hole technique" for use in the multispecific antibodies described herein.

[0500] In one embodiment of the multispecific antibody as reported herein, the method described in EP 1 870 459 is used to support heterodimerization of the first and second heavy chains of the multispecific antibody. This method is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3-domain interface between the first and second heavy chains.

[0501] Thus, this embodiment relates to a multispecific antibody as 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 between the CH3 domains of the respective antibodies, wherein the amino acid sequence of each of the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain comprises a set of amino acids located within the 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 of one heavy chain is substituted 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 of the other heavy chain is substituted 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 that the amino acids with opposite charges are introduced into the respective CH3 domains).

[0502] In one embodiment of the CH3(+ / -) engineered multispecific antibody as 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 as 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 as 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 as reported herein, in the CH3 domain of one heavy chain, amino acid R at position 409 is substituted by D and amino acid K at position 409 is substituted by E, and in the CH3 domain of the other heavy chain, amino acid D at position 399 is substituted by K and amino acid E at position 357 is substituted by K (numbering according to the Kabat EU index).

[0506] In one embodiment of the multispecific antibody reported herein, the method described in WO 2013 / 157953 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of the multispecific antibody 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 the Kabat EU index). In another embodiment of the multispecific antibody 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 the Kabat EU index).

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

[0508] In one embodiment of the multispecific antibody reported herein, the method described in WO 2012 / 058768 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one embodiment of the multispecific antibody 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 the Kabat EU index). In another embodiment, in addition to the above replacements, in the CH3 domain of the other heavy chain, 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) is replaced (numbering according to the Kabat EU index). Preferred replacements are:

[0509] - the amino acid T at position 411 is substituted by an amino acid selected from the group consisting of N, R, Q, K, D, E and W (numbering according to the KabatEU index),

[0510] - the amino acid D at position 399 is substituted by an amino acid selected from the group consisting of R, W, Y and K (numbering according to the Kabat EU index),

[0511] - the amino acid S at position 400 is substituted by an amino acid selected from the group consisting of E, D, R and K (numbering according to the Kabat EU index),

[0512] - the amino acid F at position 405 is substituted by an amino acid selected from the group consisting of I, M, T, S, V and W (numbering according to the Kabat EU index),

[0513] - the amino acid N at position 390 is substituted by an amino acid selected from the group consisting of R, K and D (numbering according to the Kabat EU index); and

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

[0515] In another embodiment of the multispecific antibody as 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 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 V and the amino acid K at position 409 is replaced by F (numbering according to the Kabat EU index). In another embodiment of the multispecific antibody as reported herein, in the CH3 domain of one heavy chain, 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 the Kabat EU index). In the last embodiment mentioned above, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is replaced by E, the amino acid T at position 411 is replaced by E, the amino acid D at position 399 is replaced by R and the amino acid S at position 400 is replaced by R (numbering according to the Kabat EU index).

[0516] In one embodiment of the multispecific antibody as reported herein, the method described in WO 2011 / 143545 is used to support heterodimerization of the first and second heavy chains of the multispecific antibody. In one embodiment of the multispecific antibody as 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 antibody reported herein, the method described in WO 2011 / 090762 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. WO 2011 / 090762 relates to amino acid modifications according to the "knob-in-hole" technique. In one embodiment of the CH3 (KiH)-engineered multispecific antibody 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 antibody 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 antibody as reported herein, which is of IgG2 isotype, the method described in WO 2011 / 090762 is used to support heterodimerization of the first and the second heavy chain of the multispecific antibody.

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

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

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

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

[0523] In one embodiment of all aspects as 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 as reported herein, the multispecific antibody has the constant domain structure of an IgG type antibody. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass, or of human IgG1 subclass with mutations L234A and L235A. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG2 subclass. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG3 subclass. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG4 subclass, or of human IgG4 subclass with the additional mutation S228P. In another embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass or human IgG4 subclass. In a further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass with mutations L234A and L235A (numbering according to the Kabat EU index). In a further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG1 subclass with mutations L234A, L235A and P329G (numbering according to the Kabat EU index). In a further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG4 subclass with mutations S228P and L235E (numbering according to the Kabat EU index). In a further embodiment of all aspects as reported herein, the multispecific antibody is characterized in that the multispecific antibody is of human IgG4 subclass with mutations S228P, L235E and P329G (numbering according to the Kabat EU index).

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

[0526] 5. Antibody variants

[0527] In certain embodiments, the 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. The amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, the disappearance of residues in the amino acid sequence of the antibody, and / or insertion and / or displacement. Any combination of disappearance, insertion and displacement can be performed to obtain the final construct, as long as the final construct has desired characteristics (for example, antigen binding).

[0528] a) Substitution, insertion and deletion variants

[0529] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The 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 can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0530] Table 1

[0531] Initial residue Exemplary substitutions Conservative substitutions 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 that affect chain direction: Gly, Pro;

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

[0539] Non-conservative substitutions will entail exchanging a member of one of these classes for another.

[0540] One type of substitutional variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study has a change (e.g., improvement) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or substantially retains certain biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which can be conveniently produced, for example, using affinity maturation techniques based on phage display, as described herein. In short, one or more HVR residues are mutated, the variant antibody is displayed on phage, and screened for a specific biological activity (e.g., binding affinity).

[0541] Changes (e.g., substitutions) can be made in HVR, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots", i.e., residues encoded by codons that undergo high-frequency mutations during somatic mutation (see, e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196), and / or residues that contact antigens, to test the binding affinity of the resulting variant VH or VL. Affinity maturation is performed by constructing a second library and then selecting, as described in, e.g., Hoogenboom, HR, et al., Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity mutations, 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 variant with the desired affinity. Another approach to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. For example, using alanine scanning mutagenesis or modeling, HVR residues involved in antigen binding can be specifically identified. CDR-H3 and CDR-L3 are particularly commonly targeted.

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

[0543] A kind of useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called " alanine scanning mutagenesis ", as described in Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085.In this method, identification residue or one group of target residues (for example, charged residue, such as Arg, Asp, His, Lys and Glu) and replace with neutral or negatively charged amino acid (for example, alanine or polyalanine), to determine whether to affect the interaction of antibody and antigen.Other displacements can be introduced at the amino acid position that initial displacement demonstrates functional sensitivity.Alternatively, or additionally, the crystal structure of antigen-antibody complex can be used to identify the contact point between antibody and antigen.Such contact residues and adjacent residues can be targeted or eliminated as the candidate for displacement.Variants can be screened to determine whether they contain required characteristics.

[0544] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or to 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 increased or decreased levels of antibody glycosylation. Addition or deletion of glycosylation sites to an 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 attached thereto can be altered. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, generally linked to Asn297 of the CH2 domain of the Fc region via an N-link (see, e.g., Wright.A and Morrison SL, TIBTECH 15: (1997) 26-32). Oligosaccharides can include a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

[0548] In one embodiment, an antibody variant is provided that has a sugar structure lacking fucose attached to the Fc region (directly or indirectly). For example, the amount of fucose in the antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose can be determined by MALDI-TOF mass spectrometry, by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297, 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 antibodies, Asn297 may also be located approximately ±3 amino acid positions upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have improved ADCC function. See, for example, US2003 / 0157108; US2004 / 0093621. Published examples of "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 0845 70; 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 defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (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 CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (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] Also provided are antibody variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. These antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of these antibody variants are described in, for example, WO 2003 / 011878; US 6,602,684; and US 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. These antibody variants may have improved CDC function. These antibody variants are described in, for example, 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 an antibody provided herein to thereby generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0552] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them ideal candidates for certain applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. The primary cells that mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression 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 assays for evaluating ADCC activity of target molecules are described in US Pat. No. 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 Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays can be used (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). Suitable effector cells for these assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes, R. et al., Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656. C1q binding assays can also be performed to demonstrate that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ 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, e.g., Petkova, SB et al., Int'l. Immunol. 18 (2006) 1759-1769).

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

[0554] Certain antibody variants with improved or decreased FcR binding have been described (see, eg, US 6,737,056; WO 2004 / 056312 and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0555] In certain embodiments, the antibody variant comprises an Fc-region with one or more amino acid substitutions that increase ADCC, eg, substitutions 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), e.g., as described in US 6,194,551, WO 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0557] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593 and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005 / 0014934. These antibodies comprise an Fc-region with one or more substitutions that improve binding of the Fc-region to FcRn. Such Fc variants include those with 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, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0558] For other examples of Fc-region variants, see also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; US Pat. No. 5,648,260; US Pat. No. 5,624,821; and WO 94 / 29351.

[0559] d) Cysteine-engineered antibody variants

[0560] In certain embodiments, it may be desirable to produce cysteine ​​engineered antibodies, such as "thio MAbs," in which one or more residues of an antibody are replaced by cysteine ​​residues. In some embodiments, the replaced residues appear 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 produce 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 produced 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 in the art and readily available. Suitable moieties 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 / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have a preparation advantage 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, it may be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically for a defined condition, 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 by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength, including, but not limited to, a wavelength that does not harm normal cells but can heat the non-protein moiety to a temperature that is capable of killing cells located near the antibody-non-protein moiety.

[0564] B. Blood-brain barrier shuttle module

[0565] In one embodiment of all aspects, the antibody is a multispecific antibody having at least one binding specificity for 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 to transferrin receptor and a second antigen binding site that binds to 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 to:

[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, 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 a replacement of the amino acid residue at Kabat position 11 with any amino acid other than leucine. In one embodiment, the replacement comprises replacing the amino acid residue at Kabat position 11 with a non-polar amino acid. In a preferred embodiment, the replacement 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.

[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 a 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 and light chain variable domains of SEQ ID NO: 34 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.

[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 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: 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 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: 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 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: 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 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: 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 a 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] A monovalent binding entity that specifically binds to a blood-brain barrier receptor may have the following characteristics with respect to its binding and transcytosis properties:

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

[0590] - as a monovalent binding entity, efficient in vivo transcytosis,

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

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

[0593] i) the amount of monovalent binding entity taken up into the cells during the loading phase,

[0594] ii) basolateral amount of monovalent binding entities 4 h after loading and washing;

[0595] iii) apical amount of monovalent bound entities 4 h after loading and washing;

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

[0597] v) Total amount of monovalent binding entities 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 shuttling module reported herein, the anti-transferrin receptor antibody (e.g., as a monovalent binding entity) must i) be taken up by hCMEC / D3 cells (endocytosis), ii) be transported to the outside of hCMEC / D3 cells (exocytosis), and iii) be stable inside hCMEC / D3 cells (no or low trafficking to endosomes for degradation).

[0599] Thus, in one embodiment, in an hCMEC / D3 based assay, the monovalent binding entity is characterized by i) (substantial) uptake into hCMEC / D3 cells during a one hour loading period, ii) release to the apical and / or basolateral compartments after the loading period and wash steps and within 4 hours after washes, and iii) a low (intracellular) degradation rate.

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

[0601] It has been found that in order to be able to function as a monovalent binding entity for the blood-brain barrier shuttling module reported herein, the monovalent binding entity must exhibit the following thresholds in the hCMEC / D3-based assay described above:

[0602] i) the amount of monovalent binding entity taken up into the cells during the loading phase is 400 pg or more,

[0603] ii) 4 hours after loading and washing, the amount of monovalent binding entity on the substrate side is 100 pg or more, and

[0604] iii) 4 hours after loading and washing, the amount of the apical side of the monovalent binding entity is 150 pg or more.

[0605] The mouse anti-human transferrin receptor antibody 128.1 (for variable region sequences, see WO93 / 10819 and SEQ ID NOs: 64 and 65) can be used as a reference. In this case, in order to be able to serve as a monovalent binding entity for the blood-brain barrier shuttle module reported herein, the monovalent binding entity must show the following thresholds in the above-mentioned hCMEC / D3-based assay:

[0606] i) during the loading phase, the amount of monovalent binding entity taken up into the cells is 60% or more of the loaded amount of antibody 128.1,

[0607] ii) 4 hours after loading and washing, the basolateral content of the monovalent binding entity is 60% or more of the basolateral content of antibody 128.1, and

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

[0609] The hCMEC / D3 based assay (which is one embodiment of all aspects reported herein) was performed as follows.

[0610] Culture medium and supplements for hCMEC / D3 (see WO 2006 / 056879 and Weksler, BB et al., FASEB J. 19 (2005) 1872-1874) can be obtained from Lonza. hCMEC / D3 cells (passage 26-29) will / can be cultured to confluence on collagen-coated coverslips (microscope) or in flasks in EBM2 medium containing 2.5% FBS, one-fourth the supply of growth factors and fully supplemented with the supply of hydrocortisone, gentamicin and ascorbic acid.

[0611] For all transcytosis experiments, high-density wells (1 × 10 8 Holes / cm 2 ) PET membrane filter inserts (0.4 μm pore size, 12 mm diameter) are used / can be used in 12-well cell culture plates. Calculate the culture medium volumes to be 400 μL and 1600 μL for the apical and basolateral chambers, respectively. Rat tail collagen I (7.5 μg / cm 2 ) Then, the apical chamber of the filter insert was coated with fibronectin (5 μg / mL) and incubated at RT for 1 h. hCMEC / D3 cells were grown in EBM2 medium for 10-12 days to a confluent monolayer (~2×10 5 cells / cm 2 Before the assay, the 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] Assays were performed in serum-free EBM2 medium (or reconstituted as described herein) (for assay protocols, 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 receptors. Filter inserts with or without cells (but blocked overnight in complete culture medium) were incubated at 37°C on the apical side with the monoclonal antibody in question (monovalent binding entity) for 1 hour. The monolayer was washed three times at room temperature (RT) in serum-free medium on the apical side (400 μL) and the basolateral side (1600 μL), each for 3-5 min. Pre-warmed culture medium was added to the apical chamber, and the filter was transferred to a new 12-well plate (blocked overnight with PBS containing 1% BSA) containing 1600 μL of pre-warmed culture medium. At this point, the filter with or without cells was lysed in 500 μL RIPA buffer to determine the absorption of the specific antibody (monovalent 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 (monovalent binding entity). A highly sensitive IgG ELISA can be used to quantify the antibody content in the sample (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., a light chain and / or a heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., an expression vector) 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 a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). 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 expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0615] For the recombinant production of anti-transferrin receptor antibodies, the nucleic acid of the separated encoding antibody, for example nucleic acid as above, and inserted in one or more vectors for further cloning and / or expressing in a host cell. This nucleic acid can be easily separated and ordered using conventional procedures (for example, by using oligonucleotide probes that can be combined with the gene specificity of the encoding antibody weight and light chain).

[0616] Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in E. coli.) After expression, the antibodies can be separated from the bacterial cell paste in a soluble fraction and can be 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 fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech (2006) 24: 210-215.

[0618] Suitable host cells for expression of glycosylated antibodies 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, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing plant cells for producing antibodies in transgenic plants). TM technology).

[0620] Vertebrate cells can also be used as hosts. For example, suspension-grown adapted mammalian cell lines can be useful. Other examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293 cells, e.g., as described in Graham, FL, et al., J. Gen Virol. 36 (1997) 59); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells, as described in Mather, JP, Biol. Reprod. 23 (1980) 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, e.g., as described in Mather, JP, et al., Annals NY 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, AM, Methods in Molecular Biology, Vol. 248, Lo. BKC (ed.), Humana Press, Totowa, NJ (2004) pp. 255-268.

[0621] D. Experiment

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

[0623] 1. Binding Test

[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 blot, antibody or reverse phase arrays, and the like.

[0625] In an exemplary ELISA or αLISA assay, transferrin receptor in a solution (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 transferrin receptor or a specific conformation of transferrin receptor) and a detection antibody coupled 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 (chemiluminescence, fluorescence, energy transfer-induced luminescence, etc.).

[0626] In the case of antibody arrays, the antibody is dotted on a glass or nitrocellulose chip. Blocked and incubated with a solution containing transferrin receptor, the slide is washed to remove unbound antibodies, and the antibodies bound are detected with fluorescently labeled corresponding second antibodies. Fluorescence signal is measured by a fluorescent slide scanner. Similarly, for reversed-phase arrays, recombinant transferrin receptor, cell supernatant, cell or tissue lysate, body fluid, etc. are dotted on a glass or nitrocellulose chip. The slide is sealed and the array is incubated with antibodies targeting specific epitopes on the transferrin receptor. Unbound antibodies are washed off, and the antibodies bound are detected with fluorescently labeled corresponding second antibodies. Fluorescence signal is measured by a fluorescent 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 "detection" 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 diagnosis or detection methods. 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 as described herein under conditions allowing the anti-transferrin receptor antibody to bind to transferrin receptor, and detecting whether a complex has been 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 anti-transferrin receptor antibodies, for example, when transferrin receptor is a biomarker for patient selection.

[0630] Exemplary conditions that can be diagnosed using the antibodies of the invention include neurodegenerative 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, a labeled anti-transferrin receptor antibody is provided. Labels include, but are not limited to, markers or moieties that can be directly detected (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that can be indirectly detected (e.g., by enzymatic reactions or molecular interactions), such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes. 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 Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, enzymes coupled to the oxidation of dye precursors such as HRP using hydrogen peroxide, lactoperoxidase or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, etc.

[0632] F. Pharmaceutical Preparations

[0633] Pharmaceutical formulations of the anti-transferrin receptor antibodies described herein can be prepared, for example, by mixing the antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Science, 16th edition, Osol, A. (ed.) (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients 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 octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polyols. Peptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinyl pyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; 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 herein further include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use thereof, including rhuPH20, are described in US2005 / 0260186 and US2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, 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 as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

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

[0637] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, 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 therapeutic methods. In one aspect, an anti-TfR antibody is provided for use as a drug. For example, the present invention provides a method for transporting a therapeutic compound across the blood-brain barrier, the method comprising exposing an anti-TfR antibody (e.g., a multispecific antibody that binds both TfR and a brain antigen) coupled to a therapeutic compound to the BBB so that the antibody transports the therapeutic compound coupled thereto across the BBB. In another example, the present invention provides a method for transporting a neurological disorder drug across the blood-brain barrier, the method comprising exposing an anti-TfR antibody of the present invention (e.g., a multispecific antibody that binds both TfR and a brain antigen) coupled to a brain disorder drug to the BBB so that the antibody transports the neurological disorder drug coupled thereto across the BBB. In one embodiment, the BBB is in a mammal (e.g., a human), for example, a mammal (e.g., a human) with a 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), an eye disease or disorder, a viral or microbial infection, inflammation (e.g., of the CNS or brain), ischemia, a neurodegenerative disease, epilepsy, behavioral disorders, lysosomal storage disorders, etc. The antibodies of the invention are particularly useful for treating these neurological disorders because they are able to transport one or more attached active ingredients / conjugated therapeutic compounds across the BBB into the CNS / brain, where the molecular, cellular, or viral / microbial basis of these disorders resides. Neuropathies are diseases or disorders of the nervous system characterized by inappropriate or uncontrolled nerve signaling or absent signaling, and include, but are not limited to, chronic pain (including nociceptive pain), pain caused by damage to body tissues, including cancer-related pain, neuropathic pain (pain caused by abnormalities in the nerves, spinal cord, or brain), and psychogenic pain (related entirely or largely to a psychological disorder), headaches, migraines, neuropathies, and the symptoms and syndromes that often accompany such neuropathies, such as vertigo or nausea.

[0641] For neuropathy, neurological medications of choice include analgesics, including but not limited to narcotic / opioid analgesics (i.e., morphine, fentanyl, hydrocodone, meperidine, methadone, oxymorphone, pentazocine, propoxyphene, tramadol, codeine, and oxycodone), nonsteroidal anti-inflammatory drugs (NSAIDs) (i.e., ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketorolac, mefenamic acid), and acetaminophen. acid, meloxicam, nabumetone, oxaprozin, piroxicam, sulindac, and tolmetin), corticosteroids (i.e., cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone, and triamcinolone), antimigraine agents (i.e., sulindac, oxaprozin, piroxicam, sulindac, and tolmetin), matriptin, 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

[00135] In some embodiments, the present invention includes but is not limited to: 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 neuropathy with vertigo, the neurological drugs that can be used include anti-vertigo agents, which include but are not limited to meclizine, diphenhydramine, promethazine, and diazepam. For neuropathy with nausea, the neurological drugs that can be used include anti-nausea agents, which include but are not limited to promethazine, chlorpromazine, prochlorperazine, trimethobenzamide, and metoclopramide.

[0642] Amyloidosis is a group of diseases and disorders associated with extracellular proteinaceous deposits in the CNS, including, but 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); Guam Parkinson's-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 deposition (i.e., macular degeneration, drusen-associated optic neuropathy, and cataracts).

[0643] For amyloidosis, neurological drugs that can be selected include, but are not limited to, antibodies or other binding molecules (including, but not limited to, small molecules, peptides, aptamers, or other protein binders) that specifically bind to targets selected from the group consisting of: beta-secretase, tau, presenilin, amyloid precursor protein or portion thereof, amyloid beta peptide or oligomers or fibrils thereof, 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. 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, nonsteroidal anti-inflammatory drugs (i.e., indomethicin and other compounds listed above); hormones (i.e., estrogens, progesterone, and leuprolide); vitamins (i.e., folic acid and niacinamide); dimebolin; homotaurine (i.e., 3-aminopropanesulfonic acid; 3APS); serotonin receptor activity modulators (i.e., xaliproden); interferons, and glucocorticoids.

[0644] CNS cancers are characterized by abnormal proliferation of one or more CNS cells (i.e., nerve cells) and include, but are not limited to, gliomas, glioblastoma multiforme, meningiomas, astrocytomas, acoustic neuromas, chondromas, oligodendrogliomas, medulloblastomas, gangliogliomas, schwannomas, neurofibromas, neuroblastomas, and epidural, intramedullary, or intradural tumors, or CNS metastases of peripheral tumors such as CD20 or HER2-positive cancers.

[0645] For cancer, the neurological drugs of choice are chemotherapy agents.

[0646] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonate 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); delta-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; acid; teniposide; cryptophycins (particularly 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, and chlorambucil. 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., calicheamicins, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994); enediyne anthracyclines (dynemicins), including enediyne anthracycline A; anesperamicin; and neocarzinostatin chromophores and related pigment proteins (enediyne antibiotic chromophores), aclacinomycins, actinomycins, authramycins, azaserines, bleomycins, cactinomycins, carabicin, carminomycins, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, 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, trimethoprim-sulfamethoxazole, quinolones, quinolones, dapoxetine ... trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone and dromostanolone propionate. propionate), epitiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone;aldophosphamide glycoside; 5-aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; bestrabucil; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate 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 (paclitaxel, Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ without Cremophor, albumin-engineered paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Illinois), and Docetaxel ( -Poulenc Rorer, Antony, France); chloranbucil; 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; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine any of the above pharmaceutically acceptable salts, acids or derivatives; and combinations of two or more of the above, such as CHOP, which is an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, which is an abbreviation for oxaliplatin combined with 5-FU and leucovovin.TM ) treatment regimen.

[0647] Also included in this definition of chemotherapeutic agents are antihormonal agents, which act to regulate, reduce, block or inhibit the effects of hormones that promote cancer growth and are usually in the form of systemic or systemic treatments. These may be hormones themselves. 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 (ERDs); agents that act to suppress or shut down the ovaries, for example, 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 the aromatase enzyme, which regulates estrogen production in the adrenal glands, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate, Exemestane, formestane, fadrozole, Fuclozole ( vorozole), Letrozole ( letrozole) and Anastrozole. In addition, 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 (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, 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] Ocular diseases or disorders are diseases or disorders of the eye, and for purposes herein, the eye is considered to be a CNS organ isolated by the BBB. Ocular diseases or disorders include, but are not limited to, sclera, cornea, iris and ciliary body disorders (i.e., scleritis, keratitis, corneal ulcers, corneal abrasions, snow blindness, arc eye, Tergersen's superficial punctate keratopathy, corneal neovascularization, Fuchs' dystrophy, keratoconjunctivitis sicca, iritis and uveitis), lens disorders (i.e., cataracts), choroidal and retinal disorders (i.e., retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, age-related macular degeneration, macular degeneration (wet or dry), epiretinal membranes (epiretinal membrane), retinitis pigmentosa and macular edema), glaucoma, floaters, optic nerve and visual pathway disorders (i.e., Leber hereditary optic neuropathy and optic disc drusen), ocular muscle / binocular movement accommodation / refractive 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, river blindness, and microphthalmia / coloboma), red eye, Algyll-Robertson pupil, keratomycosis, dry eye, and andaniridia.

[0650] For ocular diseases or conditions, neurological medications of choice are: anti-angiogenic ophthalmic agents (i.e., bevacizumab, ranibizumab, and pegaptanib), ophthalmic glaucoma agents (i.e., carbachol, epinephrine, demecarium bromide, 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), 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 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, 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 species, Streptococcus species, Pseudomonas species, Proteus species, Escherichia coli, Staphylococcus aureus, Pneumococcus species, meningococcus species, Haemophilus species, and Mycobacterium tuberculosis), and other microorganisms, such as fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., Toxoplasma gondii), or amoebae, resulting in CNS pathophysiology including, but not limited to, meningitis, encephalitis, myelitis, vasculitis, and abscesses, which can be acute or chronic.

[0652] For viral or microbial diseases, neurological drugs of choice 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 alpha-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), and sirolimus. travirine, delavirdine, and nevirapine), nucleoside reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir, zalcitabine, telbivudine, telbivudine and didanosine), protease inhibitors (i.e., darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir, amprenavir, indinavir, and saquinavir), purine nucleosides (i.e., valacyclovir, clovir), famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir, and cidofovir) and miscellaneous antivirals (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), cephalosporins (i.e., cefazolin, cephalexin, cephalothin, cefamandole, and ceftriaxone) , cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cephradine, loracarbef, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin), carbapenems / penems (i.e., imipenem, meropenem, ertapenem, faropenem, and doripenem), monobactams (i.e., aztreonam, tigemonam, norcardicin A), A) and tabtoxinine-beta-lactam), beta-lactamase inhibitors in combination with another beta-lactam antibiotic (i.e., clavulanic acid, tazobactam, and sulbactam), aminoglycosides (i.e.,amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromomycin), ansamycins (i.e., geldanamycin and herbimycin), carbacephems (i.e., loracarbef), glycopeptides (i.e., teicoplanin), planin and vancomycin), macrolides (i.e., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spectinomycin), monobactams (i.e., aztreonam), quinolone e.) (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamides (i.e., sulfonamides), mide) (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). B)), antifungals (i.e., metronidazole, nitazoxanide, tinidazole, chloroquine, iodoquinol, and paromomycin), and antiparasitics (including, but not limited to, quinine, chloroquine, amodiaquine, pyrimethamine, sulphadoxine, proguanil, mefloquine, atovaquone, primaquine, artemesinin, halofantrine, doxycycline, clindamycin, mebendazole, pyrantel pamoate, pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin, amphotericin B, melarsoprol, efornithin, and albendazole).

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

[0654] For CNS inflammation, neurological medications may be chosen that address the inflammation itself (ie, nonsteroidal anti-inflammatory agents such as ibuprofen or naproxen) or that treat the underlying cause of the inflammation (ie, antiviral or anticancer agents).

[0655] As used herein, CNS ischemia refers to a group of conditions or their causes associated with 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 increase blood flow or vascular elasticity, including, for example, blood pressure medications.

[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, basal cortical degeneration, degeneration caused by or associated with amyloidosis, Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy's disease, multiple system atrophy, multiple sclerosis, primary lateral sclerosis, progressive supranuclear palsy, spinal muscular atrophy, transverse myelitis, Refsum's disease, and spinocerebellar ataxia.

[0658] For neurodegenerative diseases, the neurological drugs that can be selected are growth hormones 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-derived neurotrophic factor (GDNF), neurotrophic factor (NF-κB), glial-derived neurotrophic factor (GDNF ... Protein, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemisinin, persephin, interleukins, glial cell line-derived neurotrophic factor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone morphogenetic protein (BMP), netrin, saposin, semaphorin, and stem cell factor (SCF).

[0659] Seizure diseases and disorders of the CNS involve inappropriate and / or abnormal electrical conduction 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, 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, toniclonic seizures, tonic seizures, psychomotor seizures, limbic epilepsy, partial-onset seizuresseizures), 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 seizures, pseudo seizures seizures), sensory seizures, subtle seizures, sylvan seizures, withdrawal seizures, and visual reflex seizures. For seizure disorders, the neurological medication of choice is an anticonvulsant or orantiepileptic, including but not limited to barbiturate anticonvulsants (i.e., primidone, metharbital, mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal, barbital, brallobarbital, and phenobarbital), benzodiazepine anticonvulsants (i.e., benzodiazepines, metharbital, mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal, barbital, brallobarbital, and phenobarbital),anticonvulsants (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), acid), GABA analogs (i.e., pregabalin, gabapentin, and vigabatrin), GABA reuptake inhibitors (i.e., tiagabine), GABA 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 anticonvulsants (i.e., paramethadione and trimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam), succinimide anticonvulsants (i.e., ethosuximid and methsuximide), triazine anticonvulsants (i.e.,anticonvulsants) (ie, lamotrigine) and urea anticonvulsants (ie, phenacemide and pheneturide).

[0660] Behavioral disorders are CNS disorders characterized by abnormal behaviors exhibited by the 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., 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), disorders) (i.e., anorexis or bulimia), major mental illnesses (psychoses), developmental behavioral disorders (i.e., autism, Rett's syndrome, Aspberger's syndrome), personality disorders, and psychotic disorders (i.e., schizophrenia, delusional disorder, etc.).

[0661] For behavioral disorders, neurological drugs can be selected from behavior-modifying compounds, including but not limited to atypical antipsychotics. 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), thioxanthenes (i.e., thiothixene), miscellaneous antipsychotics (i.e., pimozide, lithium, molindon), and dapoxetine). e), haloperido, and loxapine), selective serotonin reuptake inhibitors (ie, citalopram, escitalopram, paroxetine, fluoxetine, and sertraline), serotonin-norepinephrine reuptake inhibitors (ie, 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), benzodiazepines (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), secretins (see, e.g., Ratliff-Schaub et al., Autism 9 (2005): 256-265), opioid peptides (see, for example, Cowen et al., J. Neurochem. 8 (2004) 273-285), and neuropeptides (see, for example, Hethwa et al., Am. J. Physiol. 289 (2005) E301-305).

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

[0663] For lysosomal storage diseases, the neurological drugs of choice include the enzyme itself that is impaired in the disease, or drugs that mimic 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-L20-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, asparagylase, palmitoyl-protein thioesterase 1, and tripeptidyl aminopeptidase 1).

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

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

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

[0667] In another aspect, the present invention provides a method for treating Alzheimer's disease. In one embodiment, the method comprises administering to an individual suffering from Alzheimer's disease an effective amount of a multispecific 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 additional therapeutic agent is an agent that is effective for treating the same or different neurological disorder as the neurological disorder being treated with the anti-TfR / antibody. Exemplary additional 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 beta peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors (statins), ampakine, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid beta peptide immunization, phosphodiesterase inhibitors, 5-hydroxytryptamine receptor antagonists, and anti-amyloid beta peptide antibodies. In certain embodiments, the at least one additional therapeutic agent is selected for its ability to alleviate one or more side effects of neurological drugs.

[0669] In certain other such embodiments, at least one additional therapeutic agent is selected for its ability to inhibit or prevent activation of the complement pathway upon administration of an anti-TfR antibody. Examples of such therapeutic agents include, but are not limited to, agents that interfere with the ability of an anti-TfR antibody to bind to or activate the complement pathway and 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 contents of which are expressly incorporated herein by reference.

[0670] These combination therapies noted above and herein encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case administration of the antibodies of the invention can occur before, simultaneously with, and / or after administration of the other therapeutic agents and / or adjuvants. In some embodiments, administration of the anti-TfR antibody and 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 invention can also be used in combination with other interventional 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 of the present invention (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if local treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various administration regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulsed infusion.

[0672] The antibodies of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration 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 of delivery of the active agent, the method of administration, the time course of administration, and other factors known to medical practitioners.

[0673] The antibodies need not, but may optionally, be formulated with one or more agents currently used to prevent or treat the disorder in question or to prevent, alleviate or ameliorate one or more side effects of 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 other factors discussed above. These agents are generally used in the same dosage and using the administration routes described herein, or about 1% to 99% of the dosages described herein, or in any dosage and by any route determined empirically / clinically to be appropriate.

[0674] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present 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 preventive purposes or for therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody can be suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be the initial candidate dose administered to the patient, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dose can be in the range of about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration for several days or longer, depending on the condition, treatment will generally be continued until the desired disease symptom suppression occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 40 mg / kg. Thus, one or more doses 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 (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about two to about twenty or, for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other treatment regimens may be useful. It will be appreciated that one way to mitigate the effects of anti-TfR antibody administration on the reticulocyte population is to change the amount or time course of the dose so that there is an overall lower amount of circulating antibody in the bloodstream that interacts with the reticulocytes. In a non-limiting example, a lower dose of anti-TfR antibody can be administered more frequently than a higher dose. The dosage used can be balanced between the amount of antibody that must be delivered to the CNS (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 or administered continuously with the antibody. The progress of this treatment can be readily monitored by conventional techniques and assays as described herein and as known in the art.

[0675] It will be understood that any of the above formulations or treatment methods can be practiced using an immunoconjugate of the invention in place of or in addition to an anti-TfR antibody.

[0676] III. Products

[0677] In another aspect of the present invention, an article of manufacture is provided, comprising materials useful for treating, preventing, and / or diagnosing the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition, which can exist alone or in combination with another composition that is effective for treating, preventing, and / or diagnosing the condition, and can have a sterile access port (for example, the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. In addition, the article of manufacture may include (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the present invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user standpoint, it may further comprise other desirable materials, including other buffers, diluents, filters, needles, and syringes.

[0678] It will be understood that the immunoconjugates of the invention may be included in any of the above preparations instead of or in addition to the bispecific antibodies of the invention. IV. Examples

[0679] The following are examples of methods and compositions of the present invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0680] Materials and methods

[0681] Recombinant DNA technology

[0682] 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.

[0683] Gene and oligonucleotide synthesis

[0684] The desired gene fragments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled via PCR or by annealing chemically synthesized oligonucleotides. The corresponding oligonucleotides were obtained by metabion GmbH (Planegg-Martinsried, Germany).

[0685] Reagents

[0686] If not otherwise stated, all commercial chemicals, antibodies, and kits were used according to the manufacturer's protocols.

[0687] Example 1

[0688] Immunization of rabbits and mice

[0689] Immunization of mice

[0690] NMRI mice were genetically immunized with plasmid expression vectors encoding full-length human or macaque TfR by intradermal administration of 100 μg of vector DNA followed by electroporation (2 rectangular pulses at 1000 V / cm, duration 0.1 ms, intervals 0.125 s; followed by 4 rectangular pulses at 287.5 V / cm, duration 10 ms, intervals 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 vectors encoding macaque TfR; all other immunizations were performed with vectors encoding human TfR. Blood was collected on days 36, 78 and 92, and serum was prepared and used for ELISA titration assays (see below). The animals with the highest titers were selected and boosted on day 96: 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 HEK239F cells as an N-terminal fusion with a 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).

[0691] Rabbit immunization

[0692] New Zealand white rabbits or transgenic rabbits expressing a humanized antibody library were genetically immunized with plasmid expression vectors encoding full-length human or macaque TfR by intradermal administration of 400 μg of vector DNA followed by electroporation (5 rectangular pulses at 750 V / cm, duration 10 ms, intervals 1 s). Rabbits received 6 consecutive immunizations on days 0, 14, 28, 56, 84 and 112. The fourth and sixth immunizations were performed using vectors encoding macaque TfR; vectors encoding human TfR were used for all other immunizations. Blood was collected on days 35, 63, 91 and 119 (10% of the estimated total blood volume). 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 process (see Example 2).

[0693] Determination of serum titer (ELISA)

[0694] Human recombinant soluble TfR (R&D Systems Cat. No. 2474-TR) was immobilized on 96-well NUNC Maxisorb plates at 3 μg / mL in PBS, 100 μL / well, followed by: blocking the plates with 2% Crotein C in PBS, 200 μL / well; applying serial dilutions of antisera in 0.5% Crotein C in PBS in duplicate, 100 μL / well; and detecting with: (1) HRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch / Dianova 115-036-071; 1 / 16,000) for all mouse sera, (2) HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch / Dianova 711-036-152; 1 / 16,000) for all rabbit sera, (3) rabbit anti-human IgG antibody (Pierce / ThermoScientific 31423; 1 / 5000), only for serum from transgenic rabbits, (4) biotinylated goat anti-human kappa antibody (Southern Biotech / Biozol 2063-08, 1 / 5000) and streptavidin-HRP, only for serum 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% Tween20 in PBS. The signal was generated by adding BM Blue POD Substratesoluble (Roche), 100 μL / well; and stopped by adding 1 M HCl, 100 μL / well. The absorbance was read at 450 nm relative to 690 nm as a reference. The titer was defined as the antiserum dilution that resulted in half-maximal signal.

[0695] Example 2

[0696] Rabbit-derived B-cell clones

[0697] Isolation of rabbit peripheral blood mononuclear cells (PBMC)

[0698] Blood samples were obtained from a total of 6 animals (2 wild-type (wt) rabbits and 4 transgenic (tg) rabbits. These rabbits originated 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 the Example "Immunization of rabbits"). Whole blood containing EDTA was diluted two-fold with 1× PBS (PAA, Pasching, Austria) and then subjected to density centrifugation using mammalian lympholyte (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the manufacturer's instructions. PBMCs were washed twice with 1× PBS.

[0699] EL-4 B5 medium

[0700] RPMI 1640 (Pan Biotech, Aidenbach, Germany) was 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).

[0701] Cell depletion

[0702] First Immunoassay Series: Macrophages / monocytes were depleted by nonspecific adherent and nonspecific binding lymphocytes using sterile 6-well plates (cell culture grade) coated with confluent monolayers of CHO cells.

[0703] Second Immunoassay Series: The depletion step using CHO cell-coated wells was omitted because we could not exclude that B-cells producing antibodies cross-reactive with hamster transferrin receptor antibodies were depleted. Therefore, blank sterile 6-well plates (cell culture grade) were used to deplete macrophages and monocytes by nonspecific adhesion, allowing potential B-lymphocytes producing hamster cross-reactive (and possibly mouse cross-reactive) surface antibodies to reach the next step in the workflow.

[0704] For each immunoseries: Each well can contain a maximum of 4 mL of culture medium and up to 6 × 10 6 PBMCs from immunized rabbits were isolated and allowed to bind for 1 h in an incubator at 37° C. The cells in the supernatant (peripheral blood lymphocytes (PBL)) were used for the antigen panning step.

[0705] Enrichment of B cells at the human transferrin receptor

[0706] A 6-well tissue culture plate coated with a monolayer of human transferrin receptor-positive CHO cells was seeded with up to 6 × 10 6 Place 100 PBLs / 4 mL of culture medium and allow them to bind in an incubator at 37°C for 1 hour. Carefully wash the wells 1-2 times with 1x PBS to remove non-adherent cells. Detach remaining adherent cells by trypsinization at 37°C for 10 minutes. Stop the trypsinization with EL-4B5 culture medium. Keep cells on ice until immunofluorescence staining.

[0707] Immunofluorescence staining and flow cytometry

[0708] 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 antibodies in PBS and incubated for 45 min in the dark at 4 ° C. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Prior to FACS analysis, propidium iodide (BD Pharmingen, San Diego, CA, USA) was added at a concentration of 5 μg / mL to distinguish between dead and live cells.

[0709] A Becton Dickinson FACSAria equipped with a computer and FACSDiva software (BD Biosciences, USA) was used for single cell sorting.

[0710] B-cell culture

[0711] Rabbit B-cell cultures were prepared by methods similar to those described by Zubler et al. (1985). Briefly, sorted single rabbit B-cells were incubated in 96-well plates containing 200 μL / well EL-4B5 medium containing Pansorbin Cell (1:100,000) (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 RLT buffer (Qiagen, Hilden, Germany).

[0712] Example 3

[0713] Identification of human and macaque TfR-binding antibodies by cell-based ELISA

[0714] In order to screen rabbit B-cells or mouse hybridoma supernatants to identify antibodies that recognize human and macaque TfR, a cell ELISA using stably transfected CHO-K1 cells was used. 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 a growth medium containing 500 μg / mL G418 (Life Technologies). After the growth clones appeared, the cells were isolated and stained with MEM-75 (Abcam) or 13E4 (Life Technologies) and PE-labeled human or macaque TfR secondary antibodies, and the high fluorescent cells were sorted as single cells into 96-well flat plate wells (FACS Aria). After 7 days of growth, the TfR expression of the clones was checked again, and the best expression clones were selected for cell ELISA experiments.

[0715] In brief, 15,000 cells were seeded into each well of a 384-well plate and incubated at 37°C, 5% CO2 for 18h. Automated washing apparatus (BIOTEK) was used to remove supernatant, and 30 μL of the supernatant containing the antibody was added to each well, followed by addition of 24 μL growth medium. After 2 hours of incubation, the wells were drained and 0.05% glutaraldehyde 45min was added to 30 μL of PBS at RT. Washed 3 times with PBS / 0.025% Tween20 (PBST), 30 μL of anti-rabbit-HRP or anti-mouse-HRP (Southern Biotech) diluted in blocking buffer were added and the flat plate was incubated for 1 hour at RT. Pores were washed 6 times with PBST and 30 μL of TMB / wells were used to generate signals, and absorbance was measured at 450nm.

[0716] Example 4

[0717] Cloning and expression of anti-TfR antibodies

[0718] Recombinant DNA technology

[0719] Standard methods for manipulating DNA were used 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.

[0720] Gene and oligonucleotide synthesis

[0721] The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene segments were 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).

[0722] PCR amplification of the V-domain

[0723] Total RNA was obtained from B-cell lysates using the NucleoSpin 8 / 96 RNA kit (Macherey & Nagel; 740709.4, 740698) according to the manufacturer's protocol. 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 primers rbHC.up and rbHC.do for heavy chain, rbLC.up and rbLC.do for light chain of wild-type rabbit B cells, and BcPCR_FHLC_leader.fw and BcPCR_huCkappa.rev for light chain of transgenic rabbit B- cells (see table below). AccuPrime SuperMix (Invitrogen 12344-040) and 4 μL cDNA were used in a final volume of 50 μL. All forward primers were signal peptide specific (VH or VL), while reverse primers were constant region specific (VH or VL). PCR conditions for RbVH+RbVL were as follows: 94°C hot start for 5 min; 94°C for 20 sec, 70°C for 20 sec, 68°C for 45 sec, 35 cycles, and a final extension of 7 min at 68°C. PCR conditions for HuVL were as follows: hot start at 94°C for 5 min; 40 cycles of 94°C for 20 sec, 52°C for 20 sec, 68°C for 45 sec, and a final extension at 68°C for 7 min.

[0724]

[0725] 8 μL of the 50 μL PCR solution was loaded onto a 2% 48E-gel (Invitrogen G8008-02). Positive PCR reactions were cleaned using the NucleoSpin Extract II kit (Macherey & Nagel; 740609250) according to the manufacturer's protocol and eluted in 50 μL elution buffer. All cleanup steps were performed on a Hamilton ML Starlet system.

[0726] Recombinant expression of rabbit monoclonal bivalent antibodies

[0727] For recombinant expression of rabbit monoclonal bivalent antibodies, PCR products encoding VH or VL were cloned as cDNA into expression vectors by the overhang cloning method (RS Haun et al., BioTechniques (1992) 13, 515-518; MZ Li et al., Nature Methods (2007) 4, 251-256). 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 replication origin and a β-lactamase gene conferring ampicillin resistance for amplification of the plasmid in E. 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 κLC constant regions to accept the VL region.

[0728] Linearized expression plasmids encoding the kappa or gamma constant region and the VL / VH inserts were amplified by PCR using overlapping primers.

[0729] The purified PCR product was incubated with T4 DNA polymerase to generate single-stranded overhangs. The reaction was stopped by adding dCTP.

[0730] In the next step, plasmid and insert fragment are mixed and hatched with recA, its induction site specificity is reorganized.The plasmid of reorganization is transformed in the intestinal bacteria.On the second day, the colony of growth is selected, and by plasmid preparation, restriction analysis and DNA-order-checking, correct recombinant plasmid is checked.

[0731] For antibody expression, isolated HC and LC plasmids were transiently co-transfected into HEK293 cells and supernatants were harvested after 1 week.

[0732] Generation of vectors for rabbit monoclonal monovalent antibody expression

[0733] In order to recombinantly express the selected candidates as monoclonal monovalent antibodies, the rabbit constant regions of all VH chains were converted to human constant regions including knob mutations 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 cDNA of the selected candidates was used to amplify the immunoglobulin heavy and light chain variable regions in a final volume of 50 μL using AccuPrime SuperMix (Invitrogen 12344-040) using a signal peptide-specific forward primer and a CDR3-J region-specific reverse primer (which has an overlapping sequence (20 bp) homologous to the human constant region (VH or VL) at the 3' end). PCR conditions for VH and VL chain amplification were as follows: 94°C hot start 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.

[0734] The PCR products encoding VH or VL were cloned as cDNA into expression vectors by the overhang cloning method (RS Haun et al., BioTechniques (1992) 13, 515-518; MZ Li et al., Nature Methods (2007) 4, 251-256). 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 replication origin and a β-lactamase gene that confers ampicillin resistance for plasmid amplification in E. coli. Two variants of this basic plasmid were used: one plasmid contained a human IgG constant region designed to accept the newly amplified VH chain, and the second plasmid contained a human κLC constant region to accept the VL chain.

[0735] Linearized expression plasmids encoding the kappa or gamma constant region and the VL / VH inserts were amplified by PCR using overlapping primers.

[0736] The purified PCR product was incubated with T4 DNA polymerase to generate single-stranded overhangs. The reaction was stopped by adding dCTP.

[0737] In the next step, plasmid and insert fragment are mixed and hatched with recA, its induction site specificity is reorganized.The plasmid of reorganization is transformed in the intestinal bacteria.On the second day, the colony of growth is selected, and by plasmid preparation, restriction analysis and DNA-order-checking, correct recombinant plasmid is checked.

[0738] Example 5

[0739] Transient expression of monovalent anti-TfR antibodies

[0740] Antibodies were 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, "293-Free" transfection reagent (Novagen) was used. Antibodies and antibody-based modified molecules, as described above, were expressed from separate expression plasmids. Transfection was performed according to the manufacturer's instructions. Three to seven days after transfection, cell culture supernatants containing the recombinant proteins were collected. The supernatants were stored at a reduced temperature (e.g., -80°C) until purification.

[0741] 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.

[0742] Example 6

[0743] High-throughput purification of a single-arm transferrin receptor antibody

[0744] 50mL of the clarified supernatant containing the one-armed antibody in a 96-deep-well plate was loaded onto a 200 μL MabSelectSuRe column. After a PBS wash step at pH 7.4, the protein was eluted with 2.5mM HCl using a Tecan / Atoll-system to obtain 0.5mL of eluent. The eluent was neutralized by 2M Tris at pH 8. The purified protein was quantitatively analyzed by CE-SDS and analytical SEC under denaturing and reducing conditions using a Nanodrop spectrophotometer. In order to obtain a protein with high purity (>95%), most antibodies must be further purified on size exclusion chromatography to separate from half antibodies, pestle-pestle antibodies, and larger aggregates. In the following, 500 μL samples were injected onto a Superdex200 10 / 300GL in 20mM histidine containing 140mM NaCl pH 6.0 using a Dionex UltiMate 3000. This method allows fractionation of 25-30 samples / day and thus allows for the polishing of a large number of screening hits in a single-arm mode. Fractions were pooled and analyzed again as above.

[0745] Example 7

[0746] hCMEC / D3 cell culture for transcytosis assay

[0747] Culture media and supplements for hCMEC / D3 (Weksler, BB et al., FASEB J. 19 (2005), 1872-1874) were obtained from Lonza. hCMEC / D3 cells (passage 26-29) were cultured to confluence on collagen-coated coverslips (microscope inspection) or in flasks in EBM2 medium containing 2.5% FBS, one-quarter the supplied growth factors, and fully supplemented with the supplied hydrocortisone, gentamicin, and ascorbic acid.

[0748] For all transcytosis experiments, high-density wells (1 × 10 8 Holes / cm 2 ) PET membrane filter inserts (0.4 μm, 12 mm diameter). The calculated optimal culture medium volumes were 400 μL and 1600 μL for the apical and basolateral chambers, respectively. The apical chamber of the filter insert was incubated with rat tail collagen I (7.5 μg / cm 2 ), followed by coating with fibronectin (5 μg / mL), and each incubation lasted for 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 ).

[0749] Example 8

[0750] Monovalent antibody transcytosis assay

[0751] The entire test is carried out in serum-free EBM2 culture medium (otherwise according to the reconstruction described in Example 1). With monovalent antibody (concentration: 2.67 μ g / mL), the filter insert with cells is incubated at 37 ° C for 1 hour, after which the entire top and basolateral culture medium is collected. From these values, the paracellular flux is calculated. The monolayer is washed 3 × 3-5 min each at the top (400 μ L) and basolateral (1600 μ L) in serum-free culture medium at RT. All washing solutions are collected to monitor the removal efficiency of unbound antibodies. The culture medium warmed in advance is added to the top chamber and the filter is transferred to a new 12-well plate containing 1600 μ L of the culture medium warmed in advance (blocked overnight with PBS containing 1% BSA). At this point, the cells on the filter are lysed in 500 μ L RIPA buffers to measure specific antibody absorption. The remaining filter is incubated at 37 ° C and samples are collected at different time points to measure the top and / or basolateral release of the antibody. The amount of antibody in the samples was quantified using a high sensitivity IgG ELISA (see Example 3).For each time point, data were generated from three filter cell cultures.

[0752] Example 9

[0753] Sensitive IgG ELISA after transcytosis assay

[0754] The entire procedure was performed at room temperature, using an automated washer for washing steps. 384-well plates were coated with 30 μL / well of 1 μg / mL anti-human / mouse IgG (Fcγ specific) in PBS for 2 hours, followed by incubation for 1 hour in blocking buffer PBS containing 1% BSA or 1% Crotein C (for human and mouse IgG assays, respectively). Serially diluted samples from the transcytosis assay and standard concentrations of antibodies used in the transcytosis assay were added to the plates and incubated for 2 hours. After four washes, 30 μL / well of 50 ng / mL anti-human / mouse F(ab)2-biotin in blocking buffer was added and incubated for an additional 2 hours. After six washes, 30 μL / well of 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 and incubated for 30 minutes. After 4 washes, immune complexes were detected by adding 30 μL / well of BM chemiluminescent substrate (Roche). Luminescent signals were measured using a luminescent plate reader, and concentrations were calculated using a fitted standard curve. The sensitivity range of the test was 10 pg / mL to 10 ng / mL.

[0755] Example 10

[0756] Epitope mapping by cell ELISA in CHO cells transfected with hTfR mutants

[0757] Based on the fact that despite the significant homology between human and mouse TfR (77% identity), no antibodies against the extracellular part are known to show good cross-reactivity between the two orthologs, in order to be able to determine the epitope region on the human transferrin receptor (hTfR), the position of the surface-exposed amino acid cluster with different amino acids in the aligned mouse TfR sequence (see the table below), mutations were introduced into the hTfR sequence. The cloning of plasmids with corresponding mutations is described above. In order to map the binding of human TfR binders to the epitope, CHO-K1 cells were transiently transfected with the plasmids described, and antibody binding was measured in a cell ELISA. Briefly, one day before the experiment, 10 4Cells were seeded in normal growth medium (RPMI / 10% FCS) per well of a 96-well plate. On another day, the medium was replaced with OPTI-MEM serum-reduced medium (Gibco), and after a 30-minute preincubation, 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, after which the medium was removed and TfR antibodies were added at a concentration of 1 nM to 100 nM in growth medium, followed by incubation at 4°C for 2 h. Thereafter, the antibody solution was replaced with 0.05% glutaraldehyde in PBS, and the cells were fixed at RT for 15 min, then washed twice with PBS, and incubated for 1.5 hours at RT with an HRP-conjugated anti-human-Fc secondary antibody (1:2000 in BioRad; ELISA blocking reagent (Roche)). After washing three times with PBS, 50 μL / well of TMB was used to generate a signal, and the absorbance was measured at 450 nm.

[0758]

[0759] Example 11

[0760] Surface plasmon resonance-based binding assay for human TfR-antibody interactions

[0761] Binding experiments were performed on a BIAcore B 4000 (GE Healthcare) equipped with a C1 sensor chip (GE Healthcare, cat. no. BR1005-35) previously treated with anti-human Fab antibody (GE Healthcare cat. no. 28-9583-25) using standard amine coupling chemistry according to the supplier's manual.

[0762] For kinetic measurements, at 25 ° C, in pH 7.4 phosphate buffered saline, 0.05% Tween20, using a contact time of 60 seconds and a flow velocity of 10 μ L / min, fixed sample antibody. With increasing concentrations, the human transferrin receptor (R & D systems, catalog number 2474-TR-050) of the recombinant His6-tag is applied, and the signal is monitored over time. The average time span of 150 seconds of association time and 600 seconds of dissociation time under a record 30 μ L / min flow velocity is used. 1: 1 binding model (Langmuirisotherm) is used to fit the data.

[0763] Example 12

[0764] Humanization of the VH and VL domains of mouse and rabbit anti-transferrin receptor antibodies

[0765] The non-human anti-transferrin receptor antibodies were humanized as follows: Based on the characterization of the coding sequence and amino acid sequence of the VH and VL domains of the IgG1 class non-human anti-transferrin receptor antibody with a kappa light chain, the corresponding humanized anti-transferrin receptor antibodies were generated by CDR grafting reverse / forward mutagenesis based on the combination of the human germline frameworks VH4_3 and VK1_10 used for clone 299.

[0766] Example 13

[0767] Method for determining human / cynomolgus monkey TfR receptor affinity

[0768] In this example, a method for determining affinity of human transferrin receptor for comparison of dissociation behavior is outlined.

[0769] For all analysis, the Biotin CAPture test kit from GE Healthcare (Instruction 28-9242-34AB) was used. First, by docking the chip in a BIAcore T200 instrument, it was rehydrated. After this, the chip was left overnight for standby use with running buffer. For surface preparation, Biotin CAPture reagent was diluted 1:100 in running buffer (1 × PBS, supplemented with 0.25M NaCl). This solution was injected into flow cells 1 to 4 for 360sec, using 2 μL / min flow velocity. Then, the sensor surface was adjusted with the regeneration solution provided in the three one-minute injection Biotin CAPture test kits. This must be carried out for docking procedures or for the first time or after storage. On flow cell 2, the people or macaque monobiotinylated transferrin receptor solution of 100nM should be injected, with 10 μL / min flow velocity for 30sec. For affinity determination, six concentrations (500, 250, 125, 62.5, 31.25, 15.625, and 0 nM) were injected. They were injected onto a "hu-TfR-flow cell" (e.g., Flow Cell 2 prepared as described above) with an injection time (binding) of 180 sec 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.

[0770] Kinetic data were evaluated using BIAcore T200 evaluation software. In particular, the dissociation rate constants of different human transferrin receptor binders were considered after applying a 1:1 Langmuir binding model.

[0771] Example 14

[0772] Relative ranking of B4000 for human / macaque transferrin receptor dissociation

[0773] According to the manufacturer's instructions, the CAP sensor chip (provided in the Biotin CAPture kit, serial S#28-9202-34GE) was installed in the BIAcore B4000 system, standardized and addressed in a hydrodynamic manner. In the first cycle, the CAP reagent (provided in the kit) was addressed to points 1, 2, 4, and 5, where a flow rate of 10 μL / min was used for 300 seconds. Human transferrin receptor capture occurred in points 1 (human transferrin receptor-biotinylated) and 5 (cynomolgus transferrin receptor-biotinylated) where a flow rate of 10 μL / min and a contact time of 30 seconds 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). The antibody was injected into all flow cells at a concentration series of 100 nM, 50 nM, 25 nM, and 0 nM, where a flow rate of 30 μL / min was used for 180 seconds. The dissociation time was set to 300 sec. The entire complex was regenerated from the CAP chip using the regeneration solution provided in the Biotin CAPture kit (using a flow rate of 10 μL / min for 120 sec). To control the active protein concentration, a second cycle was performed in point 5 using biotinylated protein A (#P2165-2MG, Sigma) at a flow rate of 10 μL / min and a contact time of 30 sec. In this control cycle, point 1 remained empty. Antibodies and regeneration were manipulated similarly to those in cycle 1. The relevant kinetic data were calculated using BIAcore B4000 evaluation software. Dissociation from the human transferrin receptor was determined using a 1:1 dissociation fit.

[0774] Some embodiments of the present invention:

[0775] 1. A humanized antibody that specifically binds to human transferrin receptor, wherein the antibody

[0776] In the heavy chain variable domain, HVRs comprising SEQ ID NOs: 66, 68, and 72, and

[0777] In the light chain variable domain, the HVRs comprise SEQ ID NOs: 75, 76, and 78.

[0778] 2. The humanized antibody according to embodiment 1, comprising the heavy chain variable domain of SEQ ID NO: 24 and the light chain variable domain of SEQ ID NO: 37.

[0779] 3. The humanized antibody according to any one of embodiments 1 to 2, wherein said humanized antibody is effector function-silenced.

[0780] 4. The humanized antibody according to any one of embodiments 1 to 3, wherein the humanized antibody specifically binds to human transferrin receptor and macaque transferrin receptor.

[0781] 5. The humanized antibody according to any one of embodiments 1 to 4, wherein the humanized antibody is a multispecific antibody having at least one binding specificity for human transferrin receptor and at least one binding specificity for a therapeutic target.

[0782] 6. The humanized antibody according to embodiment 5, wherein the humanized antibody comprises a first antigen binding site that binds to human transferrin receptor and a second antigen binding site that binds to a brain antigen.

[0783] 7. The humanized antibody according to embodiment 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.

[0784] 8. The humanized antibody according to any one of embodiments 5 to 7, wherein the multispecific antibody binds to:

[0785] i) human transferrin receptor and Aβ, or

[0786] ii) human transferrin receptor and CD20, or

[0787] iii) human transferrin receptor and alpha-synuclein, or

[0788] iv) human transferrin receptor and phospho-tau protein, or

[0789] v) Human transferrin receptor and glucocerebrosidase.

[0790] 9. The humanized antibody according to any one of embodiments 1 to 8, wherein the humanized antibody is a bispecific antibody comprising

[0791] 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,

[0792] and

[0793] ii) a second binding site selected from the group consisting of

[0794] a) the heavy chain variable domain of SEQ ID NO: 81 and the light chain variable domain of SEQ ID NO: 82, or

[0795] b) the heavy chain variable domain of SEQ ID NO: 83 and the light chain variable domain of SEQ ID NO: 84, or

[0796] c) the heavy chain variable domain of SEQ ID NO: 85 and the light chain variable domain of SEQ ID NO: 86, or

[0797] d) the heavy chain variable domain of SEQ ID NO: 87 and the light chain variable domain of SEQ ID NO: 88, or

[0798] e) the heavy chain variable domain of SEQ ID NO: 91 and the light chain variable domain of SEQ ID NO: 92, or

[0799] f) the heavy chain variable domain of SEQ ID NO: 89 and the light chain variable domain of SEQ ID NO: 90, or

[0800] g) the heavy chain variable domain of SEQ ID NO: 93 and the light chain variable domain of SEQ ID NO: 94, or

[0801] h) the heavy chain variable domain of SEQ ID NO: 79 and the light chain variable domain of SEQ ID NO: 80.

[0802] 10. The humanized antibody according to any one of embodiments 1 to 9, wherein the humanized antibody is

[0803] a) a full-length antibody of the human IgG1 subclass, or

[0804] b) a full-length antibody of the human IgG4 subclass, or

[0805] c) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329G,

[0806] d) a full length antibody of human IgG4 subclass with the mutations S228P, L235E and optionally P329G,

[0807] e) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329...

Claims

1. A humanized antibody that specifically binds to human transferrin receptor, wherein the antibody comprises: - a heavy chain variable domain of SEQ ID NO: 24 and a light chain variable domain of SEQ ID NO: 37; - a heavy chain variable domain of SEQ ID NO: 25 and a light chain variable domain of SEQ ID NO: 37; - a heavy chain variable domain of SEQ ID NO: 23 and a light chain variable domain of SEQ ID NO: 37; - a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57 and 58 and a light chain variable domain selected from the group consisting of SEQ ID NOs: 60, 61, 62 and 63, or - a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 and a light chain variable domain selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.

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

3. A pharmaceutical preparation comprising the humanized antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. Use of the humanized antibody according to claim 1 or 2 or the pharmaceutical preparation according to claim 3 in the preparation of a medicament.

5. The method of claim 4 , wherein the medicament is for treating a condition selected from the group consisting of Alzheimer's disease (AD), a combination of Alzheimer's disease and Parkinson's disease, 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, traumatic brain injury, secondary amyloidosis, age-related amyloidosis, mild cognitive impairment (MCI), dementia with Lewy bodies, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson's-dementia complex, cerebral amyloid angiopathy, Huntington's disease, progressive supranuclear palsy, Creutzfeldt-Jakob disease, transmissible spongiform encephalopathy, HIV-associated dementia, inclusion body myositis (IBM), and eye diseases involving β-amyloid deposition (i.e., macular degeneration, drusen-associated optic neuropathy, and cataracts).

6. The use according to claim 4 or 5, wherein the humanized antibody comprises a heavy chain variable domain of SEQ ID NO: 24 and a light chain variable domain of SEQ ID NO:

37.

7. The use according to claim 4 or 5, wherein the humanized antibody comprises a heavy chain variable domain of SEQ ID NO: 25 and a light chain variable domain of SEQ ID NO:

37.

8. The use according to any one of claims 4 to 7, wherein the humanized antibody is a multispecific antibody having at least one binding specificity for human transferrin receptor and at least one binding specificity for a therapeutic agent.

9. The use according to any one of claims 4 to 8, wherein the humanized antibody comprises a first antigen binding site that binds to human transferrin receptor and a second antigen binding site that binds to a brain antigen.

10. The use according to claim 9, wherein the brain antigen is selected from the group consisting of Aβ, epidermal growth factor receptor, human epidermal growth factor receptor 2, α-synuclein, CD20, amyloid precursor protein and glucocerebrosidase.

11. The method according to any one of claims 4 to 10, wherein the multispecific antibody binds to: i) human transferrin receptor and Aβ, or ii) human transferrin receptor and CD20, or iii) human transferrin receptor and alpha-synuclein, or iv) human transferrin receptor and phospho-tau protein, or v) Human transferrin receptor and glucocerebrosidase.

12. The use according to any one of claims 4 to 11, wherein the humanized antibody is a bispecific antibody comprising i) a first binding site, the 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, or - a heavy chain variable domain of SEQ ID NO: 25 and a light chain variable domain of SEQ ID NO: 37; and ii) a second binding site selected from the group consisting of a) the heavy chain variable domain of SEQ ID NO: 81 and the light chain variable domain of SEQ ID NO: 82, or b) the heavy chain variable domain of SEQ ID NO: 83 and the light chain variable domain of SEQ ID NO: 84, or c) the heavy chain variable domain of SEQ ID NO: 85 and the light chain variable domain of SEQ ID NO: 86, or d) the heavy chain variable domain of SEQ ID NO: 87 and the light chain variable domain of SEQ ID NO: 88, or e) the heavy chain variable domain of SEQ ID NO: 91 and the light chain variable domain of SEQ ID NO: 92, or f) the heavy chain variable domain of SEQ ID NO: 89 and the light chain variable domain of SEQ ID NO: 90, or g) the heavy chain variable domain of SEQ ID NO: 93 and the light chain variable domain of SEQ ID NO: 94, or h) the heavy chain variable domain of SEQ ID NO: 79 and the light chain variable domain of SEQ ID NO:

80.

13. The use according to any one of claims 4 to 12, 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 human IgG1 subclass having the mutations L234A, L235A and P329G, or d) a full length antibody of human IgG4 subclass with the mutations S228P, L235E and optionally P329G, or e) a full length antibody of human IgG1 subclass having the mutations L234A, L235A and P329G in both heavy chains, and having the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, or f) a full length antibody of human IgG4 subclass having the mutations S228P, L235E and optionally P329G in both heavy chains, and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain.

14. The method according to any one of claims 4 to 13, wherein the humanized antibody comprises i) a homodimeric Fc-region of human IgG1 subclass optionally with the mutations P329G, L234A and L235A, or ii) a homodimeric Fc-region of human IgG4 subclass optionally with the mutations P329G, S228P and L235E, or iii) a heterodimeric Fc-region, wherein a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or 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 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, or iv) a heterodimeric Fc-region of 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 and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or 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 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, or v) a heterodimeric Fc-region of human IgG4 subclass, wherein both Fc-region polypeptides comprise the mutations P329G, S228P and L235E, and a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or 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 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.

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