Modified antibody Fc and methods of using the same

Abstract

Biomacromolecules have great potential for treating central nervous system (CNS) diseases, however, the presence of the blood-brain barrier (BBB) ​​makes it extremely challenging to reach therapeutically relevant antibody concentrations. Antibodies with enhanced neutral pH affinity for neonatal Fc receptors show improved accumulation in the brain. Variants disclosed herein also enhance exposure in engineered mouse models. By using anti-BACE1 antibodies, these Fc variants significantly reduce the level of brain Aβ.

Description

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 782,904, filed on December 20, 2018, which is incorporated herein by reference in its entirety for any purpose. Technical Field

[0002] The present invention relates to modified Fc and methods of using the modified Fc to cross the blood-brain barrier. Background Art

[0003] There are many central nervous system (CNS) therapeutic targets of significant medical interest - including amyloid β (Aβ), β-secretase 1 (BACE1), Tau and α-synuclein (Spillantini, Schmidt et al. 1997; Hardy and Selkoe 2002; Ghosh, Gemma et al. 2008; Thinakaran and Koo 2008; Mandelkow and Mandelkow 2012). However, the blood-brain barrier (BBB) ​​normally limits the penetration of antibodies into the brain (Abbott, Ronnback et al. 2006; Pardridge 2016).

[0004] Efforts have been made to improve antibody penetration into the brain by enhanced receptor-mediated transcytosis (RMT) (Fishman, Rubin et al. 1987), such as RMT utilizing the transferrin receptor (TfR) (Yu, Zhang et al. 2011; Yu, Atwal et al. 2014; Pardridge 2016). TfR is enriched on brain endothelial cells and rapidly transcytosed to facilitate the transport of transferrin in the brain and other tissues (Ponka and Lok 1999).

[0005] However, targeting TfR has disadvantages. For example, to generate therapeutic antibodies that utilize TfR to cross the BBB, the second binding domain must be introduced into traditional IgG via one of many multispecific technologies, including, for example, knobs and sockets, dual variable domains, or cross-mAb technologies. These technologies add expense and complexity to the development of therapeutic antibodies. In addition, TfR is expressed on many tissues, and targeting this receptor can introduce many potential safety liabilities. For example, targeting TfR can cause reticulocytopenia (Couch, Yu et al. 2013). This risk can be reduced by using various effector attenuation techniques (Couch, Yu et al. 2013; Lo, Kim et al. 2017). However, since it has been proposed that antibody effector functions play a role in the mechanism of action of some Aβ-targeting antibodies, effect attenuation can limit the efficacy of amyloid-targeting antibodies as well as antibodies targeting other antigens.

[0006] Numerous biological antibody receptors can interact with IgG molecules, such as Fc-γ receptors and neonatal Fc receptors (FcRn) (Ghetie and Ward 2002; Challa, Velmurugan et al. 2014). FcRn is a heterodimeric protein complex consisting of two subunits - FCGRT, also known as FcRnα chain, and β-2 microglobulin (β2M). In humans, FcRn is expressed on some hematopoietic cells, renal cells, intestinal and upper airway epithelial cells, and normal endothelial cells, including those located at the BBB (Roopenian and Akilesh 2007; Challa, Velmurugan et al. 2014). During development, FcRn contributes to the transport of IgG molecules across the placental barrier, and in infants, FcRn contributes to the transport of IgG from bovine milk across intestinal epithelial cells. In adults, the main function of FcRn is to mediate the endosomal recycling of IgG and thus the persistence of IgG serum half-life. FcRn is expressed in a variety of tissues and cell types, including placenta, liver (including hepatocytes and Kupffer cells), small intestine (including apical enterocytes, goblet cells, and crypt enterocytes), large intestine (including apical enterocytes, goblet cells, crypt enterocytes, colon, and rectum), oral epithelium, nasopharynx, upper airway (including lung epithelial cells), renal epithelial cells, endothelial cells, brain endothelial cells, spinal cord, cerebral cortex, choroid plexus, arachnoid villi, bone, lymph nodes, tonsils, spleen, thyroid, monocytes, macrophages, dendritic cells, B lymphocytes, NK cells, adrenal glands, breast, pancreas, islets of Langerhans, gallbladder, prostate, urinary bladder, skin, uterus, ovary, testis, seminal vesicles, and adipose tissue.

[0007] IgG binding to FcRn is regulated by pH. At physiological pH, such as in serum, there is almost no binding of IgG to FcRn, while at acidic pH, such as in endosomes, the affinity of IgG for FcRn is enhanced (Kuo and Aveson 2011). This enhanced binding to FcRn in endosomes at acidic pH causes clearance of pinocytic antibodies to prevent lysosomal degradation and maintain antibody levels in serum (Ghetie and Ward 2002). Research efforts have identified antibody Fc variants with improved pharmacokinetic properties due to enhanced binding to FcRn at acidic pH (Hinton, Johlfs et al. 2004; Dall'Acqua, Kiener et al. 2006; Hinton, Xiong et al. 2006; Petkova, Akilesh et al. 2006; Datta-Mannan, Witcher et al. 2007; Yeung, Leabman et al. 2009; Zalevsky, Chamberlain et al. 2010). Some Fc variants have been identified that improve binding to FcRn at physiological and acidic pH (Hinton, Johlfs et al. 2004; Dall'Acqua, Kiener et al. 2006; Hinton, Xiong et al. 2006; Yeung, Leabman et al. 2009; Igawa, Maeda et al. 2013). However, several of these Fc variants can also lead to enhanced antibody clearance (Dall'Acqua, Woods et al. 2002; Vaccaro, Zhou et al. 2005; Igawa, Maeda et al. 2013; Borrok, Wu et al. 2015).

[0008] Previous studies have suggested that FcRn has limited function in antibody transport into the brain (Abuqayyas and Balthasar 2013), and indeed, experiments using direct intracranial antibody injections showed that FcRn functions to facilitate antibody export out of the brain (Cooper, Ciambrone et al. 2013). Summary of the invention

[0009] Provided herein are antibodies and Fc conjugates comprising a modified Fc and having improved brain uptake.

[0010] In some embodiments, a method of treating a neurological disorder in a subject is provided, the method comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein the antibody is active in an in vitro transcytosis assay. In some embodiments, the neurological disorder is selected from a neuropathic disorder, a neurodegenerative disease, a brain disorder, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation. In some embodiments, the neurological disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from Lewy body disease, post-polio syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, amyloidosis, Parkinson's disease, multiple system atrophy, striatonigral degeneration, amyloidosis, tauopathy, Alzheimer disease, supranuclear palsy, prion disease, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia.

[0011] In some embodiments, a method of delivering an antibody to the brain of a subject is provided, the method comprising administering to the subject an antibody comprising a modified IgG Fc to the subject in need thereof, wherein the antibody is active in an in vitro transcytosis assay.

[0012] In some embodiments, methods of increasing brain exposure to an antibody are provided, the method comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein the antibody is active in an in vitro transcytosis assay.

[0013] In some embodiments, methods of increasing transport of an antibody across the blood-brain barrier (BBB) ​​are provided, the method comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein the antibody is active in an in vitro transcytosis assay.

[0014] In some embodiments, an isolated antibody is provided, wherein the antibody comprises a modified IgG Fc, wherein the antibody is active in an in vitro transcytosis assay.

[0015] In some embodiments, the antibody exhibits a transcytosis activity of at least 50 in an in vitro transcytosis assay when normalized to the same antibody comprising a wild-type IgG Fc. In some embodiments, the antibody exhibits a transcytosis activity of at least 60, at least 70, at least 80, at least 90, or at least 100 in an in vitro transcytosis assay. In some embodiments, the in vitro transcytosis assay comprises cells expressing FcRn. In some embodiments, the FcRn is human FcRn. In some embodiments, the cell is an MDCK II cell.

[0016] In some embodiments, the antibody comprising a modified IgG Fc has a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference antibody of the same species and isotype with an unmodified IgG Fc. In some embodiments, the antibody comprising a modified IgG Fc has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference antibody of the same species and isotype with an unmodified IgG Fc. In some embodiments, the antibody comprising a modified IgG Fc has a binding affinity for FcRn at pH 7.4 of ≤10 μM, ≤5 μM, ≤4 μM, ≤3 μM, ≤2 μM, ≤1 μM, ≤900 nM, ≤800 nM, ≤700 nM, ≤600 nM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, or ≤100 nM. In some embodiments, the antibody comprising a modified IgG Fc has a binding affinity for FcRn at pH 6 of ≤1 μM, ≤900 nM, ≤800 nM, ≤700 nM, ≤600 nM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, ≤100 nM, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, or ≤10 nM. In some embodiments, the ratio of the affinity of the antibody comprising a modified IgG Fc for FcRn at pH 7.4 to the affinity of the antibody comprising a modified IgG Fc for FcRn at pH 6 is at least 5, at least 10, at least 20, at least 50, or at least 100; or 5 to 200, 5 to 100, 10 to 200, 10 to 100, 20 to 100, or 20 to 200.

[0017] In some embodiments, an antibody comprising a modified IgG Fc comprises one or more mutations selected from the group consisting of 252W, 252Y, 286E, 286Q, 307Q, 308P, 310A, 311A, 311I, 428L, 433K, 434F, 434W, 434Y, and 436I according to EU numbering. In some embodiments, the modified IgG Fc comprises 252Y and 434Y. In some embodiments, the modified IgG Fc comprises 252Y and 434Y and one or two other mutations selected from the group consisting of 286E, 286Q, 307Q, 308P, 311A, 311I, 428L, 433K, and 436I. In some embodiments, the modified IgG Fc further comprises 307Q and 311A, or further comprises 286E. In some embodiments, the modified IgG Fc comprises a group of mutations selected from the group of mutations in Table 4, Table 5, and Table 6. In some embodiments, the modified IgG Fc comprises one or more modifications of a sequence selected from SEQ ID NOs: 1-4. In some embodiments, the IgG Fc is an IgG1 Fc. In some embodiments, the IgG Fc is an IgG4 Fc.

[0018] In some embodiments, the antibody binds to a brain antigen. In some embodiments, the antibody binds to a brain antigen selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (prion protein, PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6R), interleukin 1β (IL1β), caspase 6, triggering receptor expressed on myeloid cells 2 (TREM2), C1q, paired immunoglobulin-like type 2 receptor alpha (PILRA), CD33, interleukin 6 (IL6), tumor necrosis factor alpha (TNFα), tumor necrosis factor receptor superfamily member 1A (TNFR1), tumor necrosis factor receptor superfamily member 1B (TNFR2), and apolipoprotein J (ApoJ).

[0019] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is an antibody fragment.

[0020] In some embodiments, the antibody is conjugated to an imaging agent. In some embodiments, the antibody is conjugated to a neurological disorder drug. In some embodiments, the neurological disorder drug is selected from an aptamer, an inhibitory nucleic acid, a ribozyme, and a small molecule.

[0021] In some embodiments, a method of treating a neurological disorder is provided, the method comprising administering to a subject in need thereof an Fc conjugate comprising a modified IgG Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay. In some embodiments, the neurological disorder is selected from a neuropathic disorder, a neurodegenerative disease, cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation. In some embodiments, the neurological disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies, Alzheimer's disease, supranuclear palsy, prion diseases, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Strauss-Schwann disease, chronic wasting disease, and fatal familial insomnia.

[0022] In some embodiments, a method of delivering an Fc conjugate to the brain of a subject is provided, the method comprising administering to the subject an antibody comprising a modified IgG Fc to the subject in need thereof, wherein the Fc conjugate is active in an in vitro transcytosis assay.

[0023] In some embodiments, methods of increasing brain exposure to an Fc conjugate are provided, the method comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay.

[0024] In some embodiments, methods of increasing transport of an Fc conjugate across the blood-brain barrier (BBB) ​​are provided, the method comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay.

[0025] In some embodiments, an Fc conjugate is provided, wherein the Fc conjugate comprises a modified IgG Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay.

[0026] In some embodiments, the Fc conjugate exhibits a transcytosis activity of at least 50 in an in vitro transcytosis assay when normalized to the same Fc conjugate comprising a wild-type IgG Fc. In some embodiments, the Fc conjugate exhibits a transcytosis activity of at least 60, at least 70, at least 80, at least 90, or at least 100 in an in vitro transcytosis assay. In some embodiments, the in vitro transcytosis assay comprises cells expressing FcRn. In some embodiments, the FcRn is human FcRn. In some embodiments, the cell is an MDCK II cell.

[0027] In some embodiments, the Fc conjugate comprising a modified IgG Fc has a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference Fc conjugate with an unmodified IgG Fc of the same species and isotype. In some embodiments, the Fc conjugate comprising a modified IgG Fc has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference Fc conjugate with an unmodified IgG Fc of the same species and isotype. In some embodiments, the Fc conjugate comprising a modified IgG Fc has a binding affinity for FcRn at pH 7.4 that is less than 1 mM, or less than 750 nM, or less than 500 nM, or less than 400 nM, or less than 300 nM, or less than 200 nM, or less than 100 nM, or between 50 nM and 1 mM, or between 100 nM and 1 mM, or between 100 nM and 500 nM. In some embodiments, the Fc conjugate comprising a modified IgG Fc has a binding affinity for FcRn at pH 6 of less than 100 nM, or less than 90 nM, or less than 80 nM, or less than 70 nM, or less than 60 nM, or less than 50 nM, or less than 40 nM, or less than 30 nM, or less than 20 nM, or less than 10 nM, or between 1 nM and 200 nM, or between 10 nM and 200 nM. In some embodiments, the ratio of the affinity of the Fc conjugate comprising a modified IgG Fc for FcRn at pH 7.4 to the affinity of the Fc conjugate comprising a modified IgG Fc for FcRn at pH 6 is at least 5, at least 10, at least 20, at least 50, or at least 100; or 5 to 200, 5 to 100, 10 to 200, 10 to 100, 20 to 100, or 20 to 200.

[0028] In some embodiments, the modified IgG Fc comprises one or more mutations selected from the group consisting of 252W, 252Y, 286E, 286Q, 307Q, 308P, 310A, 311A, 311I, 428L, 433K, 434F, 434W, 434Y, and 436I according to EU numbering. In some embodiments, the modified IgG Fc comprises 252Y and 434Y. In some embodiments, the modified IgG Fc comprises 252Y and 434Y and one or two other mutations selected from the group consisting of 286E, 286Q, 307Q, 308P, 311A, 311I, 428L, 433K, and 436I. In some embodiments, the modified IgG Fc further comprises 307Q and 311A, or further comprises 286E. In some embodiments, the modified IgG Fc comprises a group of mutations selected from the group of mutations in Table 4, Table 5, and Table 6. In some embodiments, the modified IgG Fc comprises one or more modifications of a sequence selected from SEQ ID NOs: 1-4. In some embodiments, the IgG Fc is an IgG1 Fc. In some embodiments, the IgG Fc is an IgG4 Fc.

[0029] In some embodiments, the Fc conjugate comprises a modified IgG Fc fused to a therapeutic protein. In some embodiments, the therapeutic protein is selected from a receptor extracellular domain and an enzyme. In some embodiments, the receptor extracellular domain is selected from a TNF-R1 extracellular domain (ECD), a CTLA-4 ECD, and an IL-1R1 ECD. In some embodiments, the enzyme is selected from α-L-iduronidase, iduronic acid-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, asparagine transferase, palmitoyl protein thioesterase 1, and tripeptidyl aminopeptidase 1.

[0030] In some embodiments, the Fc conjugate comprises a modified IgG Fc conjugated to a neurological disorder drug. In some embodiments, the neurological disorder drug is selected from an aptamer, an inhibitory nucleic acid, a ribozyme, and a small molecule. In some embodiments, the Fc conjugate comprises a modified IgG Fc conjugated to an imaging agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures 1A-1D. Pharmacokinetics and pharmacodynamics of anti-BACE1 hIgG1 antibody (anti-BACE1), anti-BACE1 hIgG1 antibody with modified Fc (anti-BACE1-YTE), or control anti-gD hIgG1 antibody (anti-gD) in wild-type mice. A) Plasma antibody pharmacokinetics; B) Brain pharmacodynamics as measured by brain Aβ levels; C) Brain antibody pharmacokinetics. D) Relative antibody uptake in the brain of human IgG1YTE-modified antibodies compared to wild-type IgG1, including maximum concentration (Cmax) and area under the curve (AUC).

[0032] Figures 2A-2D .Anti-BACE1 hIgG1 antibody (anti-BACE1), anti-BACE1 hIgG1 antibody with modified Fc (anti-BACE1-YTE), or anti-gD hIgG1 antibody in the presence of human FCGRT (hFcRnα chain) and lacking mouse Fcgrt (i.e., FCGRF + / + Fcg - / - Pharmacokinetics and pharmacodynamics in transgenic (Tg32) mice expressing hFCGRT and lacking mFCGRT. A) Plasma antibody pharmacokinetics; B) Brain pharmacodynamics measured by brain Aβ levels; C) Brain antibody pharmacokinetics; D) In ​​vitro human and mouse FcRn binding properties of human IgG1 YTE-modified antibodies or human IgG1 wild-type antibodies.

[0033] Figure 3 A graph of the binding affinity of certain hIgG1 antibodies comprising mutations in the Fc to hFcRn at two different pH values ​​as described in Example 3. The X-axis shows the pH 6 affinity of the antibody, while the Y-axis shows the pH 7.4 affinity of the antibody.

[0034] Figure 4 . Graph of normalized transcytosis values ​​for certain Fc-modified antibodies as described in Example 3. Fc-modified antibodies above the dashed line displayed significantly improved transcytosis.

[0035] Figures 5A-5C Improved brain exposure characteristics of anti-gD hIgG1 antibodies containing modified Fc in transgenic (Tg32) mice expressing hFCGRT and lacking mFCGRT. A) Serum pharmacokinetics (PK); B) Brain PK; C) Summary of single-dose PK data and affinity data.

[0036] Figures 6A-6EPharmacokinetics and pharmacodynamics of an anti-BACE1 hIgG1 antibody with a modified Fc (anti-BACE1-YQAY) in transgenic (Tg32) mice expressing hFCGRT and lacking mFCGRT. A) Serum antibody concentration; B) Brain pharmacokinetics (PK); C) Brain pharmacodynamics (PD) measured by brain Aβ levels; D) Summary of hFcRn affinity and brain PK data; E) Summary of brain PD data.

[0037] Figures 7A-7D . Transgenic Tg32 expressing hFCGRT and lacking mFCGRT (FCGRT + / + Fcg - / - ) mice, expressing both hFCGRT and mFCGRT hemizygous (FCGRT + / - Fcg + / - ) mice, or wild-type (Fcgrt + / + ) mice. A) Plasma PK in homozygous Tg32 mice; B) Plasma PK in hemizygous Tg32 (FCGRT + / - Fcg + / - ) plasma PK in mice; C) wild-type (Fcgrt + / + ) Plasma PK in mice; D) Summary of FcRn binding affinity data and the ratio of plasma AUC of each FC-modified antibody relative to the wild-type antibody.

[0038] Figures 8A-8C . Brain pharmacokinetics and brain antibody concentrations of anti-gD hIgG1 antibodies containing modified Fc in transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT. A) Brain PK data; B) Brain antibody concentrations 7 days after dosing; C) Summary of affinity data and ratios of brain AUC of each Fc-modified antibody relative to the wild-type antibody.

[0039] Figures 9A-9C . Liver exposure of anti-gD hIgG1 antibodies containing modified Fc in transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT. A) Liver PK data; B) Liver antibody concentrations 7 days after dosing; C) Summary of affinity data and ratio of liver AUC for each Fc-modified antibody relative to wild-type antibody.

[0040] Figures 10A-10CLarge intestinal exposure of anti-gD hIgG1 antibodies containing modified Fc in transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT. A) Large intestinal PK data; B) Large intestinal antibody concentrations 7 days after dosing; C) Summary of affinity data and the ratio of large intestinal AUC for each Fc-modified antibody relative to the wild-type antibody.

[0041] Figures 11A-11C . Lung exposure of anti-gD hIgG1 antibodies containing modified Fc in transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT. A) Lung PK data; B) Lung antibody concentrations 7 days after dosing; C) Summary of affinity data and ratio of lung AUC for each Fc-modified antibody relative to wild-type antibody.

[0042] Fig.12 . Aligned sequences of human IgG subclasses IgG1, IgG2, IgG3, and IgG4 (SEQ ID NOs: 1-4, respectively). Differences in the sequence from IgG1 are highlighted in grey, and the presence of two residues separated by a slash indicates a common polymorphic variant. Positions are numbered according to the EU index as described in Kabat (SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th ed., NIH Publication No. 91-3242, EA Kabat et al.). The EU index or EU numbering scheme refers to the numbering of EU antibodies as described in Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85.

[0043] Figures 13A-13D Pharmacokinetics and pharmacodynamics of anti-BACE1 antibodies with modified Fc (anti-BACE1-YQAY and anti-BACE1-YY) in transgenic (Tg32) mice expressing hFCGRT and lacking mFCGRT. A) Serum antibody concentration; B) Brain pharmacokinetics (PK); C) Brain pharmacodynamics (PD) measured by brain Aβ levels; D) Summary of hFcRn affinity, brain PK data, and brain PD data.

[0044] Figures 14A-14C . Pharmacokinetics and pharmacodynamics of anti-BACE1 hIgG1 antibodies (anti-BACE1 WT) and anti-BACE1 hIgG1 antibodies with modified Fc (anti-BACE1-YEY, YQAY, YPY) in cynomolgus monkeys. A) CNS pharmacodynamics as measured by sAPPβ / α ratio in CSF at various times after antibody administration. B) Serum antibody concentrations at various times after antibody administration. C) Antibody serum exposure from day 0 to day 7 based on the data in (B).

[0045] Figures 15A-15J . Pharmacokinetics and pharmacodynamics of anti-BACE1 hIgG1 antibodies (anti-BACE1 WT or "WT") and anti-BACE1 hIgG1 antibodies with modified Fc (anti-BACE1-YQAY, YY, YLYI, YIY) in cynomolgus monkeys. A) CNS pharmacodynamics as measured by sAPPβ / α ratios in CSF at various times after antibody administration. B) Brain pharmacodynamics as measured by sAPPβ / α ratios from brain tissue on days 2 and 7. C) CSF versus brain pharmacodynamics as measured by sAPPβ / α ratios on days 2 and 7. D) Brain antibody concentrations (cortex and hippocampus) on days 2 and 7 after antibody administration. E) Fold changes in brain antibody concentrations compared to hIgG1 wild-type on days 2 and 7 based on the data in (B). F) Correlation of brain pharmacokinetics with brain pharmacodynamics. G) CSF antibody concentrations at various times after antibody administration. H) Ratio of antibody concentrations in CSF and serum at various times after antibody administration. I) Serum antibody concentrations at various times after antibody administration. J) Antibody serum exposure based on the data in (I).

[0046] Figures 16A-16F . Concentrations of anti-AβhIgG4 antibodies and anti-AβhIgG4 antibodies with modified Fc (anti-AβhIgG4-YTE) in plasma (A) and cerebellum (B) after administration at the indicated doses to PS2APP mice expressing mFCGRT. C) Binding of anti-AβhIgG4 ("Aβ"), anti-AβhIgG4 YTE ("AβYTE"), and anti-gD hIgG4 to oligomeric Aβ in the mossy fiber hippocampal tract of PS2APP mice after administration at the indicated doses to mice. D) Bar graph of the observed staining levels in (C). Binding of anti-AβhIgG4 and anti-AβhIgG4 YTE to target Aβ plaques in the subclavian (E) and prefrontal cortex (F) of PS2APP mice.

[0047] Figures 17A-17F.A) Mean brain concentrations of anti-AβhIgG4 wild-type ("WT") and anti-AβhIgG4 YY, YQAY, and YEY antibodies on days 2 and 7 after a single administration to cynomolgus monkeys. B) Fold changes in brain antibody concentrations compared to hIgG4 wild-type on days 2 and 7 based on the data in (A). C) CSF concentrations of anti-AβhIgG4 wild-type ("WT") and anti-AβhIgG4 YY, YQAY, and YEY antibodies after a single administration to cynomolgus monkeys. D) Ratios of antibody concentrations in CSF and serum after a single administration of anti-AβhIgG4 wild-type ("WT") and anti-AβhIgG4 YY, YQAY, and YEY to cynomolgus monkeys. E) Serum pharmacokinetics of anti-AβhIgG4 wild-type ("WT") antibody and anti-AβhIgG4 antibodies with modified Fc (YY, YQAY, and YEY) after administration to cynomolgus monkeys. F) Antibody serum exposure based on data in (E).

[0048] Figures 18A-18B .A) Correlation between antibody serum exposure and antibody affinity at pH 7.4 for both anti-BACE1 hIgG1 and anti-Aβ hIgG4 antibodies with modified Fc. B) Correlation between antibody brain partitioning relative to serum and antibody affinity at pH 7.4 for both anti-BACE1 hIgG1 and anti-Aβ hIgG4 antibodies with modified Fc. DETAILED DESCRIPTION

[0049] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated by the accompanying structures and formulae. Although the present invention will be described in conjunction with the enumerated embodiments, it will be understood that the present invention is not intended to be limited to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined by the scope of the claims.

[0050] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.The present invention is in no way limited to the methods and materials described.

[0051] Unless defined otherwise, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art and are in accordance with: Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons, New York, NY; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th ed., Garland Publishing, New York.

[0052] When a trade name is used herein, applicant intends to include independently the trade name product formulation, the generic drug, and one or more active pharmaceutical ingredients of the trade name product.

[0053] definition

[0054] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings:

[0055] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen, or IgG constant region or Fc and FcRn). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D , which is the ratio of the dissociation rate of X from Y (kd or koff) to the association rate of X with Y (ka or kon). An alternative measure of the affinity of one or more antibodies for their target is their half-maximal inhibitory concentration (IC50), which is a measure of how much antibody is required to inhibit the binding of a known ligand to the antibody target by 50%. Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.

[0056] "Blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord (i.e., CNS), which is formed by tight junctions within the plasma membrane of brain capillary endothelial cells, thereby establishing a tight barrier that limits the transport of molecules into the brain, even very small molecules, such as urea (60 Daltons). The blood-brain barrier in the brain, the blood-spinal barrier in the spinal cord, and the blood-retinal barrier in the retina are connected capillary barriers in the CNS and are collectively referred to herein as the blood-brain barrier or BBB. The BBB also encompasses the blood-CSF barrier (choroid plexus), in which the barrier comprises ependymal cells rather than capillary endothelial cells.

[0057] The terms "amyloid β", "β-amyloid", "Aβ", "amyloid β" and "Aβ" used interchangeably herein refer to fragments of amyloid precursor protein ("APP") produced after β-secretase 1 ("BACE1") and γ-secretase cleavage of APP, as well as modifications, fragments and any functional equivalents thereof, including but not limited to Aβ1-40 and Aβ1-42. Aβ is known to exist in monomeric form, as well as to associate to form oligomers and fibril structures, which can be found as constituent members of amyloid plaques. The structure and sequence of such Aβ peptides are well known to those of ordinary skill in the art, and methods for producing the peptides or extracting the peptides from brain and other tissues are described, for example, in Glenner and Wong, Biochem Biophys Res. Comm. 129: 885-890 (1984). In addition, Aβ peptides are also commercially available in various forms.

[0058] "Anti-Aβ immunoglobulin", "anti-Aβ antibody" and "antibody that binds to Aβ" are used interchangeably herein and refer to an antibody that specifically binds to human Aβ. A non-limiting example of an anti-Aβ antibody is crenezumab. Other non-limiting examples of anti-Aβ antibodies are solanezumab, bapineuzumab, gantenerumab, aducanumab, ponezumab, and any of the anti-Aβ antibodies disclosed in the following publications: WO2000162801, WO2002046237, WO2002003911, WO2003016466, WO2003016467, WO2003016468, WO2003016469, WO2003016461, WO2003016462, WO2003016463, WO2003016464, WO2003016465, WO2003016466, WO2003016467, WO2003016468, WO2003016467, WO2003016468, WO2003016469 ... O2003016467, WO2003077858, WO2004029629, WO2004032868, WO2004032868, WO2004108895, WO2005028511, WO2006039470, WO2006036291, WO2006066089, WO2006066171, WO2006066049, WO2006095041, WO2009027105.

[0059] The terms "clenizumab" and "MABT5102A" are used interchangeably herein and refer to a specific anti-Aβ antibody that binds to monomeric, oligomeric and fibrillar forms of Aβ and is associated with CAS Registry No. 1095207.

[0060] As used herein, the term "amyloidosis" refers to a group of diseases and disorders caused by or associated with amyloid or amyloid-like proteins and includes, but is not limited to, diseases and disorders caused by the presence or activity of amyloid-like proteins in a monomeric, fibrillar or polymeric state or any combination of the three, including those caused by amyloid plaques. Such diseases include, but are not limited to, secondary amyloidosis and age-related amyloidosis, such as, but not limited to, the following diseases: neurological disorders, such as Alzheimer's Disease (Alzheimer's Disease, "AD"); diseases or conditions characterized by loss of cognitive memory abilities, such as mild cognitive impairment (MCI), Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam Parkinson-Dementia complex; and other diseases based on or associated with amyloid-like proteins, such as progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease (Creutzfeld Jacob disease), Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), adult-onset diabetes, endocrine tumors and senile cardiac amyloidosis; and various eye diseases, including macular degeneration, choroidal drusen-related optic neuropathy, glaucoma and cataracts due to β-amyloid deposition.

[0061] Glaucoma is a group of optic nerve diseases involving the loss of retinal ganglion cells (RGCs) in a characteristic pattern of optic neuropathy. RGCs are nerve cells that transmit nerve signals from the eye to the brain. Two major enzymes in the apoptotic process, caspase-3 and caspase-8, are activated in the process, leading to RGC apoptosis. Caspase-3 cleaves amyloid precursor protein (APP) to produce neurotoxic fragments, including Aβ. Without the protective effect of APP, Aβ accumulation in the retinal ganglion cell layer causes RGC death and irreversible visual loss.

[0062] Glaucoma is often, but not always, accompanied by an increase in intraocular pressure, which may be the result of a blockage in the circulation of the aqueous humor or its drainage. Although elevated intraocular pressure is a significant risk factor for developing glaucoma, it is not possible to define a critical value of intraocular pressure that will be decisive for causing glaucoma. Damage can also be caused by poor blood supply to the vital optic nerve fibers, weakening of the nerve structure, and / or health problems with the nerve fibers themselves. Untreated glaucoma leads to permanent damage to the optic nerve and resulting visual field loss, which can progress to blindness.

[0063] "Central nervous system" or "CNS" refers to the complex of nervous tissue that controls body functions and includes the brain and spinal cord.

[0064] As used herein, "neurological disorder" refers to a disease or condition that affects the CNS and / or has a source of disease in the CNS. Exemplary CNS diseases or conditions include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or conditions, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, epileptic seizures, behavioral disorders, and lysosomal storage diseases. For purposes of this application, the CNS will be understood to include the eye, which is typically isolated from the rest of the body by the blood-retinal barrier. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Strauss-Schönlein disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and heterogeneous degenerative disorders of the nervous system (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar disorders), cancer (e.g., CNS cancer, including brain metastases caused by cancer elsewhere in the body).

[0065] "Neurodisorder drugs" are drugs or therapeutic agents for treating one or more neurological disorders. Neurodisorder drugs of the present invention include, but are not limited to, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes and small molecules, or active fragments of any of the foregoing. Exemplary neurodisorder drugs of the present invention are described herein and include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules and active fragments of any of the foregoing, which themselves or specifically recognize and / or act on (i.e., inhibit, activate or detect) CNS antigens or target molecules, such as but not limited to amyloid precursor protein or a portion thereof, amyloid β, β-secretase, γ-secretase, Tau, α-synuclein, Parkin, Huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins. Non-limiting examples of neurological disorder drugs and conditions they can be used to treat are provided in Table 1 below:

[0066] Table 1: Non-limiting examples of neurological disorder drugs and corresponding conditions they can be used to treat

[0067]

[0068] An "imaging agent" is a compound that has one or more properties that permit direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins and small molecule compounds that incorporate a labeled moiety that permits detection.

[0069] "CNS antigens" or "brain antigens" are antigens expressed in the CNS, including the brain, which can be targeted with antibodies or small molecules. Examples of such antigens include, but are not limited to, β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6R), interleukin 1β (IL1β), caspase 6, triggering receptor expressed on myeloid cells 2 (TREM2), C1q, paired immunoglobulin-like type 2 receptor alpha (PILRA), CD33, interleukin 6 (IL6), tumor necrosis factor alpha (TNFα), tumor necrosis factor receptor superfamily member 1A (TNFR1), tumor necrosis factor receptor superfamily member 1B (TNFR2) and apolipoprotein J (ApoJ). In one embodiment, the antigen is BACE1.

[0070] Unless otherwise indicated, the term "BACE1" as used herein refers to any native β-secretase 1 (also known as β-site amyloid precursor protein cleaving enzyme 1, membrane-associated aspartic protease 2, membrane aspartic protease (memapsin) 2, aspartyl protease 2 or Asp2) from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed BACE1 and any form of BACE1 produced by processing in cells. The term also encompasses naturally occurring variants of BACE1, such as splice variants or allelic variants. The amino acid sequence of an exemplary BACE1 polypeptide is the sequence of human BACE1 isoform A as reported in Vassar et al., Science 286: 735-741 (1999), which is incorporated herein by reference in its entirety. There are several other isoforms of human BACE1, including isoforms B, C and D. See UniProtKB / Swiss-ProtEntry P56817, incorporated herein by reference in its entirety.

[0071] The terms "anti-β-secretase antibody", "anti-BACE1 antibody", "antibody that binds to β-secretase" and "antibody that binds to BACE1" refer to an antibody that is capable of binding to BACE1 with sufficient affinity to make the antibody useful as a diagnostic and / or therapeutic agent in targeting BACE1. In one embodiment, the extent of binding of the anti-BACE1 antibody to unrelated non-BACE1 proteins is less than about 10% of the binding of the antibody to BACE1 as measured by, for example, radioimmunoassay (RIA). In certain embodiments, the antibody that binds to BACE1 has an equilibrium dissociation constant (K) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10-8 M or less, e.g., 10-8 M to 10-13 M, e.g., 10-9 M to 10-13 M) D ). In certain embodiments, the anti-BACE1 antibody binds to an epitope of BACE1 that is conserved among BACE1 from different species and isoforms. In other embodiments, an antibody is provided that binds to an exosite within BACE1 that is outside the catalytic domain of BACE1. In one embodiment, an antibody is provided that competes for binding to BACE1 with the peptides identified in Kornacker et al., Biochem. 44: 11567-11573 (2005), which is incorporated herein by reference in its entirety (i.e., peptides 1, 2, 3, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 4, 5, 6, 5-10, 5-9, scrambled, Y5A, P6A, Y7A, F8A, I9A, P10A, and L11A). Non-limiting exemplary anti-BACE1 antibodies are described, for example, in WO 2012 / 064836 and 2016 / 081639.

[0072] A "native sequence" protein herein refers to a protein comprising the amino acid sequence of a protein found in nature, including naturally occurring variants of the protein. The term as used herein includes proteins isolated from their natural sources or produced recombinantly.

[0073] 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 (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0074] A "reference antibody" herein is an antibody that lacks the characteristics of a test antibody. In some embodiments, a reference antibody for an Fc-modified antibody is an antibody having the same variable regions and belonging to the same isotype, but lacking one or more Fc modifications. In some embodiments, a reference antibody for an Fc-modified antibody is an antibody having one or more of the same variable regions and isotype, but having a wild-type Fc.

[0075] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments are well known in the art (see, e.g., Nelson, MAbs (2010) 2(1):77-83) and include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, and Fv; diabodies; linear antibodies; single-chain antibody molecules, including but not limited to single-chain variable fragments (scFv); fusions of light and / or heavy chain antigen-binding domains with or without a linker (and optionally in series); and monospecific or multispecific antigen-binding molecules formed from antibody fragments (including but not limited to multispecific antibodies constructed from multiple variable domains lacking an Fc).

[0076] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies constituting the population are identical and / or bind to the same epitope, differing in that they contain, for example, naturally occurring mutations or possible variants that may occur during the production of monoclonal antibodies, such variants generally being present in trace amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be made by a variety of techniques, including but not limited to the hybridoma method (see, e.g., Kohler et al., Nature, 256: 495 (1975)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display methods (e.g., using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies are described herein. Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies, including antigen-binding fragments thereof.

[0077] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0078] "Acceptor human framework" for the purposes of this article 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. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, 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 sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0079] "Human consensus framework" is a framework that represents the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. In general, the subgroup of sequences is a subgroup such as Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Vol. 1-3. In one embodiment, for VL, the subgroup is a subgroup κI such as Kabat et al., supra. In one embodiment, for VH, the subgroup is a subgroup III such as Kabat et al., supra.

[0080] The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human antibody. For the most part, humanized antibodies are human antibodies (receptor antibodies) in which residues from the hypervariable region of the recipient are replaced by residues from non-human species (donor antibodies), such as mice, rats, rabbits, or non-human primates, and have the desired specificity, affinity, and ability. For example, in certain embodiments, a humanized antibody will include substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all of the framework regions (FRs) correspond to those of human antibodies. In some cases, the FR residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. 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 hypervariable regions correspond to those of a non-human antibody and all or substantially all of the FRs are those of a human antibody, except for one or more FR substitutions as mentioned above. A humanized antibody will optionally also comprise at least a portion of an antibody constant region, typically that of a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0081] "Human antibody" herein is an antibody comprising an amino acid sequence structure corresponding to that produced by a human or human cell or derived from an antibody of a non-human origin utilizing a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Such antibodies can be identified or made by a variety of techniques, including, but not limited to, production by transgenic animals (e.g., mice) capable of producing human antibodies in the absence of endogenous immunoglobulin production upon immunization (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369, and 5,545,807); selection from phage display libraries expressing human antibodies or human antibody fragments (see, e.g., McCafferty et al., Nature, 348:552-553 (1990); Johnson et al., Current Opinion in Structural Biology 3:564-571 (1993); Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991); Griffith et al., EMBO J. 12:725-734 (1993); U.S. Pat. Nos. 5,565,332 and 5,573,905); produced by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275); and isolated from hybridomas producing human antibodies.

[0082] "Multispecific antibodies" herein are antibodies that have binding specificity for at least two different epitopes. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Engineered antibodies with two, three or more (e.g., four) functional antigen binding sites are also contemplated (see, e.g., U.S. Application No. US2002 / 0004587 A1, Miller et al.). Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0083] The antibodies herein include "amino acid sequence variants" with altered antigen binding or biological activity. Examples of such amino acid changes include antibodies with enhanced affinity for the antigen (e.g., "affinity matured" antibodies), and with altered Fc (if present), such as antibodies with altered (increased or decreased) antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) (see, e.g., WO 00 / 42072, Presta, L. and WO 99 / 51642, Iduosogie et al.); and / or increased or decreased serum half-life (see, e.g., WO 00 / 42072, Presta, L.).

[0084] "Affinity-modified variants" have one or more substituted hypervariable regions or framework residues of a parent antibody (e.g., a parent chimeric, humanized, or human antibody) that changes (increases or decreases) affinity. A convenient way to generate such substitution variants uses phage display. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are presented as fusions with the gene III product of M13 encapsulated in each particle from filamentous phage particles in a monovalent manner. The phage-displayed variants are then screened for biological activity (e.g., binding affinity). To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or in addition, the crystal structure of the antigen-antibody complex can be advantageously analyzed to identify the contact points between the antibody and its target. Such contact residues and adjacent residues are candidates for replacement according to the techniques described herein. Once such variants are generated, the panel of variants is screened and antibodies with altered affinities can be selected for further development.

[0085] The modified IgG Fc herein or an antibody or fusion protein comprising the modified IgG Fc may be conjugated to, for example, a "heterologous molecule" to increase half-life or stability or otherwise improve the antibody. For example, the modified IgG Fc herein or an antibody or fusion protein comprising the modified IgG Fc may be connected to one of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. In some embodiments, the heterologous molecule is a therapeutic compound or a developer (i.e., a detectable marker), and the IgG Fc is used to transport such a heterologous molecule across the BBB. Examples of heterologous molecules include, but are not limited to, compounds, peptides, polymers, lipids, nucleic acids, and proteins.

[0086] The modified Fc herein may be a "glycosylation variant" such that any carbohydrate attached to the Fc is altered, modified in terms of presence / absence, or modified in terms of type. For example, an antibody having a mature carbohydrate structure lacking fucose attached to the antibody Fc is described in US 2003 / 0157108 (Presta, L.). See also US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies having bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the antibody Fc are mentioned in WO 2003 / 011878, Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies having at least one galactose residue in the oligosaccharide attached to the antibody Fc are reported in WO 1997 / 30087, Patel et al. See also WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.) relating to antibodies with altered carbohydrates attached to the Fc. See also US 2005 / 0123546 (Umana et al.) describing antibodies with modified glycosylation. Mutation of the consensus glycosylation sequence in Fc (Asn-X-Ser / Thr at positions 297-299, wherein X cannot be proline), for example by mutating the Asn of this sequence to any other amino acid, by substituting Pro at position 298, or by modifying position 299 to any amino acid other than Ser or Thr, should eliminate glycosylation at that position (see, e.g., Fares Al-Ejeh et al., Clin. Cancer Res. (2007) 13:5519s-5527s; Imperiali and Shannon, Biochemistry (1991) 30(18):4374-4380; Katsuri, Biochem J. (1997) 323(Part 2):415-419; Shakin-Eshleman et al., J. Biol. Chem. (1996) 271:6363-6366).

[0087] The term "hypervariable region" or "HVR", as used herein, refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). In general, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0088] Exemplary HVRs herein include:

[0089] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0090] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0091] (c) antigenic contacts occurring 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

[0092] (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).

[0093] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0094] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4. In certain embodiments, one or more FR residues may be modified to modulate the stability of the antibody or to modulate the three-dimensional positioning of one or more HVRs of the antibody, for example to enhance binding.

[0095] A "full-length antibody" is an antibody comprising an antigen-binding variable region and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

[0096] 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 having heavy chains that contain an Fc as defined herein.

[0097] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety or a radiolabel). Naked antibodies can be present in pharmaceutical formulations.

[0098] "Native antibodies" refer to naturally occurring immunoglobulin molecules with variable structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be designated as one of two types based on the amino acid sequence of its constant domain, called kappa (κ) and lambda (λ).

[0099] "Effector function" refers to those biological activities of an antibody that cause immune system activation other than complement pathway activation. Such activities are primarily found in the Fc of an antibody (such as the modified Fc herein). Examples of effector functions include, for example, Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the modified Fc herein substantially lacks effector function. In another embodiment, the modified Fc herein retains minimal effector function. Methods for modifying or eliminating effector function are well known in the art and include, but are not limited to, modifying the Fc at one or more amino acid positions to eliminate effector function (affecting Fc binding: positions 238, 239, 248, 249, 252, 254, 256, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 311, 322, 324, 327, 329, 333, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365 and modifying the glycosylation of Fc (including but not limited to producing the antibody in an environment that does not allow wild-type mammalian glycosylation, removing one or more carbohydrate groups from an already glycosylated antibody, and modifying the Fc at one or more amino acid positions to eliminate the ability of the antibody to be glycosylated at those positions (including but not limited to N297G and N297A and D265A)).

[0100] "Complement activation" function or antibody properties capable of or triggering "complement pathway activation" are used interchangeably and refer to those biological activities of an antibody that engage or stimulate the complement pathway of the immune system in a subject. Such activities include, for example, C1q binding and complement dependent cytotoxicity (CDC), and may be mediated by both the Fc portion and the non-Fc portion of the antibody. Methods for modifying or eliminating complement activation function are well known in the art and include, but are not limited to, modifying the Fc at one or more amino acid positions to eliminate or reduce interaction with complement components or the ability to activate complement components, such as positions 270, 322, 329, and 321 known to be involved in C1q binding; and modifying or eliminating a portion of the non-Fc responsible for complement activation (i.e., eliminating or modifying the CH1 region at position 132 (see, e.g., Vidarte et al., (2001) J. Biol. Chem. 276(41):38217-38223)).

[0101] Based on the amino acid sequence, the Fc domain and the antibodies of which the Fc domain is a part can be designated as different "classes". There are five major classes of Fc domains: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art. The heavy chain constant region includes a CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0102] The term "recombinant antibody" as used herein refers to an antibody (eg, a chimeric, humanized or human antibody or antigen-binding fragment thereof) expressed by a recombinant host cell comprising nucleic acid encoding the antibody.

[0103] The term "recombinant protein" as used herein refers to a protein expressed by a recombinant host cell comprising a nucleic acid encoding the protein (such as an Fc conjugate comprising a modified Fc herein).

[0104] 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," which include the primary transformed cell and progeny derived therefrom regardless of the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the original transformed cell are included herein. Examples of "host cells" for producing recombinant antibodies and proteins include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect cells, such as sf9, sf21 and Tn5; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells, etc.

[0105] As used herein, "specifically binds" or "specifically binds to" means that the antibody selectively or preferentially binds to the antigen. Binding affinity is generally determined using standard assays such as Scatchard analysis or surface plasmon resonance techniques (e.g., using ) to determine.

[0106] An "antibody that binds to the same epitope" as a reference antibody is one that blocks binding of the reference antibody to its antigen by 50% or greater in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or greater in a competition assay.

[0107] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed herein.

[0108] An "effective amount" of an agent, such as a pharmaceutical preparation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0109] The term "Fc region" or "Fc" herein is used to define the C-terminal region of an immunoglobulin heavy chain comprising the heavy chain constant domains CH2 and CH3, or a portion of the heavy chain constant domains CH2 and CH3 sufficient to bind to FcRn at pH 6, pH 7.4, or pH 6 and pH 7.4. The term includes native sequence Fc and modified Fc. In some embodiments, the human IgG heavy chain Fc extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc or constant region is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991, also known as the EU index. Exemplary human IgG1, IgG2, IgG3 and IgG4 Fc amino acid sequences are shown in Fig.12 and SEQ ID NO: 1-4. In some embodiments, the Fc-containing antibody also comprises a heavy chain constant domain CH1 and a hinge.

[0110] As used herein, the term "FcRn receptor" or "FcRn" refers to an Fc receptor known to be involved in the transfer of maternal IgG to the fetus via the human or primate placenta or yolk sac (rabbit), and from colostrum via the small intestine to the neonate ("n" indicates neonate). FcRn is also known to be involved in maintaining constant serum IgG levels by binding IgG molecules and recycling them into the serum.

[0111] A "conjugate" is an antibody or Fc conjugated to one or more heterologous molecules. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules. An "Fc conjugate" is an Fc conjugated to one or more heterologous molecules. Non-limiting examples of such heterologous molecules include proteins, enzymes, labels, and cytotoxic agents. Optionally, such conjugation is via a linker. In some embodiments, the Fc conjugate is an "Fc fusion" in which the Fc is fused to a heterologous protein into a continuous amino acid sequence.

[0112] As used herein, a "linker" is a structure that covalently or non-covalently connects a first molecule to a second molecule. In certain embodiments, the linker is a peptide. In other embodiments, the linker is a chemical linker.

[0113] "Label" is a marker coupled to the antibody herein and used for detection or imaging. Examples of such labels include: radiolabels, fluorophores, chromophores, or affinity tags. In one embodiment, the label is a radiolabel for medical imaging, such as tc99m or I123; or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc.

[0114] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0115] An "isolated" antibody is one that has been separated from the 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 reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0116] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain 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.

[0117] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such (such) nucleic acid molecules in a single vector or separate vectors, and such (such) nucleic acid molecules present at one or more locations in a host cell.

[0118] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0119] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in various ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One 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 sequences being compared. For purposes herein, however, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was compiled by Genentech, Inc., and the source code has been filed with user files in the US Copyright Office, Washington DC, 20559, registered under US 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 UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0120] In the case of employing ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows:

[0121] 100 times the fraction X / Y

[0122] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be appreciated 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 be equal to 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.

[0123] The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0124] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical preparation 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.

[0125] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed for prevention or during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease condition, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease development or slow disease progression.

[0126] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). 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 from antibodies that bind to the antigen using VH or VL domains, respectively, to screen libraries of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0127] The term "vector" as used herein refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. The term includes vectors that are autonomously replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0128] 1. Compositions and methods

[0129] A. Antibodies and Fc Conjugates

[0130] In one aspect, the present invention is based in part on a modified Fc that can be used to transport desired molecules across the BBB. In certain embodiments, antibodies comprising a modified Fc are provided. In certain embodiments, Fc conjugates comprising a modified Fc are provided. In some embodiments, the Fc conjugate comprises a modified Fc provided herein fused to a protein, such as a therapeutic protein and / or a detectable protein. In such embodiments, the Fc conjugate may be referred to as an "Fc fusion". The antibodies and Fc conjugates of the present invention are suitable for use in, for example, the diagnosis or treatment of diseases that affect the brain and / or CNS.

[0131] A. Exemplary Modified Fc

[0132] Provided herein are antibodies and Fc conjugates comprising a modified Fc, wherein these antibodies and Fc conjugates are active in an in vitro transcytosis assay. In some embodiments, antibodies and Fc conjugates comprising a modified Fc have improved brain uptake. In some embodiments, the modified Fc can be used to improve the delivery of antibodies or Fc conjugates to the brain or central nervous system of a subject. In some embodiments, the modified Fc herein improves transport across the blood-brain barrier (BBB).

[0133] In some embodiments, certain antibodies and Fc conjugates comprising a modified Fc provided herein exhibit a transcytosis activity of at least 50 in an in vitro transcytosis assay. In some embodiments, certain antibodies and Fc conjugates comprising a modified Fc provided herein exhibit a transcytosis activity of at least 30, or at least 40, or at least 50 in an in vitro transcytosis assay when normalized to the same antibody or Fc conjugate comprising a wild-type IgG Fc. In some embodiments, certain antibodies and Fc conjugates comprising a modified Fc provided herein exhibit a transcytosis activity of at least 60, at least 70, at least 80, at least 90, or at least 100 in an in vitro transcytosis assay. Non-limiting exemplary transcytosis assays are described in the Assay section of this article. In some embodiments, the in vitro transcytosis assay comprises cells expressing FcRn. In some embodiments, the FcRn is a human FcRn. In some embodiments, the cell is an MDCK II cell.

[0134] In some embodiments, certain antibodies and Fc conjugates provided herein comprising a modified Fc have a binding affinity for FcRn (e.g., human FcRn) at pH 7.4 that is greater than the binding affinity of a reference antibody or Fc conjugate of the same class and isotype with an unmodified IgG Fc. In some embodiments, certain antibodies and Fc conjugates provided herein comprising a modified Fc have a binding affinity for FcRn (e.g., human FcRn) at pH 6 that is greater than the binding affinity of a reference antibody or Fc conjugate of the same class and isotype with an unmodified IgG Fc. In some embodiments, certain antibodies and Fc conjugates comprising a modified Fc provided herein have a binding affinity for FcRn (e.g., human FcRn) at pH 7.4 of ≤10 μM, ≤5 μM, ≤4 μM, ≤3 μM, ≤2 μM, ≤1 μM, ≤900 nM, ≤800 nM, ≤700 nM, ≤600 nM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, or ≤100 nM. In some embodiments, certain antibodies and Fc conjugates comprising a modified Fc provided herein have a binding affinity for FcRn (e.g., human FcRn) at pH 6 of ≤1 μM, ≤900 nM, ≤800 nM, ≤700 nM, ≤600 nM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, ≤100 nM, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, or ≤10 nM. In some embodiments, the ratio of the affinity of the antibody or Fc conjugate comprising a modified IgG Fc for FcRn, e.g., human FcRn, at pH 7.4 to the affinity of the antibody or Fc conjugate comprising a modified IgG Fc for FcRn, e.g., human FcRn, at pH 6 is at least 5, at least 10, at least 20, at least 50, or at least 100; or 5 to 200, 5 to 100, 10 to 200, 10 to 100, 20 to 100, or 20 to 200.

[0135] In various embodiments, the modified Fc provided herein comprises one or more mutations selected from the following: 252W, 252Y, 286E, 286Q, 307Q, 308P, 310A, 311A, 311I, 428L, 433K, 434F, 434W, 434Y and 436I according to EU numbering. In some embodiments, the modified Fc comprises 252Y and 434Y. In some embodiments, the modified Fc comprises 252Y and 434Y and one or two other mutations selected from the following: 286E, 286Q, 307Q, 308P, 311A, 311I, 428L, 433K and 436I. In some embodiments, the modified Fc further comprises 307Q and 311A, or further comprises 286E. In some embodiments, the modified Fc comprises a group of mutations selected from the mutation groups in Table 4, Table 5 and Table 6. In some embodiments, the modified Fc comprises one or more modifications of an IgG sequence selected from SEQ ID NO: 1-4. In some embodiments, the modified Fc is an IgG1 Fc. In some embodiments, the modified Fc is an IgG4 Fc. In some embodiments, the IgG Fc is an IgG2 or IgG3 Fc.

[0136] In some embodiments, a modified Fc is provided which, in the context of an antibody or Fc conjugate, has a normalized transcytosis score of at least 30. Non-limiting examples of such modified Fcs include modified Fcs comprising the following sets of mutations: 252W / 434W; 252Y / 434Y; 252Y / 286E / 434Y; 252Y / 307Q / 434Y; 252Y / 308P / 434Y; 252Y / 311A / 434Y; 252Y / 311I / N434Y; 252Y / 428L / 434Y; 252Y / 433K / 434Y; 252Y / 434Y / 436I; 286E / 311A / 434Y; 286E / 311I / 434Y; 286E / 433K / 434Y; 286E / 434Y / 436I; 307Q / 286E / 434Y; 307Q / 311A / 434Y; 3 07Q / 311I / 434Y; 307Q / 433K / 434Y; 307Q / 434Y / 436I; 311A / 428L / 434Y; 311A / 43 3K / 434Y; 311I / 433K / 434Y; 433K / 434Y / 436I; 252Y / 307Q / 311A / 434Y; 252Y / 307 In some embodiments, the modified Fc listed above is a modified IgG1 Fc. In some embodiments, the modified Fc listed above is a modified IgG4 Fc. In some embodiments, the modified Fc listed above is a modified IgG2 or IgG3 Fc.

[0137] In some embodiments, a modified Fc is provided which has a normalized transcytosis score of at least 70 in the context of an antibody or Fc conjugate. Non-limiting examples of such modified Fcs include modified Fcs comprising the following sets of mutations: 252W / 434W; 252Y / 434Y; 252Y / 286E / 434Y; 252Y / 307Q / 434Y; 252Y / 308P / 434Y; 252Y / 311A / 434Y; 252Y / 311I / N434Y; 252Y / 428L / 434Y; 252Y / 433K / 434Y; 252Y / 434Y / 436I; 286E / 311A / 434Y; 286E / 311I / 434Y; 286E / 434Y / 436I; 307Q / 286E / 434Y; 307Q / 311A ... 07Q / 311I / 434Y; 307Q / 433K / 434Y; 307Q / 434Y / 436I; 311A / 428L / 434Y; 311 I / 433K / 434Y; 433K / 434Y / 436I; 252Y / 307Q / 311A / 434Y; 252Y / 307Q / 311I / 4 In some embodiments, the modified Fc listed above is a modified IgG1 Fc. In some embodiments, the modified Fc listed above is a modified IgG4 Fc. In some embodiments, the modified Fc listed above is a modified IgG2 or IgG3 Fc.

[0138] In some embodiments, a modified Fc is provided which, in the context of an antibody or Fc conjugate, has a normalized transcytosis score of at least 100. Non-limiting examples of such modified Fcs include modified Fcs comprising the following sets of mutations: 252Y / 307Q / 434Y; 252Y / 311A / 434Y; 252Y / 311I / N434Y; 252Y / 428L / 434Y; 252Y / 433K / 434Y; 252Y / 434Y / 436I; 286E / 311A / 434Y; 307Q / 311I / 434Y; 307Q / 434Y / 436I; 311A / 428L / 434Y; In some embodiments, the modified Fc listed above is a modified IgG1 Fc. In some embodiments, the modified Fc listed above is a modified IgG4 Fc. In some embodiments, the modified Fc listed above is a modified IgG2 or IgG3 Fc.

[0139] Non-limiting additional modified Fcs are provided and can be selected using, for example, an in vitro transcytosis assay described herein. Various exemplary modified Fcs are known in the art and can be selected using an in vitro transcytosis assay for use in the antibodies and Fc conjugates herein. Non-limiting examples of such modified Fcs that can be assayed for transcytosis activity include, for example, those described in U.S. Publication Nos. 2015 / 0050269 and 2013 / 0131319, which are incorporated herein by reference in their entirety for any purpose.

[0140] 1. Modified Fc affinity

[0141] In some embodiments, provided herein are modified Fcs having an equilibrium dissociation constant (K) for FcRn at pH 7.4 of D )≤10μM, ≤5μM, ≤4μM, ≤3μM, ≤2μM, ≤1μM, ≤900nM, ≤800nM, ≤700nM, ≤600nM, ≤500nM, ≤400nM, ≤300nM, ≤200nM or ≤100nM. In some embodiments, provided herein is a modified Fc having an equilibrium dissociation constant (K) for FcRn at pH 7.4. D) is between 100 nM and 10 μM, or between 100 nM and 5 μM, or between 100 nM and 2 μM, or between 100 nM and 1 μM. In some embodiments, provided herein is a modified Fc having an equilibrium dissociation constant (K) for FcRn at pH 6. D )≤1 μM, ≤900nM, ≤800nM, ≤700nM, ≤600nM, ≤500nM, ≤400nM, ≤300nM, ≤200nM, ≤100nM, ≤90nM, ≤80nM, ≤70nM, ≤60nM, ≤50nM, ≤40nM, ≤30nM, ≤20nM or ≤10nM. In some embodiments, provided herein is a modified Fc having an equilibrium dissociation constant (K) for FcRn at pH 6. D ) is between 10 nM and 1 μM, or between 10 nM and 750 nM, or between 10 nM and 500 nM, or between 10 nM and 200 nM, or between 10 nM and 100 nM.

[0142] In some embodiments, the modified Fc provided herein binds to FcRn at pH 7.4 and binds to FcRn at pH 6, wherein the K at pH 7.4 is D With K at pH 6 D The ratio of K to FcRn is at least 5, at least 10, at least 20, at least 50, or at least 100. In some embodiments, the modified Fc provided herein binds to FcRn at pH 7.4 and binds to FcRn at pH 6, wherein the K at pH 7.4 is D With K at pH 6 D The ratio is 5 to 200, 5 to 100, 10 to 200, 10 to 100, 20 to 100 or 20 to 200.

[0143] In various embodiments, the FcRn is human FcRn.

[0144] In some embodiments, K is measured using surface plasmon resonance. D In some such embodiments, the K was measured using a BIAcore-2000 device (BIAcore, Inc., Piscataway, NJ). D. For example, the modified Fc is immobilized via protein-L binding at a surface density of 400-1000 RU, or by using an anti-human Fab capture chip at a surface density of 10-100 RU. Neutral pH binding can be determined, for example, in HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% v / v surfactant P20). Acidic pH binding can be determined, for example, in MBS-P (0.01 M MESS pH 7.5, 0.15 M NaCl, 0.005% v / v surfactant P20). A one-to-one Langmuirbinding model ( The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams using the ELISA software version 4.1. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). Alternatively, Kd can be determined from the dependence of the steady-state binding level on the analyte concentration. D In some embodiments, so-called steady-state analysis is particularly suitable for the measurement of weak to moderate interactions.

[0145] 2. Modified Fc variants

[0146] In certain embodiments, the amino acid sequence variants of the modified Fc provided herein are contemplated. For example, it may be necessary to improve the binding affinity and / or other biological properties of the modified Fc variants. The amino acid sequence variants of Fc can be prepared by introducing appropriate modifications into the nucleotide sequence encoding Fc, or by peptide synthesis. Such modifications include, for example, the deletion and / or insertion and / or substitution of residues in the amino acid sequence of Fc. Any combination of deletion, insertion and substitution can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0147] a) Substitution, insertion and deletion variants

[0148] In certain embodiments, Fc variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in Table 2A under the heading "Preferred Substitutions". More substantial changes are provided in Table 2A under the heading "Exemplary Substitutions" and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the target Fc, and the products can be screened for desired activities, such as reduced immunogenicity or improved ADCC or CDC.

[0149] Table 2A

[0150] Original residue Exemplary substitutions Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

[0151] Amino acids can be grouped according to common side chain properties:

[0152] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0153] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0154] (3) Acidic: Asp, Glu;

[0155] (4) Basic: His, Lys, Arg;

[0156] (5) Residues that affect chain orientation: Gly, Pro;

[0157] (6) Aromatic: Trp, Tyr, Phe.

[0158] Non-conservative substitutions will require exchanging a member of one of these classes for another class.

[0159] A useful method for identifying Fc residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether Fc interacts with FcRn. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitutions. Alternatively or in addition, the crystal structure of the Fc-FcRn complex is used to identify contact points between Fc and FcRn. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0160] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include Fc with an N-terminal methionyl residue. Other insertion variants of Fc molecules include fusion of the N- or C-terminus of Fc with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of Fc.

[0161] b) Glycosylation variants

[0162] In certain embodiments, the modified Fc provided herein is altered to increase or decrease the extent of Fc glycosylation. The addition or deletion of glycosylation sites of Fc can be conveniently achieved by altering the amino acid sequence so as to create or remove one or more glycosylation sites.

[0163] The native Fc produced by mammalian cells typically comprises a branched, biantennary oligosaccharide generally connected to the Asn297 of the CH2 domain of the Fc by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose connected to the GlcNAc in the "trunk" of the biantennary oligosaccharide structure. In some embodiments, modifications of oligosaccharides in the Fc of the present invention may be made to create Fc variants with certain improved properties.

[0164] In one embodiment, an Fc variant is provided that has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc. For example, the amount of fucose in such an Fc may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures (e.g., complex, mixed, and high mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc residues) in Fc; however, due to minor sequence variations in Fc, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications on "defucosylated" or "fucose-deficient" Fc variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated Fc include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO 2003 / 085107).

[0165] Fc variants further have bisecting oligosaccharides, for example, wherein the biantennary oligosaccharides attached to Fc are bisected by GlcNAc. Such Fc variants may have reduced fucosylation and / or improved ADCC function. Examples of such Fc variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Fc variants having at least one galactose residue in the oligosaccharide attached to Fc are also provided. Such Fc variants may have improved CDC function. Such Fc variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0166] c) Cysteine ​​engineered Fc variants

[0167] In certain embodiments, it may be desirable to create a cysteine ​​engineered Fc variant in which one or more residues of the modified Fc are replaced with cysteine ​​residues. In particular embodiments, the substituted residues occur at accessible sites of the modified Fc. As further described herein, by replacing these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the modified Fc and can be used to conjugate the modified Fc to other moieties, such as drug moieties or linker-drug moieties, to create Fc conjugates. For example, a cysteine ​​engineered Fc can be produced as described in U.S. Patents Nos. 7,521,541 and 9,000,130.

[0168] d) Modified Fc derivatives

[0169] In certain embodiments, the modified Fc provided herein may be further modified to contain additional non-proteinaceous parts known in the art and readily available. Suitable parts for modified Fc derivatization 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, polypropylene glycol homopolymers, polyoxypropylene / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing 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 modified Fc can vary, and if more than one polymer is attached, they can 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 specific properties or functions of the modified Fc to be improved, whether the modified Fc derivative will be used in therapy under defined conditions, etc.

[0170] In another embodiment, a conjugate of a modified Fc and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the nonproteinaceous moiety to a temperature that kills cells that are closest to the modified Fc-nonproteinaceous moiety.

[0171] B. Exemplary Antibodies

[0172] In various embodiments, antibodies comprising a modified Fc herein are provided. In certain aspects, the modified Fc can improve the transport of antibodies across the BBB. In some embodiments, antibodies comprising a modified Fc herein bind to brain antigens. Non-limiting examples of such brain antigens include β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E (ApoE), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p 75 neurotrophin receptor (p75NTR), interleukin 6 receptor (IL6R), interleukin 1β (IL1β), caspase 6, triggering receptor expressed on myeloid cells 2 (TREM2), C1q, paired immunoglobulin-like type 2 receptor alpha (PILRA), CD33, interleukin 6 (IL6), tumor necrosis factor alpha (TNFα), tumor necrosis factor receptor superfamily member 1A (TNFR1), tumor necrosis factor receptor superfamily member 1B (TNFR2) and apolipoprotein J (ApoJ).

[0173] In yet another aspect, the antibody comprising the modified Fc herein may incorporate any of the features described in the following sections 1-7, alone or in combination:

[0174] 1. Antibody affinity

[0175] In certain embodiments, the equilibrium dissociation constant (K) of an antibody provided herein for its antigen is D )≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 - 13 M, for example 10 -9 M to 10 -13 M).

[0176] In one embodiment, K is measured by radiolabeled antigen binding assay (RIA). D In one embodiment, RIA is performed using a Fab version of an antibody of interest and its antigen. For example, by using a minimal concentration of ( 125I) Labeling antigen equilibration of Fab, followed by capturing bound antigen with an anti-Fab antibody-coated plate to measure solution binding affinity of Fab for antigen (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the assay, 50 mM sodium carbonate (pH 9.6) containing 5 μg / ml capture anti-Fab antibody (Cappel Labs) was coated. Multiwell plates (Thermo Scientific) were blocked overnight and then blocked for two to five hours at room temperature (about 23°C) with PBS containing 2% (w / v) bovine serum albumin. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with serial dilutions of the target Fab (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and washed with 0.1% polysorbate 20 The plates were washed eight times with PBS. When the plates were dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and place the plate on TOPCOUNT TM The concentrations of each Fab that gave less than or equal to 20% of maximal binding were selected for use in the competition binding assay.

[0177] In one aspect, the RIA is a Scatchard assay. For example, the target antibody can be iodinated using the lactoperoxidase method (Bennett and Horuk, Methods in Enzymology 288 pp. 134-148 (1997)). The free 125 I-Na purified radiolabeled antibodies and measured their specific activity. 50 μL of competition reaction mixture containing a fixed concentration of iodinated antibody and decreasing concentrations of serially diluted unlabeled antibody was placed in a 96-well plate. Incubate at 37°C with 5% CO 2Cells transiently expressing antigen were cultured in growth medium consisting of Dulbecco's modified eagle's medium (DMEM) (Genentech) supplemented with 10% FBS, 2 mM L-glutamine, and 1× penicillin-streptomycin. Cells were detached from the culture dish using Sigma cell dissociation solution and washed with binding buffer (DMEM containing 1% bovine serum albumin, 50 mM HEPES pH 7.2, and 0.2% sodium azide). Washed cells were added to a 96-well plate containing a 50-μL competition reaction mixture at an approximate density of 200,000 cells in 0.2 mL binding buffer. The final concentration of unlabeled antibody in the competition reaction containing cells was varied, starting at 1000 nM, then reduced by 1:2-fold dilutions for 10 concentrations and including a zero-added buffer-only sample. The competition reaction containing cells was assayed in triplicate for each concentration of unlabeled antibody. The competition reaction containing cells was incubated for 2 hours at room temperature. After 2 hours incubation, the competition reaction was transferred to the filter plate and washed four times with binding buffer to separate free from bound iodinated antibody. The filtrate was counted by a gamma counter and the binding data were evaluated using the fitting algorithm of Munson and Rodbard (1980) to determine the binding affinity of the antibody.

[0178] In some embodiments, surface plasmon resonance is used to determine K was measured using an anti-human Fc kit (BiAcore Inc., Piscataway, NJ) at 25°C using a BIAcore-2000 apparatus (BIAcore, Inc., Piscataway, NJ). D . Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Anti-human Fc antibodies were diluted to 50 μg / ml with 10 mM sodium acetate, pH 4.0, and then injected at a flow rate of 5 μl / min to achieve approximately 10,000 response units (RU) of coupled protein. After injection of the antibody, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, antibodies were injected in HBS-P to achieve approximately 220 RU, followed by two-fold serial dilutions of the antigen injected into HBS-P at a flow rate of approximately 30 μl / min at 25°C. A simple 1:1 Langmuir binding model ( The association rate (kon) and dissociation rate (koff) were calculated by simultaneously fitting the association and dissociation sensorgrams using the ELISA software version 3.2. The equilibrium dissociation constant (Koff) was calculated as the ratio koff / kon. D). See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0179] Several methods of determining the IC50 of a given compound are known in the art; a common method is to perform a competitive binding assay, such as the assay described herein. In general, a high IC50 indicates that more antibody is needed to inhibit the binding of a known ligand, and therefore the antibody has a relatively low affinity for the ligand. Conversely, a low IC50 indicates that less antibody is needed to inhibit the binding of a known ligand, and therefore the antibody has a relatively high affinity for the ligand.

[0180] 2. Chimeric and humanized antibodies

[0181] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). 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. The chimeric antibodies of interest herein include "primatization" antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences (U.S. Pat. No. 5,693,780). In another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0182] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally speaking, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies optionally also include at least a portion of a human constant region. In certain embodiments, some FR residues in humanized antibodies are 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.

[0183] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method for FR shuffling).

[0184] 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 et al. J. Immunol. 151:2296 (1993)); 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 et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (199 3)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0185] 3. Human Antibodies

[0186] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0187] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or a portion of human immunoglobulin loci that replace endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, describing XENOMOUSE TM Technology; U.S. Patent No. 5,770,429, describing technology; U.S. Patent No. 7,041,870, describing KM Technology; and U.S. Patent Application Publication No. US 2007 / 0061900, describing Technology). Human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with different human constant regions.

[0188] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0189] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0190] 4. Antibodies from the library

[0191] The antibodies of the present invention can be isolated by screening the library for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed in, for example, Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described in, for example, the following documents: McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo et al., ed., Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0192] In certain phage display methods, repertoires of VH and VL genes are independently cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), natural repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without any immunization. Finally, natural libraries can also be produced synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and achieve in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Pat. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0193] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0194] 5. Multispecific Antibodies

[0195] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites. In certain embodiments, bispecific antibodies can bind to two different epitopes of the same antigen. In certain embodiments, bispecific antibodies can bind to two different antigens. Bispecific antibodies can be prepared into full-length antibodies or antibody fragments.

[0196] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimers (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for making bispecific Heterospecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0197] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimers (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for making bispecific Heterospecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0198] Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" or "Dual Variable Domain Immunoglobulins" (DVDs) are also included herein (see, e.g., US 2006 / 0025576 A1, and Wu et al. Nature Biotechnology (2007)).

[0199] 6. Antibody variants

[0200] In certain embodiments, amino acid sequence variants of antibodies provided herein are contemplated. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, the deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution may be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0201] a) Substitution, insertion and deletion variants

[0202] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in Table 2B under the heading "Preferred Substitutions". More substantial changes are provided in Table 2B under the heading "Exemplary Substitutions" and are further described below with respect 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 reduced or improved ADCC or CDC.

[0203] Table 2B

[0204]

[0205]

[0206] Amino acids can be grouped according to common side chain properties:

[0207] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0208] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0209] (3) Acidic: Asp, Glu;

[0210] (4) Basic: His, Lys, Arg;

[0211] (5) Residues that affect chain orientation: Gly, Pro;

[0212] (6) Aromatic: Trp, Tyr, Phe.

[0213] Non-conservative substitutions will require exchanging a member of one of these classes for another class.

[0214] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, one or more resulting variants selected for further study will have modifications (e.g., improvements) of certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently produced using, for example, affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0215] Changes (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such changes may be made in HVR "hot spots," i.e., residues encoded by codons that undergo high frequency mutations during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from secondary libraries has been described, e.g., in Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0216] 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 antigen. For example, conservative changes (such as conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may, for example, be outside of antigen contact residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged, or does not contain more than one, two or three amino acid substitutions.

[0217] A suitable method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or in addition, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0218] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred 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 with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0219] b) Glycosylation variants

[0220] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. The addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence so as to create or remove one or more glycosylation sites.

[0221] In the case where the antibody comprises Fc, the carbohydrate connected thereto can be changed. Natural antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide of Asn297 generally connected to the CH2 domain of Fc by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of the GlcNAc in the "trunk" of the biantennary oligosaccharide structure. In certain embodiments, the modification of oligosaccharides in the antibody of the present invention may be made to create antibody variants with certain improved properties.

[0222] In one embodiment, an antibody variant is provided that has a carbohydrate structure lacking fucose attached (directly or indirectly) to Fc. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures (e.g., complex, mixed, and high mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc residues) in Fc; however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications on "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0223] The antibody variant further has a bisecting oligosaccharide, for example, wherein the biantennary oligosaccharide attached to the antibody Fc is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0224] c) Fc variants

[0225] In various embodiments, the antibody comprises a modified Fc as provided herein. Thus, in some embodiments, one or more amino acid modifications may be introduced into the Fc of an antibody, thereby generating a modified Fc. The modified Fc may comprise a human Fc sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0226] In certain embodiments, the present invention encompasses antibody variants with some but not all effector functions, making them ideal candidates for applications in which in vivo antibody half-life is important and certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells used to 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 U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Suitable effector cells for such 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 et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot 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 may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0227] Non-limiting examples of antibodies with reduced effector function include those with substitutions of one or more of Fc residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such 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 (U.S. Pat. No. 7,332,581).

[0228] Certain antibody variants with improved or decreased binding to FcRs are described. See, e.g., U.S. Pat. Nos. 6,737,056 and 8,969,526; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0229] In certain embodiments, the antibody variant comprises an Fc with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc (EU numbering of residues).

[0230] In some embodiments, changes are made in the Fc such that Clq binding and / or complement dependent cytotoxicity (CDC) are altered (ie, improved or reduced), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0231] For other examples of Fc variants, see also Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351.

[0232] d) Cysteine ​​engineered antibody variants

[0233] In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. As further described herein, by replacing these 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 create immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; K149 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc. For example, cysteine ​​engineered antibodies may be produced as described in U.S. Pat. Nos. 7,521,541 and 9,000,130.

[0234] e) Antibody derivatives

[0235] In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous parts known in the art and readily available. Suitable parts for antibody derivatization 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, polypropylene glycol homopolymers, polyoxypropylene / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers connected to the antibody may vary, and if more than one polymer is connected, 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 specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0236] In another embodiment, a conjugate of an antibody and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the nonproteinaceous moiety to a temperature that kills cells that are closest to the antibody-nonproteinaceous moiety.

[0237] C. Recombinant Methods and Compositions

[0238] Recombinant methods and compositions known in the art can be used to produce antibodies, modified Fc and Fc fusions. See, for example, U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody, modified Fc or Fc fusion as described herein is provided. In the case of an antibody, such a nucleic acid may 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 another embodiment, one or more vectors (e.g., an expression vector) comprising such a nucleic acid are provided. In another embodiment, a host cell comprising such a nucleic acid is provided. In some embodiments for expressing an antibody, the host cell comprises (e.g., has been transformed with the following): (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 various embodiments, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphocyte-like cell (e.g., a Y0, NS0, Sp20 cell). In a certain embodiment, a method of making an antibody, a modified Fc or an Fc fusion is provided, wherein the method comprises incubating a host cell comprising a nucleic acid encoding the antibody, a modified Fc or an Fc fusion as provided above under conditions suitable for expression of the antibody, a modified Fc or an Fc fusion, and optionally recovering the antibody, modified Fc or an Fc fusion from the host cell (or host cell culture medium).

[0239] For recombinant production of antibodies, modified Fc or Fc fusions, nucleic acids encoding antibodies, modified Fc or Fc fusions, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. For antibodies, such nucleic acids can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains.

[0240] Host cells suitable for cloning or expression of protein encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, Fc-containing proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of polypeptides in bacteria, see, for example, U.S. Patents Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli (E. coli). After expression, the protein can be separated from the bacterial cell paste as a soluble portion and can be further purified.

[0241] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for protein encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce proteins with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0242] Suitable host cells for expressing glycosylated proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0243] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0244] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be used. Other examples of suitable mammalian host cell lines are monkey kidney CV1 lines transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); 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 liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0245] D. Determination

[0246] The modified Fc provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0247] 1. Binding assays and other assays

[0248] Various techniques can be used to determine the binding of an agent (such as an antibody or Fc conjugate, including an Fc fusion) comprising a modified Fc provided herein to FcRn. One such assay is an enzyme-linked immunosorbent assay (ELISA) for confirming the ability to bind to human FcRn and various pH values, such as pH 7.4 and pH 6. Various techniques can also be used to determine the binding of an antibody to its antigen, also including an enzyme-linked immunosorbent assay (ELISA). According to this assay, a plate coated with FcRn or antigen is incubated with a sample containing an antibody or other agent containing a modified Fc, and the binding of the antibody or agent containing a modified Fc to the target antigen or FcRn is determined.

[0249] In one aspect, the antigen binding activity of the antibodies of the present invention is tested, for example, by known methods such as ELISA, Western blot, etc. In another aspect, the binding activity of the modified Fc-containing agent to FcRn is tested, for example, by known methods such as ELISA, Western blot, etc.

[0250] On the other hand, competition assays can be used to identify antibodies that compete with any of the antibodies of the present invention for binding to an antigen. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) bound by any of the antibodies of the present invention, more specifically any of the epitopes specifically bound by antibodies in Class I, Class II, Class III, or Class IV as described herein (see, e.g., Example 1 and Table 4). Detailed illustrative methods for locating epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ).

[0251] In an exemplary competition assay, an immobilized antigen is incubated in a solution comprising a first labeled antibody (e.g., one or more of the antibodies disclosed herein) that binds to the antigen and a second unlabeled antibody that is tested for the ability to compete with the first antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0252] 2. Activity Assay

[0253] In one aspect, assays are provided for identifying antibodies and Fc conjugates having biological activity. Biological activity may include, for example, the ability to cross the blood-brain barrier into the brain and / or CNS and the ability to transport compounds associated with a modified Fc across the BBB into the brain and / or CNS. Antibodies and Fc conjugates having such biological activity in vivo and / or in vitro are provided.

[0254] In certain embodiments, the antibodies or Fc conjugates of the invention are tested for such biological activity. In some such embodiments, the antibodies or Fc conjugates are tested for such biological activity in an in vitro transcytosis assay. An exemplary transcytosis assay is as follows. FCGRT (UniProtKB-P55899, FCGRTN_HUMAN) and β-FcRn (e.g., separated by the P2A sequence (Kim et al. PLoS one 2011; 6(4): e18556) are transfected to express human FcRn (e.g., separated by the P2A sequence (Kim et al. PLoS one 2011; 6(4): e18556)). 2 m(UniProtKB-P61769,B2MG_HUMAN)) MDCK II cells (American Type Culture Collection; Manassas, VA) were cultured in a 0.4-μm pore size The cells were seeded in permeable support plates (Corning Inc., Corning, NY) for 3 days. On day 3, fresh medium containing the test antibody and a fluorescent marker dye, such as Lucifer Yellow (Molecular Probes, Eugene, OR) was added to the apical compartment. Fresh medium without the test antibody and Lucifer Yellow was added to the basolateral compartment. The pH of both chambers was 7.4. The cells were incubated at 37°C with 5% CO 2 The plates are incubated overnight in a humidified incubator. On day 4, the culture medium is collected from the apical and basolateral compartments, and the antibody concentration in both compartments is determined, for example, by ELISA. The integrity of junction formation in the cell monolayer is monitored by measuring the relative fluorescence units of Lucifer Yellow in the basolateral compartment. The data can be normalized by dividing the transcytosis concentration of each antibody or Fc conjugate by the transcytosis concentration of a reference antibody that typically comprises a wild-type Fc.

[0255] In some embodiments, the antibodies or Fc conjugates provided herein exhibit a transcytosis activity in an in vitro transcytosis assay of at least 60, at least 70, at least 80, at least 90, or at least 100. In some embodiments, the antibodies or Fc conjugates provided herein exhibit a transcytosis activity in an in vitro transcytosis assay of at least 60, at least 70, at least 80, at least 90, or at least 100 when normalized to the same antibody or Fc conjugate comprising a wild-type Fc.

[0256] E. Immunoconjugates and Fc conjugates

[0257] The present invention also provides a conjugate comprising an antibody or modified Fc herein, which is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof), or radioactive isotopes. When such a conjugate comprises an antibody, it may be referred to as an immunoconjugate in some embodiments.

[0258] In one embodiment, the antibody or modified Fc herein is conjugated to a neurological disorder drug, chemotherapeutic agent, and / or imaging agent to more efficiently transport the drug, chemotherapeutic agent, and / or imaging agent across the BBB.

[0259] Covalent conjugation can be direct or via a linker. In certain embodiments, direct conjugation is by constructing a protein fusion (i.e., by genetic fusion of two genes encoding a modified Fc and, for example, a neurological disorder drug and expressing them as a single protein). In certain embodiments, direct conjugation is by forming a covalent bond between a reactive group on a modified Fc or antibody and, for example, a corresponding group or receptor on a neurological drug. In certain embodiments, direct conjugation is by modifying (i.e., genetically modifying) one of the two molecules to be conjugated to include a reactive group (as a non-limiting example, a sulfhydryl or carboxyl group), which forms a covalent bond with the other molecule to be conjugated under appropriate conditions. As a non-limiting example, a molecule (i.e., an amino acid) having a desired reactive group (i.e., a cysteine ​​residue) can be introduced into an antibody or modified Fc and form a disulfide bond with, for example, a neurological drug. Methods for covalent conjugation of nucleic acids to proteins are also known in the art (ie, photocrosslinking, see, eg, Zatsepin et al. Russ. Chem. Rev. 74:77-95 (2005)).

[0260] As will be readily appreciated by one of ordinary skill in the art, non-covalent conjugation may be by any non-covalent attachment means, including hydrophobic bonds, ionic bonds, electrostatic interactions, and the like.

[0261] Conjugation can also be performed using a variety of linkers. For example, antibodies and neuropharmaceuticals or modified Fc and neuropharmaceuticals can be conjugated using a variety of bifunctional protein coupling agents 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 hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido 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). For example, as in Vitetta et al., Science 238:1098 (1987). The ricin immunotoxin can be prepared. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies, modified Fc or Fc conjugates. See WO94 / 11026. A peptide linker comprising one to twenty amino acids connected by peptide bonds can also be used. In certain such embodiments, the amino acids are selected from twenty naturally occurring amino acids. In certain other such embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. The linker can be a "cleavable linker" that helps release the neuropharmaceutical 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 Res. 52:127-131 (1992); US Pat. No. 5,208,020) can be used.

[0262] The present invention specifically contemplates, but is not limited to, conjugates prepared with cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB ((4-vinyl sulfone) benzoic acid succinimidyl ester), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0263] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235). B1); auristatin, such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 53:3336-3342 (1993); Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; and CC1065. In some embodiments, an Fc conjugate is provided comprising a modified Fc herein conjugated to one or more of the aforementioned drugs.

[0264] In another embodiment, the conjugate comprises an antibody or Fc described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor. inhibitor), gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0265] In another embodiment, the conjugate comprises an antibody or Fc as described herein, conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may include radioactive atoms such as tc99m or I123 for scintigraphic studies; or spin labels such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri).

[0266] In some embodiments, the Fc conjugate comprises a modified Fc provided herein fused to a protein, such as a therapeutic protein and / or a detectable protein. In such embodiments, such Fc conjugates may be referred to as Fc fusions. Non-limiting exemplary therapeutic proteins that can be conjugated to the modified Fc provided herein include TNF-R1, CTLA-4, IL-1R1, α-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, α-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, β-galactosidase, arylsulfatase B, β-glucuronidase, acid α-glucosidase, glucocerebrosidase, α-galactosidase A, hexosaminidase A, acid sphingomyelinase, β-galactocerebrosidase, β-galactosidase, arylsulfatase A, acid ceramidase, asparagyl transferase, palmitoyl protein thioesterase 1, and tripeptidyl aminopeptidase 1. In some embodiments, the extracellular domain of a therapeutic protein is conjugated to a modified Fc provided herein, such as the extracellular domain of TNF-R1, CTLA-4, or IL-1R1.

[0267] F. Methods and compositions for diagnosis and detection

[0268] In some embodiments, antibodies or Fc conjugates for use in diagnostic or detection methods are provided.

[0269] Exemplary disorders that can be diagnosed using the antibodies or Fc conjugates of the invention include disorders of the central nervous system (CNS), including the brain. In some embodiments, the antibodies and Fc conjugates of the invention can be used, for example, to detect antigens in the CNS (such as in the brain) to diagnose diseases or disorders associated with the presence or elevated levels of antigens.

[0270] In certain embodiments, labeled antibodies and Fc conjugates are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as indirect, for example, moieties detected via 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 131I; 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 (U.S. Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; carbohydrate oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as uricase and xanthine oxidase, coupled to enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase or microperoxidase); biotin / avidin; spin labels; bacteriophage labels; stable free radicals, and the like.

[0271] In some embodiments, the antibody or Fc conjugate lacks effector function. In some embodiments, the antibody or Fc conjugate has reduced effector function. In another embodiment, the antibody or Fc conjugate is engineered to have reduced effector function. In some aspects, the antibody or Fc conjugate has one or more Fc mutations that reduce or eliminate effector function. On the other hand, the antibody or Fc conjugate has modified glycosylation, which is attributed to, for example, producing the antibody, Fc conjugate or modified Fc in a system lacking normal human glycosylation enzymes. On the other hand, the Ig backbone is modified to a backbone that naturally has limited or no effector function.

[0272] Various techniques can be used to determine the binding of an antibody or Fc conjugate to a target protein. One such assay is an enzyme-linked immunosorbent assay (ELISA) for confirming the ability to bind to a target protein. According to this assay, a plate coated with an antigen is incubated with a sample containing an antibody or Fc conjugate, and the binding of the antibody or Fc conjugate to the target protein of interest is determined.

[0273] Assays for evaluating the uptake of systemically administered antibodies or Fc conjugates and other biological activities of the antibodies or Fc conjugates can be performed as disclosed in the Examples or as known in the art for CNS target proteins of interest.

[0274] In one aspect, an assay for identifying an anti-BACE1 antibody with biological activity is provided. Biological activity may include, for example, inhibition of BACE1 aspartyl protease activity. Antibodies with such biological activity in vivo and / or in vitro are also provided, for example, as determined by homogeneous time-resolved fluorescence HTRF or microfluidic capillary electrophoresis (MCE) using synthetic substrate peptides, or in vivo in a cell line expressing a BACE1 substrate such as APP.

[0275] F. Pharmaceutical preparations

[0276] Pharmaceutical preparations of antibodies or Fc conjugates as described herein are prepared by mixing such antibodies or Fc conjugates having the desired degree of purity with one or more optionally present pharmaceutical carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized preparations or aqueous solutions. Pharmaceutical carriers, excipients or stabilizers are generally non-toxic 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, chloride); phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl 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 pharmaceutical carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs including rHuPH20 and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0277] Exemplary lyophilized antibody or Fc conjugate formulations are described in US Pat. No. 6,267,958. Aqueous antibody or Fc conjugate formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0278] The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide one or more active ingredients for treating neuropathy disorders, neurodegenerative diseases, cancer, eye disorders, epileptic seizure disorders, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, or CNS inflammation. Examples of such drugs are discussed herein. Such active ingredients are suitable for being present in an amount combination effective for the intended purpose.

[0279] The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as 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, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980). One or more active ingredients can be encapsulated in liposomes coupled to an antibody or Fc conjugate described herein (see, e.g., U.S. Patent Application Publication No. 20020025313).

[0280] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies or Fc conjugates, which are in the form of shaped articles, such as films or microcapsules. Non-limiting examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl L-glutamate, non-degradable ethylene-vinyl acetate, such as LUPRON DEPOT TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) of degradable lactic acid-glycolic acid copolymer and poly-D-(-)-3-hydroxybutyric acid.

[0281] Preparations to be used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0282] G. Methods of treatment and compositions

[0283] Any of the antibodies or Fc conjugates provided herein can be used in a method of treatment. In one aspect, an antibody or Fc conjugate for use as a drug is provided. For example, the present invention provides a method of transporting a therapeutic compound across the blood-brain barrier, the method comprising exposing an antibody or Fc conjugate of the present invention to the BBB so that a modified Fc allows the transport of the antibody or Fc conjugate across the BBB, wherein the antibody or Fc conjugate comprises a therapeutic compound. In another example, the present invention provides a method of transporting a neurological disorder drug across the blood-brain barrier, the method comprising exposing an antibody or Fc conjugate of the present invention to the BBB so that a modified Fc allows the transport of the antibody or Fc conjugate across the BBB, wherein the antibody or Fc conjugate comprises a neurological disorder drug. In one embodiment, the BBB is in a mammal (e.g., a human), such as a mammal suffering from 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's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, traumatic brain injury, etc.

[0284] In one embodiment, the neurological disorder is selected from: neuropathy, amyloidosis, cancer (e.g., involving the CNS or brain), an ocular disease or disorder, a viral or microbial infection, inflammation (e.g., inflammation of the CNS or brain), ischemia, a neurodegenerative disease, epileptic seizures, behavioral disorders, lysosomal storage diseases, etc. The antibodies and Fc conjugates of the present invention are particularly suitable for treating such neurological disorders due to their ability to transport one or more associated active ingredients / coupled therapeutic compounds across the BBB and into the CNS / brain where the molecular, cellular or viral / microbial basis of such disorders is found.

[0285] Neuropathic disorders are diseases or disorders of the nervous system characterized by inappropriate or uncontrolled nerve signaling, or lack of nerve 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 (wholly or primarily associated with a psychological disorder), headaches, migraines, neuropathy; and symptoms and syndromes that often accompany such neuropathic disorders, such as vertigo or nausea.

[0286] For neuropathy, neurological drugs may be selected, which are 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 phentermine). acid, meloxicam, nabumetone, oxaprozin, piroxicam, sulindac, and tolmetin), corticosteroids (i.e., cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone, and triamcinolone), antimigraine agents ( that is, sumatriptin, almotriptan, frovatriptan, sumatriptan, rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan and ergotamine), acetaminophen, salicylates (i.e., aspirin, choline salicylate, magnesium salicylate, diflunisal and salsalate), anticonvulsants (i.e.,carbamazepine, clonazepam, gabapentin, lamotrigine, pregabalin, tiagabine, and topiramate), anesthetics (i.e., isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, propoxycaine, procaine, novocaine, proparacaine, caine, 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 disorders involving vertigo, neurological drugs can be selected, which are anti-vertigo agents, including but not limited to meclizine, diphenhydramine, promethazine and diazepam. For neuropathy disorders involving nausea, neurological drugs can be selected, which are anti-nausea agents, including but not limited to promethazine, chlorpromazine, prochlorperazine, trimethobenzamide and metoclopramide. ,

[0287] 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); Parkinson's-dementia complex of Guam type, 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 associated with β-amyloid deposition (i.e., macular degeneration, choroidergic optic neuropathy, and cataracts).

[0288] For amyloidosis, neurological drugs may be selected, including but 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 a target selected from the group consisting of: β-secretase, Tau, presenilin, amyloid precursor protein or a portion thereof, amyloid β peptide or oligomer or fibril 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 (i.e., ergoloid methylsulfonate); mesylate; anticholinergic antiparkinsonian agents (i.e., procyclidine, diphenhydramine, trihexylphenidyl, benztropine, biperiden, and trihexyphenidyl); dopaminergic antiparkinsonian agents (i.e., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirole, rasagiline, apomorphine, omorphine), carbidopa, levodopa, pergolide, tolcapone, and amantadine); tetrabenazine; anti-inflammatory agents (including but not limited to nonsteroidal anti-inflammatory drugs (i.e., indomethicin and other compounds listed above)); hormones (i.e., estrogen, progesterone, and leuprolide); vitamins (i.e., folic acid and niacinamide); dimebolin; homotaurine (i.e., 3-aminopropane sulfonic acid; 3APS); serotonin receptor activity modulators (i.e., xaliproden); interferon; and glucocorticoids.

[0289] 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, enchondromas, oligodendritic gliomas, medulloblastomas, gangliogliomas, schwannomas, neurofibromas, neuroblastomas, and epidural, intramedullary, or intradural tumors.

[0290] For cancer, neurological drugs may be selected, which are chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimine and methylamelamines, including altretamine, tretamine, triethylphosphoramide, triethylthiophosphoramide and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, ); beta-lapachone; lapachol; colchicine; 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; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolestadin; 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. hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine,. Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid (such as mycophenolic acid), acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiabendazole, thiamethoxazole, oxazolidinone ... miprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid, leucovorin, dapoxetine ... acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansines, such as maytansine and ansamitocin; 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; arabinoside ("Ara-C"); thiotepa; taxoids, such as 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); Chlorambucil; Gemcitabine 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Oxaliplatin; leucovovin; vinorelbine novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine A pharmaceutically acceptable salt, acid or derivative of any of the above; and a combination of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prazosin, and FOLFOX, an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

[0291] Also included in this definition of chemotherapeutic agents are antihormonal agents, which are used to regulate, reduce, block or inhibit the action of hormones that can promote cancer growth, and are often in the form of systemic or systemic treatment. Antihormonal agents can 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; antiprogestins; estrogen receptor downregulators (ERDs); agents that act to suppress or shut down the ovaries, such as luteinizing hormone-releasing hormone (LHRH) agonists, such as and leuprolide acetate, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, Vorozole, Letrozole and Anastrozole. In addition, this definition of chemotherapeutic agent includes bisphosphonates, such as clodronate (e.g. or ), Etidronate, NE-58095, Zoledronic acid / zoledronate, alendronate, Pamidronate, Tiludronate or risedronate; and troxacitabine (1,3-dioxolane nucleoside cytosine analogs); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as 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 inhibitors; 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 of the foregoing.

[0292] Another group of compounds that can be used as neurological drugs for cancer treatment or prevention are anti-cancer immunoglobulins (including but not limited to trastuzumab, pertuzumab, bevacizumab, alemtuxumab, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, panitumumab and rituximab). In some cases, antibodies combined with toxic markers or conjugates can be used to target and kill desired cells (i.e., cancer cells), including but not limited to antibodies with 131 I radiolabeled tositumomab, or trastuzumab emtansine.

[0293] An ocular disease or disorder is a disease or disorder of the eye, which for purposes herein is considered to be a CNS organ isolated by the BBB. Ocular diseases or disorders include, but are not limited to, disorders of the sclera, cornea, iris, and ciliary body (i.e., scleritis, keratitis, corneal ulcers, corneal abrasions, snow blindness, arc eye, Thygeson's superficial punctate keratopathy, corneal neovascularization, Fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca, iritis, and uveitis), disorders of the lens (i.e., cataracts), disorders of the choroid and retina (i.e., retinal detachment, retinal schisis, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, age-related macular degeneration, macular degeneration (wet or dry), epiretinal membrane, retinitis pigmentosa, and macular edema), glaucoma, floaters, disorders of the optic nerve and visual pathways (i.e., Leber's hereditary optic neuropathy neuropathy and optic disc drusen), disorders of ocular muscle / binocular movement accommodation / refraction (i.e. strabismus, ophthalmoplegia, progressive extraocular muscle palsy, esotropia, exotropia, hyperopia, farsightedness, myopia, astigmatism, anisometropia, presbyopia and ophthalmoplegia), visual impairment and blindness (i.e. amblyopia, Lever's congenital amaurosis, scotoma, color blindness, achromatopsia, night blindness, blindness, river blindness and microphthalmia / coloboma), red eye, Argyll Robertson pupil, corneal fungus, dry eye and aniridia.

[0294] For ocular diseases or disorders, neurological drugs may be selected, which 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, phenylephrine, and tetrahydrozoline), ophthalmic lubricants, ophthalmic steroids (i.e., fluorometholone, prazosin, loteprednol, dexamethasone, difluprednate, rimexolone, fluocinolone, medrysone, and triamcinolone), ophthalmic anesthetics (i.e., lidocaine, proparacaine, and tetracaine), ophthalmic anti-infectives (i.e.,levofloxacin, gatifloxacin, ciprofloxacin, moxifloxacin, chloramphenicol, bacitracin / polymyxin b, sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin, sulfisoxazole, gentamicin, idoxuridine, erythromycin, natamycin, gramicidin, neomycin, ofloxacin, trifluridine, ganciclovir, vidarabine), ophthalmic anti-inflammatory agents (i.e., nepafenac, ketorolac, flubaprofen, suprofen, cyclosporine, triamcinolone, diclofenac, and bromfenac), and ophthalmic antihistamines or decongestants (i.e., ketotifen, olopatadine, epinastine, naphazoline, cromolyn, tetrahydrozoline, pemirolast, bepotastine, naphazoline, phenylephrine, nedocromil, lodoxamide, phenylephrine, emedastine, and azelastine).

[0295] Viral or microbial infections of the CNS include, but are not limited to, infections caused by viruses (i.e., influenza, HIV, polio, rubella), bacteria (i.e., Neisseria sp., Streptococcus sp., Pseudomonas sp., Proteus sp., Escherichia coli, S. aureus, Pneumococcus sp., Meningococcus sp., Haemophilus sp., and Mycobacterium tuberculosis), and other microorganisms such as fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., toxoplasma gondii), gondii) or amebiasis, which causes CNS pathophysiology including, but not limited to, meningitis, encephalitis, myelitis, vasculitis, and abscesses (either acute or chronic).

[0296] For viral or microbial diseases, neurological drugs may be selected, including but not limited to antiviral compounds (including but not limited to adamantane antivirals (i.e., rimantadine and amantadine), antiviral interferons (i.e., peginterferon 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, delavirdine, and nevirapine), nucleoside reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir ir), zalcitabine, telbivudine, and didanosine), protease inhibitors (i.e., darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir, amprenavir, indinavir, and saquinavir) , purine nucleosides (i.e., valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir, and cidofovir), and miscellaneous 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, 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., loracarb), glycopeptides (i.e., teicoplanin and vancomycin), macrolides (i.e., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spectinomycin), spectinomycin), monoamines (i.e., ampicillin), quinolones (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamides (i.e., mafenide, sulfonamidochrysoidine), acetylcholine, sulfacycline, sulfadiazine, sulfamethizole, sulfanilamide, sulfasalazine, isoxazolidinone, trimethoprim, trimethoprim, and sulfamethoxazole), tetracycline (i.e., tetracycline, demeclocycline, doxycycline, minocycline, and oxytetracycline) ne)), antineoplastic or cytotoxic antibiotics (i.e., doxorubicin, mitoxantrone, bleomycin, daunomycin, actinomycin D, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin), and miscellaneous antibacterial compounds (i.e., subtilisin, colistin, and polymyxin B)), antifungal agents (i.e., metronidazole, nitazoxanide, tinidazole, chloroquine, iodoquinol, and paromomycin),and antiparasitic agents (including but not limited to quinine, chloroquine, amodiaquine, pyrimethamine, sulphadoxine, proguanil, mefloquine, atovaquone, primaquine, artemesinin, halofantrine, doxycycline, clindamycin, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin, amphotericin B, melarsoprol, efornithine, and albendazole).

[0297] CNS inflammation includes, but is not limited to, inflammation resulting from injury to the CNS, which may 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).

[0298] For CNS inflammation, neurological drugs that address the inflammation itself (ie, nonsteroidal anti-inflammatory agents such as ibuprofen or naproxen) or that address the underlying cause of the inflammation (ie, antiviral or anticancer agents) may be selected.

[0299] As used herein, CNS ischemia refers to a group of disorders associated with abnormal blood flow or vascular behavior in the brain or their causes, and includes, but is not limited to, focal cerebral ischemia, global cerebral ischemia, stroke (ie, subarachnoid hemorrhage and intracerebral hemorrhage), and aneurysm.

[0300] For ischemia, neurological drugs may be selected, including but 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 used to improve blood flow or vascular elasticity, including, for example, blood pressure drugs.

[0301] Neurodegenerative diseases are a group of diseases and disorders associated with loss of function or death of nerve cells in the CNS and include, but are not limited to, adrenoleukodystrophy, Alexander's disease, Alper's disease, amyotrophic lateral sclerosis, ataxia-telangiectasia, Batten disease, Cockayne syndrome, corticobasal 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 disorders.

[0302] For neurodegenerative diseases, neurological drugs can be selected, which 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), neurturin, platelet-derived growth factor (PD-1), and glial-derived neurotrophic factor (GDNF). PDGF, heregulin, neuregulin, artemin, persephin, interleukins, glial cell line-derived neurotrophic factor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, netrin, cardiotrophin-1, hedgehog, leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone morphogenetic protein (BMP), spindle protein, saposin, semaphoring and stem cell factor (SCF).

[0303] 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 seizures, clonic seizures, complex partial seizures, frontal lobe epilepsy, febrile seizures, infantile spasms, juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner Syndrome, Dravet's syndrome, Otahara syndrome, West syndrome, myoclonic seizures, mitochondrial disorders, progressive myoclonic epilepsy, psychogenic seizures, reflex epilepsy, Rasmussen's syndrome, Syndrome), simple partial seizure, secondarily generalized seizure, temporal lobe epilepsy, tonic-clonic seizure, tonic seizure, psychomotor seizure, limbic epilepsy, partial-onset seizure, generalized-onset seizure, status epilepticus, abdominal epilepsy, akinetic seizure, autonomic seizure, massive bilateral myoclonus, catamenial epilepsy, drop seizure, emotional seizure, focal seizure, gelastic seizure, Jacksonian March, Lafora's disease, motor seizure, multifocal seizure, nocturnal seizure, photosensitivity seizure, pseudoseizure, sensory seizure, microseizure, sylvan seizure, withdrawal seizure, and visual reflex seizure).

[0304] For seizure disorders, neurological drugs may be selected that are anticonvulsants or antiepileptics, including but not limited to barbiturate anticonvulsants (i.e., primidone, metharbital, mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal, barbital, brallobarbital, and phenobarbital). The anticonvulsants include phenobarbital, benzodiazepine anticonvulsants (i.e., diazepam, quinolones, and lorazepam), carbamate anticonvulsants (i.e., felbamate), carbonic anhydrase inhibitor anticonvulsants (i.e., acetazolamide, topiramate, and zonisamide), dibenzazapine anticonvulsants (i.e., rufinamide, carbamazepine, and oxcarbazepine), fatty acid derivative anticonvulsants (i.e., divalproex and valproic acid), and phenobarbital anticonvulsants. 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 ( The anticonvulsants include oxazolidinone (i.e., paramethadione and trimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam), succinimide anticonvulsants (i.e., ethosuximide and methsuximide), triazine anticonvulsants (i.e., lamotrigine), and urea anticonvulsants (i.e., phenacemide and pheneturide).

[0305] Behavioral disorders are CNS disorders characterized by abnormal behavior on the part of the afflicted subject and include, but are not limited to, sleep disorders (i.e., insomnia, parasomnias, night terrors, circadian rhythm sleep disorders, and hypersomnia), mood disorders (i.e., depression, suicidal depression, anxiety, chronic affective disorders, phobias, panic attacks, obsessive-compulsive disorder, attention deficit hyperactivity disorder (ADHD), attention deficit disorder (ADD), chronic fatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolar disorder), eating disorders (i.e., anorexia or bulimia), psychosis, developmental behavioral disorders (i.e., autism, Rett's syndrome, Aspberger's syndrome), personality disorders, and psychotic disorders (i.e., schizophrenia, delusional disorder, and the like).

[0306] For behavioral disorders, the neurological drug may be selected from behavior-modulating compounds, including but not limited to atypical antipsychotics (i.e., risperidone, olanzapine, aripiprazole, quetiapine, paliperidone, asenapine, clozapine, iloperidone, and ziprasidone), phenothiazine antipsychotics (i.e., prochlorperazine, chlorpromazine, fluphenazine, perphenazine), perphenazine, trifluoperazine, thioridazine, and mesoridazine), thioxanthenes (i.e., thiothixene), miscellaneous antipsychotics (i.e., pimozide, lithium, molindone, haloperidol, and loxapine), selective serotonin reuptake inhibitors (i.e., citalopram, escitalopram, and am, paroxetine, fluoxetine, and sertraline), serotonin-norepinephrine reuptake inhibitors (i.e., duloxetine, venlafaxine, desvenlafaxine), tricyclic antidepressants (i.e., doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine, pramine), 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, canazapine, lorazepam, and diazepam), norepinephrine-dopamine reuptake inhibitors (i.e., bupropion), CNS stimulants (i.e., phentermine, diethylpropion, methamphetamine, dextroamphetamine, amphetamine, methylphenidate, dexmethylphenidate, lisdexamfetamine tamine, 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 mebbutal), benzodiazepines (as described above), and miscellaneous anxiolytics / sedatives / hypnotics (i.e., diphenhydramine, sodium oxybate, oxybate), zaleplon, hydroxyzine, chloral hydrate, aolpidem, buspirone, doxepin, eszopiclone, ramelteon, meprobamate, and ethclorvynol), secretins (see, e.g., Ratliff-Schaub et al. Autism 9:256-265 (2005)), opioid peptides (see, e.g., Cowen et al., J. Neurochem. 89:273-285 (2004)), and neuropeptides (see, e.g., Hethwa et al. Am. J. Physiol. 289:E301-305 (2005)).

[0307] Lysosomal storage disorders are metabolic disorders that, in some instances, are associated with the CNS or have CNS-specific symptoms; such conditions include, but are not limited to, Tay-Sachs disease, Gaucher's disease, Fabry disease, mucopolysaccharidoses (type I, II, III, IV, V, VI, and VII), glycogen storage diseases, GM1-gangliosidosis, metachromatic leukodystrophy, Farber's disease, Canavan's leukodystrophy, and neuronal ceroid lipofuscinosis types 1 and 2, Niemann-Pick disease, Pompe disease, and Krabbe's disease.

[0308] For lysosomal storage diseases, neurological drugs can be selected that themselves or otherwise mimic the enzyme activity that is impaired in the disease. Exemplary recombinant enzymes for treating lysosomal storage disorders include, but are not limited to, those set forth in, for example, U.S. Patent Application Publication No. 2005 / 0142141 (i.e., α-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, α-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, β-galactosidase, arylsulfatase B, β-glucuronidase, acid α-glucosidase, glucocerebrosidase, α-galactosidase A, hexosaminidase A, acid sphingomyelinase, β-galactocerebrosidase, β-galactosidase, arylsulfatase A, acid ceramidase, asparagyl transferase, palmitoyl protein thioesterase 1, and tripeptidyl aminopeptidase 1).

[0309] In one aspect, the antibodies or Fc conjugates of the invention are used to detect neurological disorders prior to the onset of symptoms and / or to assess the severity or duration of a disease or disorder. In one aspect, the antibodies or Fc conjugates allow detection and / or imaging of neurological disorders, including imaging by radiography, tomography, or magnetic resonance imaging (MRI).

[0310] In one aspect, an antibody or Fc conjugate of the present invention for use as a medicament is provided. In yet other aspects, an antibody or Fc conjugate for treating a neurological disease or disorder (e.g., Alzheimer's disease) is provided. In certain embodiments, an antibody or Fc conjugate for use in a method of treating a subject suffering from a neurological disease or disorder is provided. In certain embodiments, the present invention provides an antibody or Fc conjugate for use in a method of treating a subject suffering from a neurological disease or disorder, the method comprising administering an effective amount of an antibody or Fc conjugate to the subject. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the subject. In other embodiments, the present invention provides an antibody or Fc conjugate for reducing or inhibiting the formation of amyloid plaques in a patient at risk of or suffering from a neurological disease or disorder (e.g., Alzheimer's disease). An "individual" according to any of the above embodiments is optionally a human. In certain aspects, an antibody or Fc conjugate comprising a modified Fc of the present invention used in the method of the present invention improves the absorption of a neurological disorder drug compared to an antibody or Fc conjugate comprising a wild-type Fc.

[0311] In yet another aspect, the invention provides the use of an antibody or Fc conjugate of the invention to manufacture or prepare a medicament. In one embodiment, 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.

[0312] In yet 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 an antibody of the present invention that binds BACE1 or Aβ. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments may be a human.

[0313] The antibodies and Fc conjugates of the present invention can be used alone or in combination with other agents in therapy. For example, the antibodies or Fc conjugates of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a therapeutic agent that is effective for treating the same or different neurological disorder as the antibody or Fc conjugate is used to treat. 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, serotonin receptor antagonists, and anti-amyloid beta peptide antibodies. In certain embodiments, at least one additional therapeutic agent is selected for its ability to reduce one or more side effects of the neurological drug.

[0314] The various combination therapies mentioned 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 the administration of the antibody or Fc conjugate of the invention may be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. In one embodiment, the administration of the antibody or Fc conjugate and the administration of the additional therapeutic agent may be performed within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The antibodies and Fc conjugates of the invention may also be used in combination with other interventional therapies, such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and suitable for the neurological disorder to be treated or prevented.

[0315] The antibody or Fc conjugate of the present invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, 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, in part according to the administration as short-term or long-term. Various dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration and pulse infusion.

[0316] The antibodies and Fc conjugates of the present invention are formulated, dosed and applied in a manner consistent with good medical practice. Considerations in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the administration schedule and other factors known to practicing physicians. The antibody or Fc conjugate is not required, but is optionally formulated with one or more agents that are currently used to prevent or treat the disorder in question or to prevent, alleviate or improve one or more side effects of the administration of the antibody or Fc conjugate. The effective amount of such other agents depends on the amount of the antibody or Fc conjugate 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 administration route as described herein, or in about 1% to 99% of the dosage described herein, or in any dosage and any route determined to be appropriate empirically / clinically.

[0317] For the prevention or treatment of a disease, the appropriate dosage of the antibody or Fc conjugate 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 or Fc conjugate, the severity and course of the disease, whether the antibody or Fc conjugate is used for prevention or treatment purposes, previous therapy, the patient's clinical history and response to the antibody or Fc conjugate, and the judgment of the attending physician. The antibody or Fc conjugate is suitable for administration to the patient once or over a series of treatments. Depending on the type and severity of the disease, an antibody or Fc conjugate of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be an initial candidate dose for administration to the patient, for example, by one or more independent administrations or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may be in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dosage of an antibody or Fc conjugate will be in the range of about 0.05 mg / kg to about 40 mg / kg. Therefore, 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) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about two to about twenty, or for example, about six doses of the antibody or Fc conjugate). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be applicable. The progress of this therapy is easily monitored by conventional techniques and assays as described herein and as known in the art.

[0318] H.Products

[0319] In another aspect of the present invention, a product is provided, which contains materials suitable for the treatment, prevention and / or diagnosis of the above-mentioned conditions. The product includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds a composition alone or in combination with another composition that is effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody comprising a modified Fc of the present invention or an Fc conjugate of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container having a composition contained therein, wherein the composition comprises an antibody comprising a modified Fc of the present invention or an Fc conjugate of the present invention; and (b) a second container having a composition contained therein, wherein the composition comprises another cytotoxic or other therapeutic agent. In this embodiment of the present invention, the product may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further include 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. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0320] Examples

[0321] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the invention in any way.

[0322] Example 1 - Method

[0323] Plasmid Construction and Antibody and FcRn Production - Antibodies, antibody Fc, and human and mouse FcRn complexes were expressed using standard techniques, including cloning sequences encoding antibody heavy and light chains or Fc, or in the case of FcRn, FCGRT or Fcgrt (which encode human and mouse FcRn α chains, respectively) and β-2 microglobulin (β2M) into mammalian expression vectors by standard molecular biology techniques as previously described (Eaton, Wood et al. 1986). FcRn was purified by expression with a His tag and by purification by immobilized metal ion chromatography (IMAC) or by purification on an immobilized IgG column (e.g., Sigma A0919 for mouse IgG agarose, or A2909 for rabbit IgG agarose). Antibodies were expressed as transient transfected cultures in CHO cells (Wong, Baginski et al. 2010, Biotechol. Bioeng., 106(5):751-63) and affinity purified on a GE MabSelect SuRe column (GE Healthcare, Pittsburgh, PA) followed by Superdex-200 size exclusion chromatography (GE Healthcare, Pittsburgh, PA).

[0324] Antibody-FcRn affinity measurements - The affinity of antibodies comprising a modified Fc for human or mouse FcRn was determined by surface plasmon resonance using a Biacore T200 (GE Healthcare) instrument. All experiments were performed at 25°C. The modified Fc antibodies were immobilized at a surface density of 1000RU by protein-L binding. Neutral pH binding was determined in HBS-P (0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% v / v surfactant P20). Acidic pH binding was determined in MBS-P (0.01M MES pH 6.0, 0.15M NaCl, 0.005% v / v surfactant P20). The association rate (ka) and dissociation rate (kd) were calculated using a 1:1 Langmuir binding model by simultaneously fitting the association and dissociation sensorgrams (BIA Evaluation Version 4.1). All fits had chi-square / Rmax values ​​less than 10%, meeting the generally accepted goodness-of-fit criteria. The equilibrium dissociation constant (K) was calculated as the kd / ka ratio. D Alternatively, the equilibrium binding K is determined from the dependence of the steady-state binding level on the analyte concentration. D Values. So-called steady-state analysis is suitable for the measurement of weak to moderate interactions. Undetectable binding or binding too weak for accurate affinity analysis is set as >10 uM in the tables herein.

[0325] Transcytosis assay - An in vitro assay was used to measure the transcytosis activity of modified Fc antibodies. In this dual chamber invasion chamber (trans-well) assay, a layer of epithelial cells expressing hFcRn or mFcRn is established on a membrane separating two chambers. Tight junctions formed between cells exclude antibody diffusion, and therefore antibody transfer from one chamber to the other is only possible via intracellular transport. Therefore, antibody transport from one chamber to the other across this cell layer serves as a model for transcytosis. Similar assays are described, for example, in Claypool et al. Journal of Biological Chemistry 2002 Aug 2;277(31):28038-50. The cells were incubated at 37°C, 5% CO 2 MDCK II cells (American Type Culture Collection; Manassas, VA) were grown in Dulbecco's modified minimum essential medium (Invitrogen, Gaithersburg, MD) containing 10% fetal bovine serum (Clontech, Mountain View, CA), 100 units / mL penicillin, 100 μg / mL streptomycin, and 0.292 mg / mL L-glutamine (ThermoFisher Scientific, Waltham, MA) in a humidified incubator. First, FCGRT (UniProtKB-P55899, FCGRTN_HUMAN) encoding hFcRn (separated by the P2A sequence (Kim et al. PLoS one 2011; 6(4):e18556) and β 2 m(UniProtKB-P61769,B2MG_HUMAN)) gene was transfected into MDCK II cells, and then the transfected cell lines were expanded from isolated single colonies selected by FACS using anti-FCGRT antibody (ADM31, Aldevron, Fargo, ND) and secondary anti-mouse PE-conjugated antibody (Thermo Fisher Scientific, Waltham, MA). All clones were maintained under constant antibiotic selection (5 μg / mL puromycin). Based on the use of FITC anti-human β-catenin by flow cytometry, the transfected cell lines were isolated and then expanded from isolated single colonies selected by FACS using anti-FCGRT antibody (ADM31, Aldevron, Fargo, ND) and secondary anti-mouse PE-conjugated antibody (Thermo Fisher Scientific, Waltham, MA). All clones were maintained under constant antibiotic selection (5 μg / mL puromycin). 2 FCGRT and β evaluated by M (BioLegend) 2 M cell surface expression to select the final clones.

[0326] The transcytosis assay was performed as follows. FcRn-expressing cells were plated in a 0.4-μm pore The cells were seeded in permeable support plates (Corning Inc., Corning, NY) for 3 days. On day 3, fresh medium containing the test antibody and the fluorescent marker dye Lucifer Yellow (Molecular Probes, Eugene, OR) was added to the apical compartment. Fresh medium without the test antibody and Lucifer Yellow was added to the basolateral compartment. The pH of both chambers was 7.4. The cells were incubated at 37°C with 5% CO 2 The plates were incubated overnight in a humidified incubator. On day 4, culture media was collected from the apical and basolateral compartments, and the antibody concentrations in both compartments were determined by ELISA as described below. The data were normalized by dividing the concentration of the test antibody in the basolateral compartment by the concentration of the reference antibody, typically the wild-type antibody, in the same compartment. The integrity of junction formation in the cell monolayer was monitored by measuring the relative fluorescence units of Lucifer Yellow in the basolateral compartment. Data from wells with elevated Lucifer Yellow levels above the control level were discarded, as this would indicate an incomplete epithelial barrier.

[0327] Wild-type and transgenic mice PK and PD studies - Wild-type C57B / 6 mice aged 6 to 8 weeks were used for mFcRn studies. For mouse studies to model hFcRn, a transgenic mouse expressing the human FcRn α chain (FCGRT) under the control of a human promoter and harboring a mouse FcRn α chain (Fcgrt tm1Dcr )-B6.Cg-Fcgrt tm1Dcr Tg(FCGRT)32Dcr / DcrJ (JAX Reserve No. 014565) is a transgenic Tg32 mouse with knockout alleles (Petkova SB 2006 Int. Immunol 18(2): 1759-69, Roopenian, DC, 2010, Methods Mol. Biol. 602: 93-104).

[0328] Dosing, sample collection for mouse studies - mice were injected intravenously with a specified dose of antibody as outlined below. After multiple times after dosing, blood was collected and plasma or serum was separated for antibody concentration measurement. For plasma collection, whole blood was collected in EDTA microcontainer tubes (BD Diagnostics) before perfusion, centrifuged at 5,000xg for 15 minutes, and the supernatant was separated for measuring plasma antibody concentration. For serum collection, whole blood was collected in serum separator microcontainer tubes (BD Diagnostics), allowed to coagulate for at least 30 minutes, and rapidly centrifuged at 5,000xg for 90 seconds. The supernatant was separated for serum antibody measurement. At the specified time, mice were perfused with D-PBS, and brains were collected for antibody concentration and / or Aβ measurement. For brain antibody concentration measurement, half brains from each mouse were homogenized in PBS containing a complete miniature EDTA-free protease inhibitor mixed tablet (Roche Diagnostics) in 1% NP-40 (Cal-Biochem). The homogenized brain samples were rotated at 4°C for 1 hour and then centrifuged at 14,000 rpm for 20 minutes. The supernatant was isolated for brain antibody measurement. 1-40 For measurement, hemibrains were homogenized in 5 M guanidine hydrochloride buffer and the samples were rotated for 3 h at room temperature before being diluted (1:10) in 0.25% casein, 5 mM EDTA (pH 8.0) in PBS containing freshly added aprotinin (20 mg / mL) and leupeptin (10 mg / ml). The diluted homogenate was centrifuged at 14,000 rpm for 20 min and the supernatant was isolated for Aβ 1-40 Measurement.

[0329] Measurement of antibody concentration in mouse plasma or serum (pharmacokinetics) - Total antibody concentration in mouse plasma or serum was measured by universal human Fc ELISA. Nunc 384-well MaxiSorp immunoplates were incubated at 4°C with donkey anti-human IgG and Fc fragment-specific polyclonal antibodies (Jackson ImmunoResearch) F(ab') 2The fragments were coated overnight. The plates were blocked with PBS and 0.5% bovine serum albumin (BSA) for 1 hour at 25°C. Each antibody (control IgG or modified Fc) was used as a standard to quantify the respective antibody concentration. The plates were washed with PBS and 0.05% Tween 20 using a microplate washer (Bio-Tek Instruments Inc.), and the standards and samples diluted in PBS containing 0.5% BSA, 0.35M NaCl, 0.25% CHAPS, 5mM EDTA, 0.05% Tween 20 and 15ppm (parts per million) Proclin were added for 2 hours at 25°C. F(ab') conjugated with HRP was added. 2 Bound antibodies were detected by goat anti-human IgG and Fc-specific polyclonal antibodies (Jackson ImmunoResearch) and developed with TMB (KPL Inc.), and absorbance (A) was measured at 450 nm on a Multiskan Ascent reader (Thermo Scientific). Concentrations were determined from the standard curve using a four-parameter nonlinear regression program.

[0330] Anti-BACE1 antibody pharmacokinetic assay - Antibody concentrations in mouse serum and brain samples were measured using ELISA. NUNC 384-well Maxisorp immunoplates (Neptune, NJ) were coated with coating agent recombinant human BACE1 ECD overnight at 4°C. The plates were then blocked with PBS containing 0.5% BSA for 1 hour at room temperature. Each antibody was used as a standard to quantify the respective antibody concentrations in serum and brain samples. After washing the plates with PBS containing 0.05% Tween 20 using a microplate washer (Bio-Tek Instruments, Inc., Winooski, VT), the standards and samples diluted in PBS containing 0.5% BSA, 0.35M NaCl, 0.25% CHAPS, 5mM EDTA, 0.05% Tween 20 and 15ppm Proclin were incubated on the plates for 2 hours at room temperature with gentle agitation. F(ab') conjugated with HRP was then blocked with PBS containing 0.5% BSA and 15ppm Proclin. 2Goat anti-human IgG Fc specific polyclonal antibody (Jackson ImmunoResearch) detects bound antibodies. Finally, the plate is developed using substrate 3,3',5,5'-tetramethylbenzidine (TMB) (KPL, Inc., Gaithersburg, MD). The absorbance is measured at a wavelength of 450nm with reference to 630nm on a Multiskan Ascent reader (Thermo Scientific, Hudson, NH). The concentration is determined from the standard curve using a four-parameter nonlinear regression program. Free anti-BACE1 mouse ELISA has a lower limit of detection (LLOD) of 0.06ng / ml.

[0331] PD assay - Aβ was measured in mouse brain samples using an ELISA similar to that described above for PK analysis 1-40 In short, the Aβ 1-40 A rabbit polyclonal antibody specific for the C-terminus of Αβ (Millipore, Bedford, MA) was coated onto the plate, and biotinylated anti-mouse Aβ monoclonal antibody M3.2 (Covance, Dedham, MA) was used for detection. The assay had a lower limit of quantification of 1.96 pg / ml in plasma and 39.1 pg / g in brain.

[0332] Radiolabeling studies - A modified Chizzonite radioiodination protocol was used to 125 I-labeled antibody (Chizzonite, Truitt et al. 1991). Wild-type or human FCGRT homozygous transgenic mice (Tg32) were administered [ 125 I] labeled antibody (5 mCi, intravenous). After injection, blood, brain and other tissues were collected at designated time points. The samples were then analyzed for total radioactivity per gram of tissue. Tissue radioactivity was corrected for the contribution from vascular blood concentration (n=2 per group).

[0333] Example 2 - Pharmacokinetics and pharmacodynamics of wild-type and Fc-modified hIgG1 antibodies in wild-type and human FCGRT transgenic mice

[0334] Two antibodies comprising modified Fc, M428L / N434A (LA) and M252Y / S254T / T256E (YTE), previously identified as having improved binding to hFcRn at pH 6, and two antibodies comprising wild-type hIgG1 and hIgG4 were expressed and purified, and the affinity to hFcRn was measured at pH 7.4 and pH 6. In addition, the transcytosis of each antibody comprising wild-type IgG or modified Fc was determined in an in vitro transcytosis assay as described in Example 1. Table 2 shows the affinity and transcytosis results of these antibodies.

[0335] Table 2: hFcRn affinity and transcytosis activity of wild-type antibodies and antibodies with improved hFcRn binding at pH 6

[0336]

[0337]

[0338] As shown in Table 2, antibodies comprising LA or YTE Fc modifications had improved FcRn binding at pH 6 and no detectable difference in binding at pH 7.4, and showed minimal improvement in transcytosis compared to the wild-type control. (>10 uM indicates that the affinity at pH 7.4 is undetectable or too weak to measure). The LA and YTE variants increased transcytosis 5.1-fold and 1.9-fold, respectively.

[0339] To evaluate whether antibodies with enhanced FcRn affinity at pH 6 can improve brain uptake, we performed a double-blind double-blind double-antibody assay in the presence of wild-type (Fcgrt + / + ) mice to determine the antibody pharmacokinetics and Aβ pharmacodynamics. As shown in Figure 1, anti-BACE1-hIgG1-YTE showed a faster clearance rate from plasma compared to anti-BACE1 hIgG1 with wild-type Fc ( Figure 1A ) and improved brain antibody concentrations ( Figure 1C ). Consistent with the improved brain concentrations of the antibody, anti-BACE1-hIgG1-YTE administration reduced brain Aβ 1-40 level( Figure 1B ). In contrast, anti-gD-hIgG1 (control) and anti-BACE1-hIgG1 antibodies were 1-40 levels, which is consistent with the low levels of those antibodies detected in the brain ( Figure 1B , 1C Compared with anti-BACE1-hIgG1, anti-BACE1-hIgG1-YTE showed significant difference in the expression of Fcgrt + / + ) mice based on both the maximum concentration (Cmax) and the area under the curve (AUC) ( Figure 1D ) Increased brain exposure.

[0340] Surprisingly, when transgenic Tg32 expressing hFCGRT and lacking mFCGRT (FCGRT + / + Fcg - / -When anti-BACE1-hIgG1-YTE was tested in 247 mice, there was no improvement in brain uptake or brain Aβ expression compared with anti-BACE1-hIgG1 1-40 The reduction in the level ( Figure 2A , 2B , 2C). To explore the reason for this, wild-type hIgG1 antibody and hIgG1 antibody containing YTE modification were tested for binding to mFcRn and hFcRn at pH 7.4 and pH 6. The data showed that YTE modification improved the binding to mFcRn by about 10-fold at both pH 7.4 and pH 6.0, while the binding to hFcRn was only improved at pH 6.0 ( Figure 2D ; Figure 2D K D The data differ slightly from those in Table 2 because they were measured in different experiments.) These results suggest that a stronger affinity for FcRn at neutral pH rather than pH 6 could increase transport into the brain.

[0341] Example 3 - Evaluation of hIgG1 and hIgG4 Fc modifications

[0342] To identify antibodies with improved brain uptake, anti-BACE1 antibodies with a series of single Fc modifications were generated. The positions of the modifications are according to EU numbering. See Fig.12 .

[0343] Antibodies comprising Fc modifications were expressed and purified and affinity for hFcRn at pH 7.4 and pH 6 was measured using Biacore, and transcytosis activity was determined as described in Example 1.

[0344] Table 3 shows antibodies with Fc modifications that improve binding to human FcRn at pH 6.0 that do not substantially improve transcytosis in vitro.

[0345] Table 3: hFcRn binding affinity and transcytosis activity of antibodies containing a single Fc modification

[0346]

[0347] As shown in Table 3, no or modest improvements in transcytosis (1.7-7.8) were observed for modified Fc antibodies with enhanced affinity for hFcRn at pH 6 but unmeasurable affinity at pH 7.4, consistent with the YTE results described in Example 2. In contrast, the weak but measurable pH 7.4 affinity (5.5 μM) of the antibody comprising the N434W Fc modification showed an increase in transcytosis (26-fold) relative to the wild-type antibody.

[0348] Single Fc modifications were combined into double, triple and quadruple Fc modifications, and anti-BACE1 antibodies comprising modified Fc were constructed, expressed and purified as described in Example 1, and tested for binding to human FcRn at pH 7.4 and pH 6 and efficacy in the transcytosis assay described in Example 1. Tables 4, 5 and 6 show antibodies comprising double, triple and quadruple Fc modifications, respectively, that have improved binding to human FcRn at pH 7.4 and have improved transcytosis activity. Figure 3 The correlation between pH 7.4 and pH 6 FcRn affinity of modified Fc antibodies is shown. Each point represents a single antibody. The open triangles represent exemplary antibodies of the present disclosure, which comprise the quadruple Fc modification M252Y / T307Q / Q311A / N434Y and are represented by Q95 (YQAY) which was further tested in vivo. Figure 4 Normalized transcytosis activities of the Fc-modified antibodies shown in Table 3, Table 4, Table 5 and Table 6 are shown.

[0349] Table 4: hFcRn binding affinity and transcytosis activity of antibodies containing dual Fc modifications

[0350]

[0351] Table 5: hFcRn binding affinity and transcytosis activity of antibodies containing triple Fc modifications

[0352]

[0353]

[0354]

[0355] Table 6: hFcRn binding affinity and transcytosis activity of antibodies containing quadruple Fc modifications

[0356]

[0357] As shown in the above table, a series of Fc modified antibodies were generated that had substantially improved transcytosis activity, with some Fc modifications promoting transcytosis more than 100-fold relative to wild-type IgG1 antibodies.

[0358] Tables 7 and 8 show the steady-state affinities of certain anti-BACE1 antibodies and certain anti-Aβ antibodies comprising a modified Fc as determined by BIACORE using an anti-human Fab capture chip.

[0359] Table 7: Steady-state affinity of selected anti-BACE1 antibodies comprising Fc modifications with improved binding to human FcRn

[0360]

[0361] Table 8: Steady-state affinity of selected anti-Aβ antibodies comprising Fc modifications with improved binding to human FcRn

[0362]

[0363]

[0364] Example 4 - Pharmacokinetics of Antibodies Comprising Modified Fc in Tg32 Mice

[0365] Transgenic Tg32 expressing hFCGRT and lacking mFCGRT (FCGRT + / + Fcg - / - ) mice were administered a single 25 mg / kg intravenous (IV) dose of an anti-gD antibody comprising wild-type human IgG1 (anti-gD-hIgG1), an anti-gD antibody comprising hIgG1 with a YY (D1, M252Y / N434Y) Fc modification (anti-gD-hIgG1-YY), an anti-gD antibody comprising hIgG1 with a YQAY (Q95, M252Y / T307Q / Q311A / N434Y) Fc modification (anti-gD-hIgG1-YQAY), or an anti-gD antibody comprising hIgG1 with a LA (M428L / N434A) Fc modification (anti-gD-IgG1-LA). Figure 5A and 5B As shown in , although the serum levels of anti-gD antibodies containing different hIgG1 Fc were similar, the brain levels of anti-gD-hIgG1-YY, anti-gD-hIgG1-YQAY, and anti-gD-hIgG1-LA were higher than anti-gD-hIgG1.

[0366] The binding affinity of anti-gD antibodies to hFcRn was measured at pH 6 and pH 7.4 as described above. As observed in the case of anti-BACE1 (Tables 4 and 6), the YY and YQAY Fc-modified antibodies provided similar hFcRn affinity enhancement in anti-gD antibodies ( Figure 5C ). The PK results and hFcRn affinity are summarized in Figure 5C In the Figure 4, it is shown that anti-gD-hIgG1-YY and anti-gD-hIgG1-YQAY have higher brain / serum ratios (measured as % AUC / AUC for brain / serum). Both Fc modifications contributed to significantly improved binding to hFcRn at pH 7.4 and pH 6 and high transcytosis scores (79 for YY and 100 for YQAY in the case of anti-BACE1 - Table 4 and Table 6).

[0367] Example 5 - Pharmacodynamics of Antibodies Containing Modified Fc in Tg32 Mice

[0368] Pharmacodynamic (PD) assays were performed to evaluate the efficacy of hFCGRT in transgenic Tg32 (FCGRT + / + Fcg - / - Aβ levels were assessed after a single intravenous administration of 50 mg / kg of anti-BACE1 antibodies comprising wild-type hIgG1 (anti-BACE1-hIgG1) and anti-BACE1 antibodies comprising hIgG1 with YQAY Fc modification (anti-BACE1-hIgG1-YQAY) to mice. Brain antibody levels and Aβ levels were determined as described in Example 1 1-40 Consistent with previous results, Fc-modified anti-BACE1-hIgG1-YQAY showed higher brain uptake than wild-type anti-BACE1-hIgG1 ( Figure 6B ). In addition, brain Aβ 1-40 levels were decreased, while anti-BACE1-hIgG1 showed little or no decrease ( Figure 6C ).like Fig.6D and 6E As shown in Figure 2, anti-BACE1-hIgG1-YQAY has a higher Cmax and AUC than anti-BACE1-hIgG1. 终末 , and greater Aβ reduction.

[0369] Similar experiments were performed with different anti-BACE1 antibodies in the form of anti-BACE1-hIgG1, anti-BACE1-hIgG1-YY, and anti-BACE1-hIgG1-YQAY. + / + Fcg - / - ) mice received a single intravenous administration of 50 mg / kg anti-BACE1-hIgG1, anti-BACE1-hIgG1-YY or anti-BACE1-hIgG1-YQAY. Brain antibody levels and Aβ were determined as described in Example 1 1-40 Although all three antibodies had similar serum concentrations ( Fig.13A ), but anti-BACE1-hIgG1-YQAY showed the highest brain uptake and anti-BACE1-hIgG1-YY tended to have enhanced brain uptake compared with anti-BACE1-hIgG1 ( Fig. 13B ). Consistent with higher brain uptake, brain Aβ 1-40 The level was most decreased, and a decreasing trend was observed after Fc-modified anti-BACE1-hIgG1-YY ( Fig. 13C ).like Fig.13DAs shown in , anti-BACE1-hIgG1-YQAY had higher brain Cmax and AUC, and anti-BACE1-hIgG1-YY trended towards higher brain Cmax and AUC, and greater Aβ reduction compared to anti-BACE1-hIgG1.

[0370] Example 6 - High transcytosis IgG1 variants in FCGRT + / + 、FCGRT + / - and Fcgrt + / + Pharmacokinetics in mice

[0371] Transgenic Tg32 expressing hFCGRT and lacking mFCGRT (FCGRT + / + Fcg - / - ) mice, expressing both hFCGRT and mFCGRT hemizygous (FCGRT + / - Fcg + / - ) mice, and wild-type (Fcgrt + / + ) mice were administered a single intravenous (IV) dose of 5 mg / kg anti-gD-hIgG1 antibody, anti-gD-hIgG1-YY, anti-gD-hIgG1-YQAY, or anti-gD antibody (anti-gD-hIgG1-YTE) containing hIgG1 with YTE (M252Y / S254T / T256E) Fc modification. 125 I labeled each antibody. All antibodies were expressed in homozygous Tg32 mice and hemizygous (FCGRT + / - Fcg + / - ) mice showed similar plasma PK. In wild-type mice, anti-gD-hIgG1-YY and anti-gD-hIgG1-YQAY showed faster clearance from plasma than anti-gD-hIgG1 or anti-gD-hIgG1-YTE ( Fig. 7A , 7B , 7C). Fig.7D Shown are the affinity of each of the anti-gD-hlgG1 and Fc-modified antibodies to hFcRn and mFcRn at pH 7.4 and pH 6, and the plasma AUC of each antibody relative to wild-type in each strain of mice.

[0372] The brain PK of anti-gD-hIgG1 and three Fc-modified antibodies in homozygous hFCGRT mice is shown in Figure 8. Anti-gD-hIgG1-YY and anti-gD-hIgG1-YQAY showed higher brain uptake than anti-gD-hIgG1 or anti-gD-hIgG1-YTE ( Fig. 8A The increased brain uptake of anti-gD-hIgG1-YY and anti-gD-hIgG1-YQAY was specific to homozygous hFCGRT Tg32 mice ( Figure 8BAs measured by AUC in mice homozygous for hFCGRT, anti-gD-hIgG1-YY provided a 2.2-fold increase in brain exposure compared to anti-gD-hIgG1, and anti-gD-hIgG1-YQAY provided an even greater increase in brain exposure, a 3.4-fold increase compared to anti-gD-hIgG1 ( Figure 8C ).

[0373] The pharmacokinetics of anti-gD-hlgG1 and the three Fc-modified antibodies were also determined in other tissues including liver, large intestine, and lung (Figures 9-11). These tissues also showed a modest improvement in tissue penetration of the Fc-modified antibodies, although this improvement was less than that seen in the brain. Without wishing to be bound by any particular theory, these tissues may have lower levels of FcRn or contain a vasculature with fenestrated capillaries that may allow a degree of antibody penetration via diffusion.

[0374] Example 7 - Pharmacokinetics and pharmacodynamics of a high transcytosis IgG1 variant in cynomolgus monkeys

[0375] Cynomolgus Monkey PK / PD Studies. For both studies, four male cynomolgus monkeys aged 3 to 5 years were used in each experimental group.

[0376] In the first study, anti-BACE1 hIgG1 wild-type antibody and anti-BACE1 hIgG1 antibodies with modified Fc (anti-BACE1-YQAY, YEY, YPY) were administered at 50 mg / kg via intravenous bolus injection into the saphenous vein on day 0. CSF and blood samples were collected at multiple time points from 7 days before dosing to 29 days after dosing. Samples were collected at the same time of the day.

[0377] In the second study, anti-BACE1 hIgG1 wild-type antibody and anti-BACE1 hIgG1 antibody with modified Fc (anti-BACE1-YQAY, YY, YLYI, YIY) were administered at 50 mg / kg via intravenous bolus injection into the saphenous vein. CSF and blood samples were collected at multiple time points from 7 days before dosing to 7 days after dosing. Brains of two animals were collected after systemic perfusion 2 days and 7 days after dosing. The brain regions were finely cut and immediately frozen. Different brain regions were homogenized in PBS containing complete micro-EDTA-free protease inhibitor mixed tablets (Roche Diagnostics) in 1% NP-40 (Cal-Biochem). The homogenized brain samples were rotated at 4°C for 1 hour, followed by spinning at 14,000 rpm for 20 minutes. The supernatant was separated for brain pharmacokinetic and pharmacodynamic (sAPPβ / α) analysis.

[0378] Pharmacokinetic Assays. Total antibody concentrations in monkey serum were measured using an LCMS method with affinity capture by pure protein body protein A magnetic beads (Millipore), followed by denaturation, reduction, alkylation, and trypsin digestion. Tag peptides from the Fc region were selected as a surrogate for total antibody concentration quantification. The MQC determined was 60 ng / mL. Total antibody concentrations in CSF and brain samples were measured using an ELISA method using monkey adsorbed sheep anti-human IgG polyclonal antibody (binding site) as a coating agent and monkey adsorbed goat anti-human IgG antibody (Bethyl) conjugated to HRP as a detector. The assay had a MQC value of 1.6 ng / ml in CSF and brain.

[0379] Pharmacodynamic Assays. sAPPβ / α Ratio. CSF and brain concentrations of sAPPα and sAPPβ were determined using a sAPPα / sAPPβ multiplex ECL assay. Anti-Aβ monoclonal antibody 6E10 was used to capture sAPPα, and an antibody against amino acids 591 to 596 of APP was used to capture APPβ. Both analytes were detected with an antibody against the N-terminus of APP. CSF was thawed on ice and then diluted 1:10 into TBS-Tween 20 containing 1% BSA. The assay had LLOQ values ​​of 0.05 and 0.03 ng / ml for sAPPα and sAPPβ, respectively.

[0380] Results. Fig.14A As shown in , in the first study, anti-BACE1 antibodies with modified Fc showed a significant reduction in cerebrospinal fluid (CSF) sAPPβ / α ratio compared to anti-BACE1 hIgG1 wild-type antibodies. Fig. 14B and 14C As shown in , FcRn affinity at neutral pH appears to correlate with increased clearance of antibodies from serum, with the anti-BACE1 hIgG1 wild-type antibody being cleared more slowly than either of the antibodies with a modified Fc.

[0381] like Fig.15A As shown in , in a second study, anti-BACE1 antibodies with modified Fc showed a significant reduction in CSF sAPPβ / α ratio compared to anti-BACE1 hIgG1 wild-type antibodies. Fig. 15B As shown in , anti-BACE1 antibodies with modified Fc showed a substantial reduction in brain sAPP β / α ratio compared to anti-BACE1 hIgG1 wild-type antibody. Fig. 15C Comparison of CSF sAPPβ / α ratio and brain sAPPβ / α ratio for each of the antibodies is shown. CSF and brain showed similar PD effects for various antibodies.

[0382] As shown in 15D, all anti-BACE1 antibodies with modified Fc were present in the brain at higher concentrations than anti-BACE1 hIgG1 wild-type antibody on day 2 (average of cortical and hippocampal values), and anti-BACE1 antibodies with YQAY, YY, and YIY Fc modifications had higher brain concentrations than anti-BACE1 hIgG1 wild-type antibody on day 7. For anti-BACE1 hIgG1YY, the fold increase in antibody concentration was 7.4 on day 2 and 4.7 on day 7. For anti-BACE1 hIgG1 YQAY, the fold increase in antibody concentration was 7.4 on day 2 and 8.1 on day 7 ( Fig.15E ).

[0383] Fig.15F A correlation between brain sAPPβ / α ratio and brain antibody concentration was demonstrated, which demonstrated that higher levels of anti-BACE1 antibodies were a result of lower sAPPβ / α ratio and stronger PD response in the brain.

[0384] Figure 15G Mean CSF concentrations of anti-BACE1 antibodies with wild-type or modified hIgG1 Fc are shown. Anti-BACE1 antibodies with hIgG1 Fc comprising YY and YQAY modifications showed higher CSF concentrations compared to anti-BACE1 antibodies with hIgG1 wild-type Fc at different time points. Fig.15H The ratio of the concentration of anti-BACE1 antibodies in CSF to the concentration of anti-BACE1 antibodies in serum is shown. All modified Fcs, YY, YQAY, YLYI and YIY, increase the proportion of anti-BACE1 antibodies in CSF. Fig.15I and 15J As shown in , anti-BACE1 antibodies with modified Fc were cleared from serum faster than anti-BACE1 hIgG1 wild-type antibodies.

[0385] Example 8 - Evaluation of anti-Aβ antibody target engagement in PS2APP transgenic mice

[0386] As discussed in Example 2, a modified Fc comprising M252Y / S254T / T256E (YTE) has improved binding to mFcRn at both pH 7.4 and pH 6.0, but only improves hFcRn at pH 6.0. In wild-type mice, anti-BACE1-hIgG1-YTE showed improved brain uptake compared to anti-BACE1-hIgG1. To determine whether improved brain uptake is observed for antibodies that bind to other brain antigens, anti-Aβ-hIgG4-YTE antibodies were administered to PS2APP mice co-expressing human APP (hAPP) with Swedish mutations K670N / M671L driven by Thy1 and PrP promoters, respectively, and human presenilin 2 with N141I mutations. See, e.g., Richards et al., J. Neurosci. 23, 8989-9003 (2003). These mice accumulate oligomeric and fibrillar amyloid deposits in the brain, including amyloid plaques, and thus can assess the target engagement of anti-Aβ antibodies. In general, after administration of anti-Aβ antibodies, antibody binding was observed along the periphery of amyloid plaques and in the mossy fiber hippocampal bundle. This specific staining pattern was not observed for isotype-matched control antibodies. (Data not shown; see, e.g., Meilandt, WJ et al., Characterization of the selective in vitro and in vivo binding properties of crenezumab to oligomeric Aβ. Alz Res Therapy 11, 97 (2019) doi: 10.1186 / s13195-019-0553-5).

[0387] In vivo administration. Transgenic PS2APP or non-transgenic (Ntg) littermates were randomized into treatment groups and received a single intravenous (iv) dose of anti-AβhIgG4 or anti-AβhIgG4-YTE (20, 40, 80 or 120 mg / kg). Antibodies were diluted in platform buffer (20 mM histidine, 240 mM sucrose; pH 5.5, 0.02% Tween 20) and injected at a volume of 5 ml / kg. Five days after administration, animals were sacrificed and peripheral plasma was collected via cardiac puncture, followed by perfusion with phosphate-buffered saline (PBS); the right hemisphere was excised and drop-fixed in 4% paraformaldehyde. The hippocampus, cortex and cerebellum were dissected from the left hemisphere, weighed and stored at -80°C.

[0388] Immunohistochemistry. The right hemibrain was drop-fixed in 4% paraformaldehyde for 48 hours and then transferred to 30% sucrose in PBS. Free-floating sagittal cryosections (35 μm) of mouse brain were washed in PBS, then PBS-Triton X100 (PBST, 0.1%), then blocked in 5% bovine serum albumin (BSA) in PBST (0.3%) and incubated overnight at 4°C with primary antibodies diluted in 1% BSA in PBST (0.3%). Goat anti-human IgG-Alexa594 (or Alexa555, 1:100-1:500; Thermo-Fisher, Waltham, MA) was used to localize the applied human antibodies. Plaques were detected using the Aβ fluorescent marker methoxy-X04.

[0389] Fluorescence microscopy. Whole-slide images were captured at 20× using a Pannoramic 250 (3D Histech, Hungary) equipped with a PCO.edge camera (Kelheim, Germany), a Lumencor SpectraX (Beaverton, OR), and Semrock filters (Rochester, NY) and optimized for 4'6-dicarboxamidino-2-phenylindole dihydrochloride (DAPI), tetramethylrhodamine isothiocyanate (TRITC), and cyanine 5 (Cy5) fluorophores. The ideal exposure for each channel was determined based on the sample with the brightest intensity and set for the entire set of slides to run as a batch. Images were also captured at 20× using a Leica DM5500B optical microscope using Leica Application Suite AdvancedFlorescence software (LAS AF4.0). Confocal images were acquired using a 20× or 40× oil objective on a Zeiss LSM800 confocal laser scanning microscope using Zen 2.3 software. Quantification of mossy fiber staining was performed by measuring the integrated density of two to four sections from each animal using ImageJ (NIH).

[0390] In vivo antibody pharmacokinetic (PK) measurements. Cerebellar samples were weighed and homogenized in 300 μl 1% NP-40 (containing Roche complete ETDA-free protease inhibitor cocktail) using Qiagen TissueLyser II (2x 3 minutes at 30 Hz). The samples were then placed on ice for 20 minutes and then centrifuged at 20,000×g for 20 minutes. The supernatant was collected and stored at −80° C. until used for PK assays. Antibody concentrations in mouse plasma and brain samples were measured using ELISA. NUNC 384-well Maxisorp immunoplates (Neptune, NJ, USA) were incubated at 4° C. with F(ab′) of a sheep anti-human IgG Fc fragment-specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA, USA). 2 The fragments were coated overnight. The plates were then blocked with PBS containing 0.5% BSA for 1 hour at room temperature. Each antibody (anti-AβhIgG4 or anti-AβhIgG4-YTE) was used as a standard to quantify the respective antibody concentration. After washing the plates with PBS containing 0.05% Tween 20 using a microplate washer (Bio-Tek Instruments, Inc., Winooski, VT), the standards and samples diluted in PBS containing 0.5% BSA, 0.35M sodium chloride (NaCl), 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 5mM EDTA, 0.05% Tween 20 and 15ppm Proclin were incubated on the plates for 2 hours at room temperature with gentle agitation. The bound antibodies were detected with horseradish peroxidase-conjugated F(ab')2 goat anti-human IgG Fc fragment-specific polyclonal antibodies (Jackson ImmunoResearch). Finally, the plate was developed using the substrate 3,3',5,5'-tetramethylbenzidine (KPL, Inc., Gaithersburg, MD, USA). The absorbance was measured at a wavelength of 450 nm with a reference of 630 nm on a Multiskan Ascent reader (Thermo Scientific, Hudson, NH, USA). The concentration was determined from the standard curve using a four-parameter nonlinear regression program. The assay had a lower limit of quantification of 13.7 ng / ml in plasma and 1.37 ng / ml in brain.

[0391] Results. Figures 16A-16BAs shown in, a dose-dependent increase in plasma PK and brain PK was observed for anti-AβhIgG4, while anti-AβhIgG4-YTE showed lower serum exposure and enhanced brain uptake at all doses. The brain: plasma ratio was approximately 0.15% for anti-AβhIgG4 and approximately 0.75% for anti-AβhIgG4 YTE.

[0392] Fig. 16C In vivo target engagement as measured by antibody binding to the mossy fiber hippocampal bundle is shown. Anti-AβhIgG4YTE showed significant binding after administration at 20 mg / kg, and increased binding with increasing doses. In contrast, anti-AβhIgG4 showed much lower staining compared to anti-AβhIgG4 YTE after administration at 20 mg / kg and 40 mg / kg. Fig.16D To show the Fig. 16C Bar graph of staining levels.

[0393] Fig.16E and 16F Binding of anti-AβhIgG4 and anti-AβhIgG4 YTE to the periphery of amyloid plaques in the peduncle (16E) and prefrontal cortex (16F) following administration at 20 mg / kg is shown. The level of binding of anti-AβhIgG4 YTE to amyloid plaques in these brain regions was much higher than that observed with anti-AβhIgG4.

[0394] Example 9 - Pharmacokinetics of a high transcytosis IgG4 variant in cynomolgus monkeys

[0395] Cynomolgus monkey PK study. Four male cynomolgus monkeys aged 3 to 5 years were used per experimental group. Anti-AβhIgG4 wild-type antibody and anti-AβhIgG4 antibodies with modified Fc (anti-Aβ-YQAY, YEY, YY) were administered at 50 mg / kg via intravenous bolus injection into the saphenous vein on day 0. The binding affinity of each of the antibodies to cynomolgus monkey FcRn at pH 7.4 and pH 6.0 is shown in Table 9. The affinities shown were measured at 25°C; the affinities at 37°C were similar (data not shown).

[0396] Table 9: Affinity of anti-AβhIgG4 antibodies to FcRn

[0397] Fc modification <![CDATA[K D pH7.4(nM)]]> <![CDATA[K D pH 6.0(nM)]]> wild type >2530 >253 YEY 369 10.1 YQ 215 8.2 YY 571 11.5

[0398] CSF and blood samples were collected at multiple time points from 7 days before dosing to 7 days after dosing. Samples were collected at the same time of the day. 2 days and 7 days after dosing, brains of two animals were collected after systemic perfusion. The brain regions were finely cut and immediately frozen. Different brain regions were homogenized in PBS containing complete micro-EDTA-free protease inhibitor mixed tablets (Roche Diagnostics) in 1% NP-40 (Cal-Biochem). The homogenized brain samples were rotated at 4°C for 1 hour, followed by a 20-minute spin at 14,000 rpm. The supernatant was separated for brain pharmacokinetic analysis.

[0399] Pharmacokinetic Assays. Total antibody concentrations in monkey serum, CSF, and brain samples were measured using an ELISA method with monkey-adsorbed sheep anti-human IgG polyclonal antibody (binding site) as coating agent and monkey-adsorbed goat anti-human IgG antibody (Bethyl) conjugated to HRP as detection agent. The assay had an MQC value of 1.6 ng / ml in CSF and brain.

[0400] result. Fig.17A The mean brain concentrations of anti-Aβ antibodies on days 2 and 7 are shown. Fig. 17B The fold increase in brain concentration observed for each modified hIgG4 Fc is summarized. Anti-Aβ antibodies with modified Fc showed a 2.7 to 4.7 fold increase in brain uptake at day 2, and a 3.7 to 4.2 fold increase at day 7 compared to wild type Fc.

[0401] Fig. 17C Mean CSF concentrations of anti-Aβ antibodies with wild-type or modified hIgG4 Fc are shown. Anti-Aβ antibodies with hIgG4 Fc comprising YEY and YQAY modifications showed higher CSF concentrations compared to anti-Aβ antibodies with hIgG4 wild-type Fc. Small improvements were also observed for Fc comprising YY modifications. Fig.17D The ratio of the concentration of anti-Aβ antibodies in CSF to the concentration of anti-Aβ antibodies in serum is shown. All modified Fcs, YY, YEY and YQAY, increased the proportion of anti-Aβ antibodies in CSF. Fig.17E and 17F As shown in , anti-Aβ antibodies with modified Fc were cleared from serum faster than anti-Aβ hIgG4 wild-type antibodies.

[0402] Fig.18A Serum exposure in cynomolgus monkeys and affinity of Fc for hFcRn at pH 7.4 (K D (7.4)). Both anti-BACE1 hIgG1 (circles) and anti-Aβ IgG4 (squares) Fc modified variants are shown on the graph. WT Fc is circled and labeled.

[0403] Fig.18B Antibody brain distribution in cynomolgus monkeys is shown (% [mAb 脑 ] / [mAb 血清 ]) and the affinity of Fc for hFcRn at pH 7.4 (K D (7.4)). Both anti-BACE1 hIgG1 (circles) and anti-Aβ IgG4 (squares) Fc modified variants are shown on the graph. WT Fc is circled and labeled.

[0404] In summary, the results demonstrate that modified Fc with increased affinity for FcRn at pH 7.4 improves brain exposure in cynomolgus monkeys in both hIgG1 and hIgG4 cases. Improvements in CSF exposure were also observed, especially compared to serum exposure. Without wishing to be bound by any particular theory, increased affinity for FcRn at pH 7.4 may increase the fraction of IgG that is transcytosed rather than recycled or degraded, thereby improving brain and CSF exposure. Alternatively or in addition, increased affinity for FcRn on the cell surface at pH 7.4 may increase the amount of IgG antibodies that are internalized into cells and transcytosed, thereby improving brain and CSF exposure.

[0405] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

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[0444] Sequence Listing

[0445] Sequence Listing <110> Hoffmann-La Roche GmbH <120> Modified antibody Fc and methods of using the same <130> 01146-0072-00PCT <150> US 62 / 782,904 <151> 2018-12-20 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 218 <212> PRT <213> Homo sapiens <220> <221> Features not yet classified <222> (127)..(127) <223> X is D or E <220> <221> Features not yet classified <222> (129)..(129) <223> X is L or M <220> <221> Features not yet classified <222> (202)..(202) <223> X is A or G <400> 1 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1 5 10 15 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25 30 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 35 40 45 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90 95 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 100 105 110 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu 115 120 125 Xaa Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 130 135 140 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 145 150 155 160 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170 175 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 180 185 190 Val Phe Ser Cys Ser Val Met His Glu Xaa Leu His Asn His Tyr Thr 195 200 205 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 2 <211> 217 <212> PRT <213> Homo sapiens <220> <221> Features not yet classified <222> (52)..(52) <223> X is V or M <400> 2 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Xaa Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 3 <211> 218 <212> PRT <213> Homo sapiens <400> 3 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1 5 10 15 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25 30 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp 35 40 45 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60 Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90 95 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 100 105 110 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 115 120 125 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 130 135 140 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn 145 150 155 160 Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 165 170 175 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 180 185 190 Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr 195 200 205 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 4 <211> 218 <212> PRT <213> Homo sapiens <400> 4 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1 5 10 15 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25 30 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 35 40 45 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90 95 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 100 105 110 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 115 120 125 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 130 135 140 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 145 150 155 160 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170 175 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 180 185 190 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 195 200 205 Gln Light See Leu See Leu See Leu Gly Light 210 215

Claims

1. Use of an antibody comprising human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method for treating a neurodegenerative disease, the method comprising administering an antibody comprising human IgG1 or IgG4 Fc to a subject in need thereof, wherein the antibody is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

2. The method according to claim 1, wherein the neurodegenerative disease is selected from the group consisting of Lewy body disease, post-polio syndrome, olivopontocerebellar atrophy, amyloidosis, multiple system atrophy, striatonigral degeneration, tauopathy, supranuclear palsy, prion disease, bovine spongiform encephalopathy, scrapie and fatal familial insomnia.

3. The use according to claim 1, wherein the neurodegenerative disease is selected from Shy-Drager syndrome, Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob syndrome and Gerstmann-Straussler-Scheinker disease.

4. The use according to claim 1, wherein the neurodegenerative disease is kuru.

5. Use of an antibody comprising human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method of delivering an antibody to the brain of a subject having a neurodegenerative disease, the method comprising administering to the subject an antibody comprising human IgG1 or IgG4 Fc, wherein the antibody is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

6. Use of an antibody comprising human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method of increasing brain exposure to an antibody in a subject having a neurodegenerative disease, the method comprising administering to the subject an antibody comprising human IgG1 or IgG4 Fc, wherein the antibody is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

7. Use of an antibody comprising human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method of increasing antibody transport across the blood-brain barrier (BBB) ​​in a subject with a neurodegenerative disease, the method comprising administering to the subject an antibody comprising human IgG1 or IgG4 Fc, wherein the antibody is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

8. The use according to any one of claims 1-7, wherein the antibody exhibits a transcytosis activity of at least 50 in the in vitro transcytosis assay when normalized to the same antibody comprising wild-type IgG Fc.

9. The use according to claim 8, wherein the antibody exhibits a transcytosis activity of at least 60 in the in vitro transcytosis assay.

10. The use according to claim 8, wherein the antibody exhibits a transcytosis activity of at least 70 in the in vitro transcytosis assay.

11. The use according to claim 8, wherein the antibody exhibits a transcytosis activity of at least 80 in the in vitro transcytosis assay.

12. The use according to claim 8, wherein the antibody exhibits a transcytosis activity of at least 90% in the in vitro transcytosis assay.

13. The use according to claim 8, wherein the antibody exhibits a transcytosis activity of at least 100 in the in vitro transcytosis assay.

14. The use according to any one of claims 1 to 13, wherein the cell is an MDCKII cell.

15. The use according to any one of claims 1-14, wherein the antibody has a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference antibody having a wild-type IgG Fc of the same species and isotype.

16. The use according to any one of claims 1 to 15, wherein the antibody is active at pH 6. The binding affinity to FcRn is greater than the binding affinity of a reference antibody having a wild-type IgG Fc of the same species and isotype.

17. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤10 μM.

18. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤5 μM.

19. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤4 μM.

20. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤3 μM.

21. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤2 μM.

22. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤1 μM.

23. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤900 nM.

24. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤800 nM.

25. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤700 nM.

26. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤600 nM.

27. The use according to any one of claims 1 to 16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤500 nM.

28. The use according to any one of claims 1-16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤400 nM.

29. The use according to any one of claims 1-16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤300 nM.

30. The use according to any one of claims 1-16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤200 nM.

31. The use according to any one of claims 1-16, wherein the antibody is active at pH 7.

4. The binding affinity to FcRn is ≤100 nM.

32. The use according to any one of claims 1 to 31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤1 μM.

33. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤900 nM.

34. The use according to any one of claims 1 to 31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤800 nM.

35. The use according to any one of claims 1 to 31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤700 nM.

36. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤600 nM.

37. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤500 nM.

38. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤400 nM.

39. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤300 nM.

40. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤200 nM.

41. according to the use described in any one of claims 1-31, wherein the antibody is at pH 6 to The binding affinity to FcRn is ≤100 nM.

42. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤90 nM.

43. according to the use described in any one of claims 1-31, wherein the antibody is at pH 6 to The binding affinity to FcRn is ≤80 nM.

44. according to the use described in any one of claims 1-31, wherein the antibody is at pH 6 to The binding affinity to FcRn is ≤70 nM.

45. according to the use described in any one of claims 1-31, wherein the antibody is at pH 6 to The binding affinity to FcRn is ≤60 nM.

46. ​​according to the use described in any one of claims 1-31, wherein the antibody is at pH 6 to The binding affinity to FcRn is ≤50 nM.

47. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤40 nM.

48. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤30 nM.

49. The use according to any one of claims 1-31, wherein the antibody is active at pH 6. The binding affinity to FcRn is ≤20 nM.

50. according to the use described in any one of claims 1-31, wherein the antibody is at pH 6 to The binding affinity to FcRn is ≤10 nM.

51. The use according to any one of claims 1-50, wherein the antibody is active at pH 7.

4. The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is at least 5.

52. The use according to any one of claims 1-50, wherein the antibody is active at pH 7.

4. The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is at least 10.

53. according to the use described in any one of claims 1-50, wherein the antibody is to The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is at least 20.

54. according to the use described in any one of claims 1-50, wherein the antibody is to The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is at least 50.

55. according to the use described in any one of claims 1-50, wherein the antibody is to The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is at least 100.

56. according to the use described in any one of claims 1-50, wherein the antibody is to The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is from 5 to 200.

57. The use according to any one of claims 1-50, wherein the antibody is active at pH 7.

4. The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is between 5 and 100.

58. The use according to any one of claims 1-50, wherein the antibody is active at pH 7.

4. The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is from 10 to 200.

59. The use according to any one of claims 1-50, wherein the antibody is active at pH 7.

4. The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is 10 to 100.

60. The use according to any one of claims 1-50, wherein the antibody is active at pH 7.

4. The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is 20 to 100.

61. according to the use described in any one of claims 1-50, wherein the antibody is to The ratio of the affinity for FcRn to the affinity of the antibody for FcRn at pH 6 is 20 to 200.

62. The use according to any one of claims 1-61, wherein the antibody is a monoclonal antibody.

63. The use according to any one of claims 1-62, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

64. The use according to any one of claims 1-63, wherein the antibody is a bispecific antibody.

65. The use according to any one of claims 1-64, wherein the wild-type IgG Fc corresponding to the human IgG1 or IgG4 Fc comprises the sequence of SEQ ID NO: 1 or 4.

66. The use according to any one of claims 1-65, wherein the antibody is conjugated to an imaging agent.

67. The use according to any one of claims 1-65, wherein the antibody is conjugated to a neurological disorder drug.

68. The use according to claim 67, wherein the neurological disorder drug is an aptamer.

69. The use according to claim 67, wherein the neurological disorder drug is an inhibitory nucleic acid.

70. The use according to claim 67, wherein the neurological disorder drug is a ribozyme.

71. The use of claim 67, wherein the neurological disorder drug is a small molecule.

72. Use of an Fc conjugate of an antibody comprising human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method of treating a neurodegenerative disease, the method comprising administering to a subject in need thereof an Fc conjugate of an antibody comprising human IgG1 or IgG4 Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

73. The use according to claim 72, wherein the neurodegenerative disease is selected from Lewy body disease, post-polio syndrome, olivopontocerebellar atrophy, amyloidosis, multiple system atrophy, striatonigral degeneration, tauopathies, supranuclear palsy, prion diseases, bovine spongiform encephalopathy, scrapie and fatal familial insomnia.

74. The use according to claim 72, wherein the neurodegenerative disease is selected from Shy-Drager syndrome, Parkinson's disease, Alzheimer's disease, Creutzfeldt-Jakob syndrome and Gerstmann-Straussler-Scheinker disease.

75. The use according to claim 72, wherein the neurodegenerative disease is kuru.

76. Use of an Fc conjugate of an antibody comprising human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method of delivering an Fc conjugate to the brain of a subject having a neurodegenerative disease, the method comprising administering to the subject an Fc conjugate of an antibody comprising human IgG1 or IgG4 Fc. conjugate, wherein the Fc conjugate is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of: β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and among them (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

77. Use of an Fc conjugate of an antibody comprising human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method of increasing brain exposure to an Fc conjugate in a subject having a neurodegenerative disease, the method comprising administering to the subject an Fc conjugate of an antibody comprising human IgG1 or IgG4 Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

78. Use of an Fc conjugate of an antibody comprising a human IgG1 or IgG4 Fc for the preparation of a medicament for use in a method of increasing transport of an Fc conjugate across the blood-brain barrier (BBB) ​​in a subject having a neurodegenerative disease, the method comprising administering to the subject an Fc conjugate of an antibody comprising a human IgG1 or IgG4 Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of: β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), and among them (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

79. The use according to any one of claims 72 to 78, wherein the Fc conjugate exhibits a transcytosis activity of at least 50 in the in vitro transcytosis assay when normalized to the same Fc conjugate comprising a wild-type IgG Fc.

80. The use according to claim 79, wherein said Fc conjugate exhibits a transcytosis activity of at least 60 in said in vitro transcytosis assay.

81. The use of claim 79, wherein said Fc conjugate exhibits a transcytosis activity of at least 70 in said in vitro transcytosis assay.

82. The use of claim 79, wherein said Fc conjugate exhibits a transcytosis activity of at least 80 in said in vitro transcytosis assay.

83. The use of claim 79, wherein said Fc conjugate exhibits a transcytosis activity of at least 90% in said in vitro transcytosis assay.

84. The use of claim 79, wherein said Fc conjugate exhibits a transcytosis activity of at least 100 in said in vitro transcytosis assay.

85. The method of any one of claims 72 to 84, wherein the cell is an MDCK II cell.

86. The use according to any one of claims 72 to 85, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is greater than that of the reference Fc conjugate with wild-type IgG Fc of the same species and isotype.

87. The use according to any one of claims 72 to 86, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is greater than that of wild-type IgG of the same species and isotype Binding affinity of the reference Fc conjugate to Fc.

88. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is less than 1 mM.

89. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is less than 750 nM.

90. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is less than 500 nM.

91. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is less than 400 nM.

92. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is less than 300 nM.

93. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is less than 200 nM.

94. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is less than 100 nM.

95. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is between 50 nM and 1 mM.

96. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 is between 100 nM and 1 mM.

97. The use according to any one of claims 72 to 87, wherein the Fc conjugate is The binding affinity to FcRn at pH 7.4 was between 100 nM and 500 nM.

98. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 100 nM.

99. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 90 nM.

100. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 80 nM.

101. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 70 nM.

102. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 60 nM.

103. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 50 nM.

104. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 40 nM.

105. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 30 nM.

106. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 20 nM.

107. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is less than 10 nM.

108. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 was between 1 nM and 200 nM.

109. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is between 10 nM and 200 nM.

110. The use according to any one of claims 72 to 97, wherein the Fc conjugate is The binding affinity to FcRn at pH 6 is between 10 nM and 100 nM.

111. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of said Fc conjugate for FcRn at pH 6 is at least 5.

112. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of said Fc conjugate for FcRn at pH 6 is at least 10.

113. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of said Fc conjugate for FcRn at pH 6 is at least 20.

114. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of said Fc conjugate for FcRn at pH 6 is at least 50.

115. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of said Fc conjugate for FcRn at pH 6 is at least 100.

116. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is between 5 and 200.

117. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is between 5 and 100.

118. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is 10 to 200.

119. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is 10 to 100.

120. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is 20 to 100.

121. The use according to any one of claims 72 to 110, wherein the Fc conjugate is The ratio of the affinity for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is 20 to 200.

122. The use according to any one of claims 72 to 121, wherein the Fc conjugate comprises the human IgG1 or IgG4 Fc fused to a therapeutic protein.

123. The use according to claim 122, wherein the therapeutic protein is selected from the group consisting of a receptor extracellular domain and an enzyme.

124. The use according to claim 123, wherein the receptor extracellular domain is selected from the group consisting of TNF-R1 extracellular domain (ECD), CTLA-4 ECD and IL-1R1 ECD.

125. The use according to claim 123, wherein the enzyme is selected from α-L-iduronidase, iduronic acid-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, arylsulfatase A, acid ceramidase, asparagyl transferase, palmitoyl protein thioesterase 1 and tripeptidyl aminopeptidase 1.

126. The use according to any one of claims 72 to 121, wherein the Fc conjugate comprises the human IgG1 or IgG4 Fc conjugated to a neurological disorder drug.

127. The use according to claim 126, wherein the neurological disorder drug is an aptamer.

128. The use of claim 126, wherein the neurological disorder drug is an inhibitory nucleic acid.

129. The use according to claim 126, wherein the neurological disorder drug is a ribozyme.

130. The use of claim 126, wherein the neurological disorder drug is a small molecule.

131. The use according to any one of claims 72 to 121, wherein the Fc conjugate comprises the human IgG1 or IgG4 Fc conjugated to an imaging agent.

132. An isolated antibody, wherein the antibody comprises a human IgG1 or IgG4 Fc, wherein the antibody is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of β-secretase 1 (BACE1), amyloid β (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

133. The isolated antibody of claim 132, wherein the antibody exhibits a transcytosis activity of at least 50 in the in vitro transcytosis assay when normalized to the same antibody comprising a wild-type IgG Fc.

134. The isolated antibody of claim 133, wherein the antibody exhibits a transcytosis activity of at least 60 in the in vitro transcytosis assay.

135. The isolated antibody of claim 133, wherein the antibody exhibits a transcytosis activity of at least 70 in the in vitro transcytosis assay.

136. The isolated antibody of claim 133, wherein the antibody exhibits a transcytosis activity of at least 80 in the in vitro transcytosis assay.

137. The isolated antibody of claim 133, wherein the antibody exhibits a transcytosis activity of at least 90% in the in vitro transcytosis assay.

138. The isolated antibody of claim 133, wherein the antibody exhibits a transcytosis activity of at least 100 in the in vitro transcytosis assay.

139. The isolated antibody of any one of claims 132 to 138, wherein the cell is a MDCKII cell.

140. The isolated antibody of any one of claims 132 to 139, wherein the antibody has a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference antibody having a wild-type IgG Fc of the same species and isotype.

141. The isolated antibody of any one of claims 132 to 140, wherein the antibody has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference antibody having a wild-type IgG Fc of the same species and isotype.

142. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 10 μM at pH 7.

4.

143. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 5 μM at pH 7.

4.

144. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 4 μM at pH 7.

4.

145. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 3 μM at pH 7.

4.

146. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 2 μM at pH 7.

4.

147. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 1 μM at pH 7.

4.

148. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤900 nM at pH 7.

4.

149. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 800 nM at pH 7.

4.

150. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤700 nM at pH 7.

4.

151. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 600 nM at pH 7.

4.

152. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤500 nM at pH 7.

4.

153. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤400 nM at pH 7.

4.

154. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 300 nM at pH 7.

4.

155. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤200 nM at pH 7.

4.

156. The isolated antibody of any one of claims 132 to 141, wherein the antibody has a binding affinity for FcRn of ≤ 100 nM at pH 7.

4.

157. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤ 1 μM at pH 6.

158. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤900 nM at pH 6.

159. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤800 nM at pH 6.

160. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤700 nM at pH 6.

161. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤ 600 nM at pH 6.

162. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤500 nM at pH 6.

163. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤400 nM at pH 6.

164. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤300 nM at pH 6.

165. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤200 nM at pH 6.

166. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤ 100 nM at pH 6.

167. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤90 nM at pH 6.

168. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤ 80 nM at pH 6.

169. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤70 nM at pH 6.

170. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤ 60 nM at pH 6.

171. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤50 nM at pH 6.

172. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤40 nM at pH 6.

173. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤30 nM at pH 6.

174. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤20 nM at pH 6.

175. The isolated antibody of any one of claims 132 to 156, wherein the antibody has a binding affinity for FcRn of ≤ 10 nM at pH 6.

176. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is at least 5.

177. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is at least 10.

178. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is at least 20.

179. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is at least 50.

180. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is at least 100.

181. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is from 5 to 200.

182. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is from 5 to 100.

183. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is from 10 to 200.

184. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is 10 to 100.

185. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is from 20 to 100.

186. The isolated antibody of any one of claims 132 to 175, wherein the ratio of the affinity of the antibody for FcRn at pH 7.4 to the affinity of the antibody for FcRn at pH 6 is from 20 to 200.

187. The isolated antibody of any one of claims 132 to 186, wherein the antibody is a monoclonal antibody.

188. The isolated antibody of any one of claims 132 to 187, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

189. The isolated antibody of any one of claims 132 to 188, wherein the antibody is a bispecific antibody.

190. The isolated antibody of any one of claims 132 to 189, wherein a wild-type IgG Fc corresponding to the human IgG1 or IgG4 Fc comprises SEQ ID NO: 1 or 4.

191. The isolated antibody of any one of claims 132 to 190, wherein the antibody is conjugated to an imaging agent.

192. The isolated antibody of any one of claims 132 to 190, wherein the antibody is conjugated to a neurological disorder drug.

193. The isolated antibody of claim 192, wherein the neurological disorder drug is an aptamer.

194. The isolated antibody of claim 192, wherein the neurological disorder drug is an inhibitory nucleic acid.

195. The isolated antibody of claim 192, wherein the neurological disorder drug is a ribozyme.

196. The isolated antibody of claim 192, wherein the neurological disorder drug is a small molecule.

197. An Fc conjugate of an antibody comprising a human IgG1 or IgG4 Fc, wherein the Fc conjugate is active in an in vitro transcytosis assay, wherein the in vitro transcytosis assay comprises cells expressing FcRn, wherein the antibody binds to a brain antigen selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta (Aβ), Tau, glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), and wherein (1) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by A, and N at position 434 is replaced by Y, according to EU numbering; (2) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (3) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, Q at position 311 is replaced by I, and N at position 434 is replaced by Y, according to EU numbering; (4) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (5) in human IgG4 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (6) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (7) in human IgG4 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (8) in human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by A, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (9) in human IgG1 Fc, M at position 252 is replaced by Y, M at position 428 is replaced by L, N at position 434 is replaced by Y, and Y at position 436 is replaced by I, according to EU numbering; (10) in human IgG1 Fc, M at position 252 is replaced by Y, T at position 307 is replaced by Q, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to EU numbering; or (11) In human IgG1 Fc, M at position 252 is replaced by Y, Q at position 311 is replaced by I, M at position 428 is replaced by L, and N at position 434 is replaced by Y, according to the EU numbering.

198. The Fc conjugate of claim 197, wherein said Fc conjugate exhibits a transcytosis activity of at least 50 in said in vitro transcytosis assay when normalized to the same Fc conjugate comprising a wild-type IgG Fc.

199. The Fc conjugate of claim 198, wherein said Fc conjugate exhibits a transcytosis activity of at least 60 in said in vitro transcytosis assay.

200. The Fc conjugate of claim 198, wherein said Fc conjugate exhibits a transcytosis activity of at least 70 in said in vitro transcytosis assay.

201. The Fc conjugate of claim 198, wherein said Fc conjugate exhibits a transcytosis activity of at least 80 in said in vitro transcytosis assay.

202. The Fc conjugate of claim 198, wherein said Fc conjugate exhibits a transcytosis activity of at least 90% in said in vitro transcytosis assay.

203. The Fc conjugate of claim 198, wherein said Fc conjugate exhibits a transcytosis activity of at least 100 in said in vitro transcytosis assay.

204. The Fc conjugate of any one of claims 197 to 203, wherein the cell is a MDCK II cell.

205. The Fc conjugate of any one of claims 197 to 204, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is greater than the binding affinity of a reference Fc conjugate having a wild-type IgG Fc of the same species and isotype.

206. The Fc conjugate of any one of claims 197 to 205, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is greater than the binding affinity of a reference Fc conjugate having a wild-type IgG Fc of the same species and isotype.

207. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 10 μM.

208. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 5 μM.

209. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 4 μM.

210. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 3 μM.

211. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 2 μM.

212. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 1 μM.

213. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 900 nM.

214. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 800 nM.

215. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 700 nM.

216. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 600 nM.

217. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 500 nM.

218. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 400 nM.

219. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 300 nM.

220. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 200 nM.

221. The Fc conjugate of any one of claims 197 to 206, wherein the binding affinity of the Fc conjugate to FcRn at pH 7.4 is ≤ 100 nM.

222. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 1 μM.

223. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 900 nM.

224. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 800 nM.

225. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 700 nM.

226. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 600 nM.

227. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 500 nM.

228. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 400 nM.

229. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 300 nM.

230. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 200 nM.

231. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 100 nM.

232. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 90 nM.

233. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 80 nM.

234. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 70 nM.

235. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 60 nM.

236. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 50 nM.

237. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 40 nM.

238. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 30 nM.

239. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 20 nM.

240. The Fc conjugate of any one of claims 197 to 221, wherein the binding affinity of the Fc conjugate to FcRn at pH 6 is ≤ 10 nM.

241. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is at least 5.

242. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is at least 10.

243. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is at least 20.

244. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is at least 50.

245. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is at least 100.

246. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is from 5 to 200.

247. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is from 5 to 100.

248. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is from 10 to 200.

249. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is from 10 to 100.

250. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is from 20 to 100.

251. The Fc conjugate of any one of claims 197 to 240, wherein the ratio of the affinity of the Fc conjugate for FcRn at pH 7.4 to the affinity of the Fc conjugate for FcRn at pH 6 is from 20 to 200.

252. The Fc conjugate of any one of claims 197 to 251, wherein the Fc conjugate comprises the human IgG1 or IgG4 Fc fused to a therapeutic protein.

253. The Fc conjugate of claim 252, wherein the therapeutic protein is selected from a receptor extracellular domain and an enzyme.

254. The Fc conjugate of claim 253, wherein the receptor extracellular domain is selected from the group consisting of TNF-R1 extracellular domain (ECD), CTLA-4 ECD, and IL-1R1 ECD.

255. The Fc conjugate of claim 253, wherein the enzyme is selected from α-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, α-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, β-galactosidase, arylsulfatase B, β-glucuronidase, acid α-glucosidase, glucocerebrosidase, α-galactosidase A, hexosaminidase A, acid sphingomyelinase, β-galactocerebrosidase, arylsulfatase A, acid ceramidase, asparagyl transferase, palmitoyl protein thioesterase 1, and tripeptidyl aminopeptidase 1.

256. The Fc conjugate of any one of claims 197 to 255, wherein the wild-type IgG Fc corresponding to the human IgG1 or IgG4 Fc comprises the sequence of SEQ ID NO: 1 or 4.

257. The Fc conjugate of any one of claims 197 to 256, wherein the Fc conjugate is conjugated to an imaging agent.

258. The Fc conjugate of any one of claims 197 to 256, wherein the Fc conjugate is conjugated to a neurological disorder drug.

259. The Fc conjugate of claim 258, wherein the neurological disorder drug is an aptamer.

260. The Fc conjugate of claim 258, wherein the neurological disorder drug is an inhibitory nucleic acid.

261. The Fc conjugate of claim 258, wherein the neurological disorder drug is a ribozyme.

262. The Fc conjugate of claim 258, wherein the neurological disorder drug is a small molecule.

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