Modified antibody fc and method for using the same

Abstract

To provide an antibody and Fc conjugate having improved incorporation into a brain.SOLUTION: In one embodiment, provided is a method for treating neurological disorder of a subject which includes a step of administering an antibody including modified IgG Fc to a subject in need thereof, where the antibody is active in an in-vitro transcytosis assay. In one embodiment, provided is a method for delivering an antibody to a brain of a subject which includes a step of administering an antibody including modified IgG Fc to a subject in need thereof, where the antibody is active in an in-vitro transcytosis assay.SELECTED DRAWING: Figure 8A

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 782,904, filed December 20, 2018, which is incorporated by reference in its entirety for all purposes.

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

[0003] There are several central nervous system (CNS) therapeutic targets with significant medical value, including amyloid beta (Abeta), beta-secretase 1 (BACE1), tau, and alpha-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) ​​limits antibody penetration into the brain under normal circumstances (Abbott, Ronnback et al. 2006; Pardridge, 2016).

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

[0005] However, TfR targeting has drawbacks. For example, to generate therapeutic antibodies that utilize TfR to cross the BBB, a second binding domain must be introduced into traditional IgG via one of a number of multispecific technologies, including, for example, knob-in-hole, dual variable domain, or cross-mAb technologies. These technologies add both cost and complexity to the development of therapeutic antibodies. Furthermore, TfR is expressed in several tissues, and targeting this receptor may introduce several potential safety liabilities. For example, targeting TfR may cause reticulocyte reduction (Couch, Yu et al. 2013). This risk may be mitigated by using various effector attenuation technologies (Couch, Yu et al. 2013; Lo, Kim et al. 2017). However, since antibody effector function has been proposed to play a role in the mechanism of action of some Abeta-targeting antibodies, effector attenuation may limit the effectiveness of amyloid-targeting antibodies, as well as antibodies targeting other antigens.

[0006] A number of biological antibody receptors can interact with IgG molecules, such as Fc-gamma receptors and fetal Fc receptors (FcRn) (Ghetie and Ward 2002; Challa, Velmurugan et al. 2014). FcRn is a heterodimeric protein complex composed of two subunits, FCGRT, also known as the FcRn alpha chain, and beta-2 microglobulin (β2M). In humans, FcRn is expressed on some hematopoietic cells, kidney cells, intestinal and upper respiratory epithelial cells, and normal endothelial cells, including those located at the BBB (Roopenian and Akilesh 2007; Challa, Velmurugan et al. 2014). During development, FcRn facilitates the transport of IgG molecules across the placental barrier, and during infancy, FcRn facilitates the transport of IgG from breast milk across intestinal epithelial cells. In adults, the primary function of FcRn is to mediate endosomal recycling of IgG and the resulting sustained serum half-life of IgG.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 respiratory tract (including lung epithelial cells), kidney 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, ovaries, testes, seminal vesicles and adipose tissue.

[0007] IgG binding to FcRn is regulated by pH. At physiological pH, such as in serum, IgG binding to FcRn is virtually nonexistent, but at acidic pH, such as in endosomes, the affinity of IgG for FcRn is enhanced (Kuo and Aveson, 2011). This enhanced binding to FcRn at acidic pH in endosomes results in trapping of pinocytosed antibodies, preventing lysosomal degradation and maintaining antibody levels in serum (Ghetie and Ward, 2002). Research efforts have identified antibody Fc variants with improved pharmacokinetic properties as a result of 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). Several Fc variants have been identified that improve binding to FcRn at both 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, some of these Fc variants may also result in 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 a limited function in the transport of antibodies to the brain (Abuqayyas and Balthasar, 2013), and indeed experiments using direct intracranial antibody infusion have shown that FcRn functions to facilitate the transport of antibodies from the brain (Cooper, Ciambrone et al., 2013). Summary of the Invention

[0009] Provided herein are antibodies and Fc conjugates that comprise a modified Fc and have improved brain uptake.

[0010] In some embodiments, a method of treating a neurological disorder in a subject is provided, 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 neuropathy disorder, a neurodegenerative disease, a brain disorder, a cancer, an eye disease disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and a 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, amyloidosis, Parkinson's disease, multiple system atrophy, striatonigral degeneration, amyloidosis, tauopathy, Alzheimer's disease, supranuclear palsy, prion disease, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-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, 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.

[0012] In some embodiments, a method of increasing brain exposure to an antibody is provided, 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, a method is provided for increasing transport of an antibody across the blood-brain barrier (BBB), 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 that comprises a modified IgG Fc and is active in an in vitro transcytosis assay.

[0015] In some embodiments, the antibody exhibits a transcytosis activity in an in vitro transcytosis assay of at least 50 when normalized to the same antibody with a wild-type IgG Fc. In some embodiments, the antibody exhibits 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 in vitro transcytosis assay comprises a cell 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 the modified IgG Fc has a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference antibody with an unmodified IgG Fc of the same species and isotype. In some embodiments, the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference antibody with an unmodified IgG Fc of the same species and isotype. In some embodiments, the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 7.4 of 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less. In some embodiments, antibodies comprising a modified IgG Fc have a binding affinity for FcRn at pH 6 of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or less than 10 nM. In some embodiments, the ratio of the affinity of the antibody comprising the modified IgG Fc for FcRn at pH 7.4 to the affinity of the antibody comprising the 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 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

[0017] In some embodiments, the antibody comprising a modified IgG Fc comprises one or more mutations selected from 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 additional mutations selected from 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 set of mutations selected from the sets of mutations in Tables 4, 5 and 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 beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p 75NTR), interleukin 6 receptor (IL6R), interleukin 1 beta (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 IB (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 neuropathic drug. In some embodiments, the neuropathic 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, comprising administering to a subject in need thereof an Fc conjugate comprising 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 neuropathy disorder, a neurodegenerative disease, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and a 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, tauopathy, Alzheimer's disease, supranuclear palsy, prion disease, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-Scheinker 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, 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.

[0023] In some embodiments, a method of increasing brain exposure to an Fc conjugate is provided, 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, a method is provided for increasing transport of an Fc conjugate across the blood-brain barrier (BBB), 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 that comprises a modified IgG Fc and is active in an in vitro transcytosis assay.

[0026] In some embodiments, the Fc conjugate exhibits a transcytosis activity in an in vitro transcytosis assay of at least 50 when normalized to the same Fc conjugate comprising a wild-type IgG Fc. In some embodiments, the Fc conjugate exhibits 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 in vitro transcytosis assay comprises a cell 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 conjugates comprising modified IgG Fc have a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference Fc conjugate with unmodified IgG Fc of the same species and isotype. In some embodiments, the Fc conjugates comprising modified IgG Fc have a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference Fc conjugate with unmodified IgG Fc of the same species and isotype. In some embodiments, the Fc conjugates comprising modified IgG Fc have a binding affinity for FcRn at pH 7.4 of 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 the 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, or between 10 nM and 100 nM. In some embodiments, the ratio of the affinity of the Fc conjugate comprising modified IgG Fc for FcRn at pH 7.4 to the affinity of the Fc conjugate comprising 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 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

[0028] In some embodiments, the modified IgG Fc comprises one or more mutations selected from 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 additional mutations selected from 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 set of mutations selected from the sets of mutations in Tables 4, 5 and 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 alpha-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, acid alpha-glucosidase, glucocerebrosidase, alpha-galactosidase A, hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase, beta-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase, palmitoyl-protein thioesterase 1, and tripeptidyl aminopeptidase 1.

[0030] In some embodiments, the Fc conjugate comprises a modified IgG Fc conjugated to a neuropathic drug. In some embodiments, the neuropathic 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] [Figure 1A-1D] Figure 1 shows the pharmacokinetics and pharmacodynamics of anti-BACE1 hIgG1 antibody (anti-BACE1), anti-BACE1 hIgG1 antibody with engineered 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 Abeta levels; C) Brain antibody pharmacokinetics. D) Relative antibody uptake in the brain of human IgG1 YTE engineered antibody compared to wild type IgG1, including maximum concentration (Cmax) and area under the curve (AUC).

[0032] [Figures 2A-2D] FIG. 1 shows the pharmacokinetics and pharmacodynamics of anti-BACE1 hIgG1 antibody (anti-BACE1), anti-BACE1 hIgG1 antibody with modified Fc (anti-BACE1-YTE), or anti-gD hIgG1 antibody in transgenic (Tg32) mice containing human FCGRT (hFcRn alpha chain) and lacking mouse Fcgrt (i.e., FCGRF+ / +Fcgrt- / - mice), expressing hFCGRT and lacking mFCGRT. A) Plasma antibody pharmacokinetics; B) Brain pharmacodynamics as measured by brain Abeta 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] [Diagram 3] Figure 1 shows plots of binding affinity of certain hIgG1 antibodies containing Fc mutations to hFcRn at two different pHs as described in Example 3. The X-axis shows the pH 6 affinity of the antibody and the Y-axis shows the pH 7.4 affinity of the antibody.

[0034] [Figure 4] 1 is a graph of normalized transcytosis values ​​for certain Fc modified antibodies, as described in Example 3. Fc modified antibodies above the dashed line showed significantly improved transcytosis.

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

[0036] [Figures 6A-6E]Figure 1 shows the pharmacokinetics and pharmacodynamics of anti-BACE1 hIgG1 antibody with modified Fc (anti-BACE1-YQAY) in transgenic (Tg32) mice expressing hFCGRT and lacking mFCGRT. A) Serum antibody concentrations; B) Brain pharmacokinetics (PK); C) Brain pharmacodynamics (PD) as measured by brain Abeta levels; D) Summary of hFcRn affinity and brain PK data; E) Summary of brain PD data.

[0037] [Figure 7A-7D] Figure 1 shows plasma pharmacokinetics and binding affinity of D1 (YY) and Q95 (YQAY) hIgG1 engineered Fc antibodies administered to transgenic Tg32 (3>FCGRT+ / +Fcgrt- / -) mice expressing hFCGRT and lacking mFCGRT; hemizygous (FCGRT+ / -Fcgrt+ / -) mice expressing both hFCGRT and mFCGRT; or wild-type (Fcgrt+ / +) mice expressing only mFCGRT. A) Plasma PK in homozygous Tg32 mice; B) Plasma PK in hemizygous Tg32 (FCGRT+ / -Fcgrt+ / -) mice; C) Plasma PK in wild-type (Fcgrt+ / +) mice; D) Summary of FcRn binding affinity data and ratio of plasma AUC of each FC engineered antibody compared to wild-type antibody.

[0038] [Figure 8A-8C] 1 shows brain pharmacokinetics and brain antibody concentrations of anti-gD hIgG1 antibodies with modified Fc in transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT: A) Brain PK data; B) Brain antibody concentrations 7 days after administration; C) Summary of affinity data and ratio of brain AUC for each Fc-modified antibody compared to wild type antibody.

[0039] [Figure 9A-9C] FIG. 1 shows liver exposure of anti-gD hIgG1 antibodies with 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 compared to wild type antibody.

[0040] [Figure 10A-10C] 1 shows colon exposure of anti-gD hIgG1 antibodies with modified Fc in transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT. A) Colon PK data; B) Colon antibody concentrations 7 days after dosing; C) Summary of affinity data and ratio of colon AUC for each Fc modified antibody compared to wild type antibody.

[0041] [Figures 11A-11C] FIG. 1 shows pulmonary exposure of anti-gD hIgG1 antibodies with 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 compared to wild type antibody.

[0042] [Figure 12] FIG. 1 shows aligned sequences of human IgG subclasses IgG1, IgG2, IgG3 and IgG4 (SEQ ID NOs: 1-4, respectively). Sequence differences 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 by Rabat (SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th ed., NIH publication, no. 91-3242, EARabat et al.). The EU index or EU numbering scheme refers to the numbering of EU antibodies as described by Edelman et al., 1969, Proc Nail Acad Sci USA 63:78-85.

[0043] [Figure 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 concentrations; B) Brain pharmacokinetics (PK); C) Brain pharmacodynamics (PD) measured by brain Abeta levels; D) Summary of hFcRn affinity, brain PK data and brain PD data.

[0044] [Figures 14A-14C] Figure 2 shows the pharmacokinetics and pharmacodynamics of anti-BACE1 hIgG1 antibody (anti-BACE1 WT) and anti-BACE1 hIgG1 antibody with modified Fc (anti-BACE1-YEY, YQAY, YPY) in cynomolgus monkeys. A) CNS pharmacodynamics as measured by sAPPβ / α ratio in CSF at various time points after antibody administration. B) Serum antibody concentrations at various time points after antibody administration. C) Antibody serum exposure from day 0 to day 7 based on data in (B).

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

[0046] [Fig. 16A-16F]Concentrations of anti-Abeta hIgG4 antibody and anti-Abeta hIgG4 antibody with modified Fc (anti-Abeta hIgG4-YTE) in plasma (A) and cerebellum (B) after administration to PS2APP mice expressing mFCGRT at the indicated doses. C) Anti-Abeta hIgG4 ("Abeta"), anti-Abeta hIgG4 YTE ("Abeta YTE") and anti-gD hIgG4 binding to oligomeric Abeta in the mossy fiber hippocampal tract of PS2APP mice after administration to mice at the indicated doses. D) Bar graph of the level of staining observed in (C). Binding of anti-Abeta hIgG4 and anti-Abeta hIgG4 YTE to target Abeta plaques in the subiculum (E) and prefrontal cortex (F) of PS2APP mice.

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

[0048] [Figure 18A-18B] A) Correlation between antibody serum exposure and antibody affinity at pH 7.4 for both anti-BACE1 hIgG1 and anti-Abeta hIgG4 antibodies with modified Fc. B) Correlation between antibody partitioning to brain and antibody affinity to serum at pH 7.4 for both anti-BACE1 hIgG1 and anti-Abeta hIgG4 antibodies with modified Fc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Reference will now be made in detail to certain specific embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by 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, and the present invention is in no way limited to the methods and materials described.

[0051] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are consistent 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 trade names are used herein, applicants intend to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product. definition

[0053] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

[0054] "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, as used herein, "binding affinity" 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 is generally determined 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 to Y (ka or kon). A surrogate measurement for the affinity of one or more antibodies to its target is its half maximal inhibitory concentration (IC50), which is a measure of how much of an antibody is required to inhibit 50% of the binding of a known ligand to the antibody target. Affinity can be measured by common methods known in the art, including those described herein. Certain illustrative and exemplary embodiments for measuring binding affinity are described herein.

[0055] "Blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord (i.e., CNS) formed by tight junctions in the brain capillary endothelial cell membrane, forming a tight barrier that limits the transport of molecules, even very small molecules such as urea (60 daltons), into the brain. The blood-brain barrier in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retina barrier in the retina are continuous 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), which is composed of ependymal cells rather than capillary endothelial cells.

[0056] The terms "amyloid beta", "beta-amyloid", "A beta", "amyloid beta", and "Aβ", as used interchangeably herein, refer to a fragment of amyloid precursor protein ("APP") produced upon beta-secretase 1 ("BACE1") and gamma-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 and to associate to form oligomeric and fibrillar structures, which may be found as constituent members of amyloid plaques. The structures and sequences of such Aβ peptides are well known to those of skill in the art, and methods for making said peptides or extracting them from brain and other tissues are described, for example, in Glenner and Wong, Biochem Biophys Res.Comm. 129:885-890 (1984). Additionally, Aβ peptides are also commercially available in various forms.

[0057] "Anti-A beta immunoglobulin", "anti-A beta antibody" and "antibody that binds to Abeta" are used interchangeably herein and refer to an antibody that specifically binds to human Abeta. A non-limiting example of an anti-A beta antibody is crenezumab. Other non-limiting examples of anti-A beta antibodies are solanezumab, bapinezumab, gantenerumab, aducanumab, ponezumab and any anti-A beta antibody disclosed in the following publications: WO 2000162801, WO 2002046237, WO 2002003911, WO 2003016466, WO 2003016467, WO 2003077858, WO 2004029629 ... No. 2004032868, International Publication No. 2004032868, International Publication No. 2004108895, International Publication No. 2005028511, International Publication No. 2006039470, International Publication No. 2006036291, International Publication No. 2006066089, International Publication No. 2006066171, International Publication No. 2006066049, International Publication No. 2006095041, International Publication No. 2009027105.

[0058] The terms "crenezumab" and "MABT5102A" are used interchangeably herein and refer to a specific anti-Abeta antibody that binds to monomeric, oligomeric and fibrillar forms of Abeta and is related to CAS Registry Number 1095207.

[0059] The term "amyloidosis" as used herein refers to a group of diseases and disorders caused by or associated with amyloid or amyloid-like proteins, including, but not limited to, diseases and disorders caused by the presence or activity of amyloid-like proteins in a monomeric, fibrillar or multimeric 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, neurological disorders such as Alzheimer's disease ("AD"), diseases or conditions characterized by loss of cognitive memory abilities such as mild cognitive impairment (MCI), dementia with Lewy bodies, Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam Parkinson-dementia complex, as well as other diseases based on or associated with amyloid-like proteins such as progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, Parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), adult-onset diabetes, endocrine tumors and senile cardiac amyloidosis, as well as various ocular diseases including macular degeneration due to beta-amyloid deposits, drusen-associated optic neuropathy, glaucoma and cataracts.

[0060] Glaucoma is a group of diseases of the optic nerve accompanied by a characteristic pattern of optic neuropathy involving loss of retinal ganglion cells (RGCs). RGCs are nerve cells that transmit visual signals from the eye to the brain. Two key enzymes in the apoptotic process, caspase-3 and caspase-8, are activated in a process that leads to apoptosis of RGCs. Caspase-3 cleaves amyloid precursor protein (APP) to produce neurotoxic fragments, including Abeta. Without the protective effect of APP, Abeta accumulation in the retinal ganglion cell layer leads to the death of RGCs and irreversible vision loss.

[0061] Glaucoma is often, but not always, accompanied by elevated intraocular pressure, which may be the result of blockage of the aqueous humor circulation or its drainage. Although elevated intraocular pressure is a significant risk factor for developing glaucoma, no threshold of intraocular pressure can be defined that is determinative for causing glaucoma. Damage may also be caused by insufficient blood supply to vital optic nerve fibers, weakness of the nerve's structure, and / or problems with the health of the nerve fibers themselves. Untreated glaucoma can lead to permanent damage to the optic nerve and consequent loss of vision, progressing to blindness.

[0062] "Central nervous system" or "CNS" refers to the complex of nervous tissue that controls bodily functions and includes the brain and spinal cord.

[0063] As used herein, "neurological disorder" refers to a disease or disorder that affects and / or has a CNS etiology. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorder, and lysosomal storage disease. For the purposes of this application, the CNS is understood to include the eye, which is normally separated from the rest of the body by the blood-retinal barrier. Specific examples of neurological disorders include 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-Sträussler-Scheinker disease, chronic wasting disease and fatal familial insomnia), bulbar palsies, motor neuron diseases, and nervous system disorders. Heterogeneous degenerative disorders (including but not limited to Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome and Unverricht-Lundborg disease), dementias (including but not limited to Pick's disease and spinocerebellar ataxia), cancers (e.g., of the CNS, including brain metastases arising from cancer elsewhere in the body).

[0064] A "neuropathy drug" is a drug or therapeutic agent that treats one or more neuropathies. Neuropathic drugs of the present invention include, but are not limited to, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified versions of natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNAs (siRNAs) and short hairpin RNAs (shRNAs)), ribozymes, and small molecules, or active fragments of any of the above. Exemplary neuropathic drugs of the invention are described herein and include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules that specifically recognize and / or act on (i.e., inhibit, agonize or detect) CNS antigens or target molecules, such as, but not limited to, amyloid precursor protein or a portion thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins, as well as active fragments of any of the foregoing. Non-limiting examples of neuropathic drugs and disorders that can be treated using them are provided in Table 1 below: TIFF2025037985000002.tif111170

[0065] An "imaging agent" is a compound that has one or more properties that allow its presence and / or location to be detected, either directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds that incorporate a label moiety that allows for detection.

[0066] A "CNS antigen" or "brain antigen" is an antigen expressed in the CNS, including the brain, that can be targeted with an antibody or small molecule. Examples of such antigens include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotransmitter (NMT), and NF-kappaB1 (NF-kappaB1) proteins. These include trophin receptor (p75NTR), interleukin 6 receptor (IL6R), interleukin 1 beta (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 IB (TNFR2), and apolipoprotein J (ApoJ). In one embodiment, the antigen is BACE1.

[0067] The term "BACE1" as used herein refers to any native beta-secretase 1 (also called 3-site amyloid precursor protein-cleaving enzyme 1, membrane-bound aspartic protease 2, 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), unless otherwise indicated. The term encompasses "full-length" unprocessed BACE1, and any form of BACE1 that results from processing in cells. The term also encompasses naturally occurring variants of BACE1, e.g., splice variants or allelic variants. An exemplary amino acid sequence of a BACE1 polypeptide is that 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. Several other isoforms of human BACE1 exist, including isoforms B, C and D. See UniProtKB / Swiss-Prot Entry P56817, which is incorporated by reference in its entirety.

[0068] The terms "anti-beta secretase antibody," "anti-BACE1 antibody," "antibody that binds to beta-secretase," and "antibody that binds to BACE1" refer to an antibody that can bind to BACE1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting BACE1. In one embodiment, the extent to which an anti-BACE1 antibody binds to an unrelated non-BACE1 protein is less than about 10% of the binding of the antibody to BACE1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to BACE1 has an equilibrium dissociation constant (K) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (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). DIn certain embodiments, the anti-BACE1 antibody binds to an epitope of BACE1 that is conserved among BACE1 of different species and isoforms. In other embodiments, an antibody is provided that binds to an exosite in BACE1 that is located outside the catalytic domain of BACE1. In one embodiment, antibodies are provided that compete with peptides identified in Kornacker et al., Biochem. 44:11567-11573 (2005), which are incorporated by reference in their entireties, for binding to BACE1 (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, 19A, P10A, and L1 1A). Non-limiting exemplary anti-BACE1 antibodies are described, for example, in WO 2012 / 064836 and WO 2016 / 081639.

[0069] A "native sequence" protein herein refers to a protein that comprises the amino acid sequence of a protein found in nature, including variants of a naturally occurring protein. As used herein, the term includes a protein that is isolated from its natural source or that is recombinantly produced.

[0070] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0071] A "reference antibody" herein is an antibody that lacks the characteristics of a test antibody. In some embodiments, the reference antibody for an Fc-modified antibody is an antibody that has the same variable region and is the same isotype, but lacks Fc modification. In some embodiments, the reference antibody for an Fc-modified antibody is an antibody that has the same variable region and isotype, but has wild-type Fc.

[0072] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments are well known in the art (see, for example, 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 (and optionally tandems) of light and / or heavy chain antigen-binding domains with or without linkers); and monospecific or multispecific antigen-binding molecules formed from antibody fragments (including, but not limited to, multispecific antibodies constructed from multiple variable domains that lack Fc).

[0073] 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, excluding possible variants, including, for example, naturally occurring mutations or mutations that may arise during the production of the monoclonal antibody. Such variants are generally present in small amounts. Compared to polyclonal antibody preparations that typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods (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 (see, e.g., 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 a portion of the human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described herein. Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies, including antigen-binding fragments thereof.

[0074] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so 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)).

[0075] An "acceptor human framework" for the purposes of this specification is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0076] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0077] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence from the non-human antibody. In most cases, a humanized antibody is a human antibody (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. For example, in certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to those of a non-human antibody, and all or substantially all of the framework regions (FRs) corresponding to those of a human antibody. In some instances, FR residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which 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 the FR substitution(s) described above. A humanized antibody also optionally comprises at least a portion of an antibody constant region, typically a human antibody constant region. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

[0078] A "human antibody" herein is an antibody that contains an amino acid sequence structure that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Such antibodies can be produced by production by transgenic animals (e.g., mice) that are capable, upon immunization, of producing human antibodies in the absence of endogenous immunoglobulin production (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); by 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 348:552-553 (1990); 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); see U.S. Pat. Nos. 5,565,332 and 5,573,905); production via in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275); and isolation from human antibody-producing hybridomas.

[0079] A "multispecific antibody" herein is an antibody that has 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. Patent Application Publication No. 2002 / 0004587 A1, Miller et al.). Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0080] The antibody herein includes "amino acid sequence variants" with altered antigen binding or biological activity. Examples of such amino acid changes include antibodies with enhanced affinity for antigen (e.g., "affinity matured" antibodies), and antibodies with altered (increased or decreased) antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), including, if present, altered Fc (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.).

[0081] "Affinity-modified variants" have one or more substituted hypervariable region or framework residues of a parent antibody (e.g., a parent chimeric, humanized or human antibody) that alter (increase or decrease) affinity. A convenient method for generating such substitution variants uses phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their 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, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the target. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening and antibodies with altered affinities selected for further development.

[0082] The modified IgG Fc or antibody or fusion protein comprising modified IgG Fc herein can be conjugated with a "heterologous molecule" to, for example, increase half-life or stability or improve the antibody. For example, the modified IgG Fc or antibody or fusion protein comprising modified IgG Fc herein can be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the heterologous molecule is a therapeutic compound or a visualization agent (i.e., a detectable label), and the IgG Fc is used to transport such heterologous molecules across the BBB. Examples of heterologous molecules include, but are not limited to, chemical compounds, peptides, polymers, lipids, nucleic acids, and proteins.

[0083] The modified Fc herein may be a "glycosylation variant" such that any carbohydrate attached to the Fc is altered, altered in presence / absence, or altered in type. For example, an antibody having a mature carbohydrate structure lacking fucose attached to the Fc of the antibody is described in US Patent Publication No. 2003 / 0157108 (Presta, L.). See also US Patent Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). An antibody having a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc of the antibody is mentioned in WO 2003 / 011878, Jean-Mairet et al., and U.S. Patent No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc of the antibody have been reported in WO 1997 / 30087, Patel et al. See also WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.) on antibodies with altered carbohydrate attached to their Fc. See also U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.), which describes antibodies with altered glycosylation. Mutation of the consensus glycosylation sequence in Fc (Asn-X-Ser / Thr at positions 297-299, where X cannot be proline), for example by mutating the Asn in this sequence to any other amino acid, by placing Pro at position 298, or by modifying position 299 to any amino acid other than Ser or Thr, should abolish glycosylation at that position (see, e.g., Fares A1-Ejeh et al., Clin. Cancer Res. (2007) 13:5519s-5527s; Imperiali and Shannon, Biochemistry (1991) 30(18):4374-4380; Katsuri, Biochem J. (1997) 323 (Pt 2):415-419; see Shakin-Eshleman et al., J. Biol. Chem. (1996) 271:6363-6366).

[0084] 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 that structurally form defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs of the invention include the following: (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)); (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) (Rabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen 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 (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).

[0085] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Rabat et al., supra.

[0086] "Framework" or "FR" residues are 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, the HVR and FR sequences generally appear in the following sequence in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. In certain embodiments, one or more FR residues can be modified to modulate the stability of the antibody or to adjust the three-dimensional position of one or more HVRs of the antibody, e.g., to enhance binding.

[0087] A "full length antibody" is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0088] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc as defined herein.

[0089] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety or a radiolabel). Naked antibodies may be present in a pharmaceutical formulation.

[0090] "Natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called 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 assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0091] "Effector function" refers to the biological activity of an antibody that results in activation of the immune system other than activation of the complement pathway. Such activity is found primarily in the Fc of the 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 essentially 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 modifying the Fc at one or more amino acid positions to eliminate effector function (Fc binding insertions: 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, 338, 340, 373, 376, 382, ​​388, 389, 414, 416, 417, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472 9, 434, 435, 436, 437, 438 and 439; modifying the glycosylation of the Fc (including, but not limited to, producing the antibody in an environment that does not permissively tolerate wild-type mammalian glycosylation, removing one or more carbohydrate groups from an antibody that is already glycosylated, 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)).

[0092] "Complement activation" function, or the property of an antibody that allows or induces "activation of the complement pathway", are used interchangeably and refer to the biological activity of an antibody that engages or stimulates the complement pathway of the subject's immune system. Such activities include, for example, C1q binding and complement-dependent cytotoxicity (CDC), and can be mediated by both the Fc and non-Fc portions 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 Fc at one or more amino acid positions (such as positions 270, 322, 329, and 321, which are known to be involved in C1q binding) to eliminate or reduce the ability to interact with or activate complement components, and modifying or eliminating the portion of 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)).

[0093] Depending on the amino acid sequence, Fc domains and the antibodies of which they are a part can be assigned to different "classes". There are five main classes of Fc domains: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma and mu, 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.

[0094] As used herein, the term "recombinant antibody" refers to an antibody (e.g., a chimeric, humanized or human antibody, or an antigen-binding fragment thereof) expressed by a recombinant host cell containing nucleic acid encoding the antibody.

[0095] As used herein, the term "recombinant protein" refers to a protein (such as an Fc conjugate comprising a modified Fc herein) expressed by a recombinant host cell comprising a nucleic acid encoding the protein.

[0096] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for the originally transformed cell are included in the invention. 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 those belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells.

[0097] As used herein, "specifically binding" or "binds specifically" refers to an antibody that selectively or preferentially binds to an antigen. Binding affinity is generally determined using standard assays such as Scatchard analysis or surface plasmon resonance technology (e.g., using BIACORE®).

[0098] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay.

[0099] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function 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, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; 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 antitumor or anticancer agents disclosed herein.

[0100] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0101] The term "Fc region" or "Fc" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes the heavy chain constant domains CH2 and CH3, or a portion of the heavy chain constant domains CH2 and CH3 sufficient to bind FcRn at pH 6, pH 7.4, or both pH 6 and pH 7.4. This term includes native sequence Fc and modified Fc. In some embodiments, a human IgG heavy chain Fc extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. Meanwhile, the C-terminal lysine (Lys447) of 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 (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Exemplary human IgG1, IgG2, IgG3 and IgG4 Fc amino acid sequences are shown in Figure 12 and in SEQ ID NOs: 1 to 4. In some embodiments, Fc-containing antibodies also comprise the heavy chain constant domain, CH1, and hinge.

[0102] The term "FcRn receptor" or "FcRn" as used herein refers to an Fc receptor (where "n" indicates neonate) known to be involved in the transfer of maternal IgG through the human or primate placenta or yolk sac (rabbit) to the fetus, and from colostrum through the small intestine to the neonate. FcRn is also known to be involved in maintaining constant serum IgG levels by binding IgG molecules and recycling them into the serum.

[0103] 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 as a contiguous amino acid sequence.

[0104] 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.

[0105] "Label" refers to a marker that is attached to the antibody herein and is used for detection or imaging. Examples of such labels include radiolabels, fluorophores, chromophores or affinity tags. In one embodiment, the label is a radiolabel used in 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 again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc.

[0106] 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.

[0107] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0108] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes nucleic acid molecules that are contained in a cell that originally contained the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0109] An "isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) are present in one or more locations within a host cell.

[0110] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, and includes information regarding the indications, uses, dosage, administration, concomitant therapy, contraindications and / or warnings regarding such therapeutic product.

[0111] "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 those in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed in the user documentation at the US Copyright Office, Washington DC, 20559, and is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from its 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.

[0112] In situations where ALIGN-2 is employed for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, against, or against a given amino acid sequence B (which can alternatively be purchased as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, against, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y (where X is the number of amino acid residues scored as identity matches by the sequence alignment program ALIGN-2 in this program's alignment of A and B, and Y is the total number of amino acid residues in B). It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then 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.

[0113] The term "pharmaceutical formulation" refers to a preparation that is in a form such that it effectively utilizes the biological activity of the active ingredients contained therein and does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0114] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0115] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, remission or alleviation of disease symptoms, and improved or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0116] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is 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 a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0117] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors integrated into the genome of a host cell into which the vector is 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." 1. Compositions and Methods A. Antibodies and Fc Conjugates

[0118] In one aspect, the present invention is based in part on modified Fc that can be used to transport desired molecules across the BBB. In certain embodiments, an antibody comprising modified Fc is provided. In certain embodiments, an Fc conjugate comprising modified Fc is provided. In some embodiments, the Fc conjugate comprises 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 useful, for example, for the diagnosis or treatment of diseases that affect the brain and / or CNS. A. Exemplary Modified Fcs

[0119] Provided herein are antibodies and Fc conjugates that comprise modified Fc, and the antibodies and Fc conjugates are active in in vitro transcytosis assays.In some embodiments, the antibodies and Fc conjugates that comprise modified Fc have improved brain uptake.In some embodiments, 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, modified Fc herein improves transport through the blood-brain barrier (BBB).

[0120] In some embodiments, certain antibodies and Fc conjugates comprising modified Fc provided herein exhibit a transcytosis activity in an in vitro transcytosis assay of at least 50. In some embodiments, certain antibodies and Fc conjugates comprising modified Fc provided herein exhibit a transcytosis activity in an in vitro transcytosis assay of at least 30, or at least 40, or at least 50, when normalized to the same antibody or Fc conjugate comprising wild-type IgG Fc. In some embodiments, certain antibodies and Fc conjugates comprising modified Fc 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. Non-limiting exemplary transcytosis assays are described in the Assays section herein. In some embodiments, the in vitro transcytosis assay comprises cells expressing FcRn. In some embodiments, the FcRn is human FcRn. In some embodiments, the cells are MDCK II cells.

[0121] In some embodiments, certain antibodies and Fc conjugates comprising modified Fc provided herein 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 having an unmodified IgG Fc of the same species and isotype. In some embodiments, certain antibodies and Fc conjugates comprising modified Fc provided herein 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 having an unmodified IgG Fc of the same species and isotype. In some embodiments, certain antibodies and Fc conjugates comprising an altered Fc provided herein have a binding affinity for FcRn (e.g., human FcRn) at pH 7.4 of 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less. In some embodiments, certain antibodies and Fc conjugates comprising an altered Fc provided herein have a binding affinity for FcRn (e.g., human FcRn) at pH 6 of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less. In some embodiments, the ratio of the affinity of an antibody or Fc conjugate comprising a modified IgG Fc for FcRn (e.g., human FcRn) at pH 7.4 to the affinity of an 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 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

[0122] In various embodiments, the modified Fc provided herein comprises one or more mutations selected from 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 additional mutations selected from 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 set of mutations selected from the set of mutations in Tables 4, 5, and 6. In some embodiments, the modified Fc comprises one or more modifications of an IgG sequence selected from SEQ ID NOs: 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.

[0123] In some embodiments, in the context of an antibody or Fc conjugate, modified Fcs are provided that have a normalized transcytosis score of at least 30. Non-limiting examples of such modified Fcs include modified Fcs that include the following sets of mutations: 252W / 434W; 252Y / 434Y; 252Y / 286E / 434Y; 252Y / 307Q / 434Y; 252Y / 308P / 434Y; 252Y / 311A / 434Y; 252Y / 311I / N4 34Y;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 / 43 4Y;307Q / 311I / 434Y;307Q / 433K / 434Y;307Q / 434Y / 436I;311A / 428L / 434Y;311A / 433K / 434Y;311I / 433K / 434Y;433K / 434Y / 436I;252Y / 307Q / 311A / 434Y;252Y / 30 7Q / 311I / 434Y; 252Y / 307Q / 434Y / 436I; 252Y / 311I / 434Y / 436I; 252Y / 311A / 434Y / 436I; 252Y / 428L / 434Y / 436I; 252Y / 307Q / 428L / 434Y; and 252Y / 311I / 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.

[0124] In some embodiments, in the context of an antibody or Fc conjugate, a modified Fc is provided that has a normalized transcytosis score of at least 70. Non-limiting examples of such modified Fc's include modified Fc's comprising the following set 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 / 43 4Y;307Q / 311I / 434Y;307Q / 433K / 434Y;307Q / 434Y / 436I;311A / 428L / 434Y;3 11I / 433K / 434Y;433K / 434Y / 436I;252Y / 307Q / 311A / 434Y;252Y / 307Q / 311I / 434Y; 252Y / 307Q / 434Y / 436I; 252Y / 311I / 434Y / 436I; 252Y / 311A / 434Y / 436I; 252Y / 428L / 434Y / 436I; 252Y / 307Q / 428L / 434Y; and 252Y / 311I / 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.

[0125] In some embodiments, in the context of an antibody or Fc conjugate, modified Fcs are provided that have a normalized transcytosis score of at least 100. Non-limiting examples of such modified Fcs include modified Fcs that include 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 / 42 8L / 434Y; 311I / 433K / 434Y; 252Y / 307Q / 311A / 434Y; 252Y / 307Q / 311I / 434Y; 252Y / 307Q / 434Y / 436I; 252Y / 311I / 434Y / 436I; 252Y / 311A / 434Y / 436I; 252Y / 428L / 434Y / 436I; 252Y / 307Q / 428L / 434Y; and 252Y / 311I / 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.

[0126] Non-limiting further modified Fc is provided, and can be selected, for example, using the in vitro transcytosis assay described herein.Various exemplary modified Fc are known in the art, and can be selected, for use in antibody and Fc conjugate herein, using the in vitro transcytosis assay.Non-limiting examples of such modified Fc that can be assayed for transcytosis activity include, for example, those described in US Patent Application Publication No. 2015 / 0050269 and US Patent Application Publication No. 2013 / 0131319, which are incorporated herein by reference in their entirety for any purpose. 1. Engineered Fc affinity

[0127] In some embodiments, the equilibrium dissociation constant (K) for FcRn at pH 7.4 is 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less. D In some embodiments, a modified Fc having an equilibrium dissociation constant (K) for FcRn at pH 7.4 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 is provided herein. D In some embodiments, provided herein is a modified Fc having an equilibrium dissociation constant (K) of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less for FcRn at pH 6. D In some embodiments, a modified Fc having an equilibrium dissociation constant (K) for FcRn at pH 6 of 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 is provided herein. D ) is provided herein.

[0128] In some embodiments, the modified Fc provided herein binds to FcRn at pH 7.4, binds to FcRn at pH 6, and has a K D , K at pH 6 D to 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, binds to FcRn at pH 6, and has a K D K at pH 6 D The ratio to is 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

[0129] In various embodiments, the FcRn is a human FcRn.

[0130] In some embodiments, K D is measured using surface plasmon resonance. In some such embodiments, K D is measured at 25° C. using a BIACORE®-2000 instrument (BIAcore, Inc., Piscataway, NJ). The modified Fc is immobilized, for example, through 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). The association rate (k) and dissociation rate (k) are calculated using a one-to-one Langmuir binding model (BIACORE® Evaluation Software version 4.1) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K) is calculated as the ratio k / k. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). Alternatively, K D Values ​​can be determined from the dependence of steady-state binding levels on analyte concentration. In some embodiments, so-called steady-state analysis is particularly suitable for measuring weak to moderate interactions. 2. Engineered Fc variants

[0131] In certain embodiments, amino acid sequence variants of modified Fc provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of modified Fc variants. 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, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequence of Fc. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct has the desired characteristics (e.g., antigen binding). a) Substitution, insertion and deletion variants

[0132] In certain embodiments, Fc variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 2A under the heading of "preferred substitutions". More substantial changes are shown in Table 2A under the heading of "exemplary substitutions" and are as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the Fc of interest and the product screened for desired activity, for example, reduced immunogenicity, or reduced or improved ADCC or CDC. Table 2A TIFF2025037985000003.tif147170

[0133] Amino acids may be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0134] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0135] A useful method for identifying residues or regions of Fc that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, vol. 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, e.g., arg, asp, his, lys, and glu) are identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of Fc with FcRn is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the Fc-FcRn complex to identify contact points between Fc and FcRn. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they have the desired properties.

[0136] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion includes an Fc with an N-terminal methionyl residue. Other insertion variants of the Fc molecule include fusions to the N- or C-terminus of the Fc with an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the Fc. b) Glycosylation variants

[0137] In certain embodiments, the modified Fc provided herein is altered to increase or decrease the extent to which the Fc is glycosylated. Addition or deletion of glycosylation sites to an Fc can be conveniently accomplished by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0138] Native Fc produced by mammalian cells typically contains branched, biantennary oligosaccharides that are generally linked to Asn297 in the CH2 domain of Fc by an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may contain a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the Fc of the invention may be made to generate Fc variants with certain improved properties.

[0139] In one embodiment, Fc variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc. For example, the amount of fucose in such Fc may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 of Fc (EU numbering of Fc residues); however, Asn297 may also be located upstream or downstream of position 297, i.e., about ±3 amino acids between positions 294 and 300, due to minor sequence variations in Fc. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications relating to "defucosylated" or "fucose-deficient" Fc variants include: U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282. US Patent Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 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 Lee 13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-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).

[0140] Further provided are Fc variants with bisected oligosaccharides, for example, 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, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Also provided are Fc variants with at least one galactose residue in the oligosaccharide attached to Fc. Such Fc variants may have improved CDC function. Such Fc variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). c) Cysteine ​​engineered Fc variants

[0141] In certain embodiments, it may be desirable to create cysteine ​​engineered Fc variants, in which one or more residues of modified Fc are replaced with cysteine ​​residues. In particular embodiments, the replaced residues occur at accessible sites of modified Fc. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of modified Fc, which can be used to conjugate modified Fc to other moieties (such as drug moieties or linker-drug moieties) to create Fc conjugates as further described herein. Cysteine ​​engineered Fc can be created, for example, as described in U.S. Patent Nos. 7,521,541 and 9,000,130. d) Modified Fc Derivatives

[0142] In certain embodiments, the modified Fc provided herein may be further modified to contain additional non-proteinaceous moieties that are known and readily available in the art. Suitable sites for derivatization of the modified Fc 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous 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 when multiple polymers are 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 particular property or function of the modified Fc to be improved, and whether the modified Fc derivative will be used therapeutically under defined conditions.

[0143] In another embodiment, a conjugate of modified Fc and non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that do not harm normal cells but heat the non-proteinaceous moiety to a temperature that kills cells proximal to the modified Fc-non-proteinaceous moiety. B. Exemplary Antibodies

[0144] In various embodiments, an antibody comprising the modified Fc herein is provided. In certain aspects, the modified Fc may improve the transport of the antibody across the BBB. In some embodiments, the antibody comprising the modified Fc herein binds to a brain antigen. Non-limiting examples of such brain antigens include beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin, and the like. These include fin receptor (p75NTR), interleukin 6 receptor (IL6R), interleukin 1 beta (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 IB (TNFR2), and apolipoprotein J (ApoJ).

[0145] In further embodiments, the antibodies comprising the modified Fc herein may incorporate any of the features described in sections 1-7 below, either alone or in combination: 1. Antibody affinity

[0146] In certain embodiments, the antibodies provided herein have a titer of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less for their antigen (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Equilibrium dissociation constant (K D ).

[0147] In one embodiment, K D is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by the minimum concentration of ( 125 I) Fab is equilibrated with labeled antigen, and then bound antigen is measured by capturing it on a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, a MICROTITER (登録商標) Multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation can be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 (TWEEN-20) in PBS for 1 hour. (登録商標) ) for 8 washes. After plates have dried, 150 μl / well of scintillate (MICROSCINT-20™, Packard) is added and plates are counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that result in 20% or less of maximal binding are selected for use in competitive binding assays.

[0148] In one embodiment, the RIA is a Scatchard analysis. For example, the antibody of interest can be iodized using the lactoperoxidase method (Bennett and Horuk, Methods in Enzymology 288 pp. 134-148 (1997)). The radiolabeled antibody can be released by gel filtration using a NAP-5 column. 125 The antibodies are purified from I-Na and their specific activity is measured. 50 μL of competition reaction mixture containing a constant concentration of iodized antibody and decreasing concentrations of serially diluted unlabeled antibodies are placed in a 96-well plate. Cells transiently expressing the antigen are incubated at 37°C, 5% CO 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. 2Cells are cultured at 4°C for 12 h at 4°C. Cells are detached from the dishes using Sigma Cell Dissociation Solution and washed with binding buffer (DMEM with 1% bovine serum albumin, 50 mM HEPES, pH 7.2 and 0.2% sodium azide). Washed cells are added to a 96-well plate containing 50 μL of competition reaction mixture at a density of approximately 200,000 cells in 0.2 mL of binding buffer. The final concentration of unlabeled antibody in the competition reaction with cells is varied, starting at 1000 nM and then decreasing in a 1:2 dilution for 10 concentrations, including a sample with zero added buffer only. The competition reaction with cells for each concentration of unlabeled antibody is assayed in triplicate. The competition reaction with cells is incubated for 2 hours at room temperature. After the 2 hour incubation, the competition reaction is transferred to a filter plate and washed 4 times with binding buffer to separate it from the bound iodinated antibody. Filters are counted in a gamma counter and the binding data evaluated using the fitting algorithm of Munson and Rodbard (1980) to determine the binding affinity of the antibody.

[0149] In some embodiments, K Dis measured using a surface plasmon resonance assay on a BIACORE®-2000 instrument (BIAcore, Inc., Piscataway, NJ) at 25° C. using an anti-human Fc kit (BiAcore Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Anti-human Fc antibodies are diluted to 50 μg / ml in 10 mM sodium acetate, pH 4.0, before injection at a flow rate of 5 μl / min to achieve approximately 10,000 response units (RU) of binding protein. After injection of the antibody, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, antibody is injected into HBS-P to reach approximately 220 RU, and then two-fold serial dilutions of antigen are injected into HBS-P at a flow rate of approximately 30 μl / min at 25° C. Association rates (k) and dissociation rates (k) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K D ) is calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0150] Several methods for determining IC50 for a given compound are known in the art; a common approach is to carry out a competitive binding assay as described herein.In general, a high IC50 indicates that more antibody is required to inhibit the binding of a known ligand, and thus the affinity of the antibody to the ligand is relatively low.Conversely, a low IC50 indicates that less antibody is required to inhibit the binding of a known ligand, and thus the affinity of the antibody to the ligand is relatively high. 2. Chimeric and humanized antibodies

[0151] In certain embodiments, the antibody provided herein is a chimeric antibody. 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 from a mouse, rat, hamster, rabbit, or a non-human primate, e.g., a monkey) and a human constant region. Chimeric antibodies of interest herein include "primatized" antibodies that comprise variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey, such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780). In a further example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody comprises an antigen-binding fragment thereof.

[0152] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVR, e.g., CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0153] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are 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) grafting); 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 "guided selection" for FR shuffling).

[0154] Human framework regions that may be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences 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 (1993)); human mature (somatically mutated) framework regions or human germline framework regions. framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of 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)). 3. Human antibodies

[0155] In certain embodiments, the antibody provided herein is a human antibody.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).

[0156] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech., 23:1117-1125 (2005). See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes the HUMAB® technology; U.S. Patent No. 7,041,870, which describes the KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes the VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0157] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, for example, 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 via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiandai Mianyixue, 26(4):265-268 (2006), which describes 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).

[0158] Human antibodies can also be made by isolating Fv clone variable domain sequences selected from a human-derived phage display library. 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. 4. Library-derived antibodies

[0159] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and are described, for example, in 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, eds., 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).

[0160] In certain phage display methods, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to obtain single-source antibodies against a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions to achieve rearrangement in vitro, as described in 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. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0161] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments. 5. Multispecific antibodies

[0162] In certain embodiments, the antibody provided herein is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In certain embodiments, a bispecific antibody can bind to two different epitopes of the same antigen. In certain embodiments, a bispecific antibody can bind to two different antigens. A bispecific antibody can be prepared as a full-length antibody or an antibody fragment.

[0163] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-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 "knobs-into-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); 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)); the use of leucine zippers to make bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 1999, 2002, 2003, 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2019, 2020, 203). nol., 148(5):1547-1553 (1992); use of "diabody" technology to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by the preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0164] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-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 "knobs-into-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); 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)); the use of leucine zippers to make bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 1999, 2002, 2003, 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2019, 2020, 203). nol., 148(5):1547-1553 (1992); use of "diabody" technology to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by the preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0165] 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., U.S. Patent Application Publication No. 2006 / 0025576A1 and Wu et al. Nature Biotechnology (2007)). 6. Antibody variants

[0166] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct has the desired characteristics (e.g., antigen binding). a) Substitution, insertion and deletion variants

[0167] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 2B under the heading of "preferred substitutions". More substantial changes are shown in Table 2B under the heading of "exemplary substitutions" and are as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for the desired activity, for example, retained / improved downstream binding, reduced immunogenicity, or reduced or improved ADCC or CDC. Table 2B TIFF2025037985000004.tif144170

[0168] Amino acids may be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0169] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0170] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing have a modified (e.g., improved) certain biological property compared to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or have a certain biological property of the parent antibody substantially retained. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0171] Modifications (e.g., substitutions) can be made to HVRs to, for example, improve antibody affinity. Such modifications can be made within HVR "hot spots," i.e., residues encoded by codons that undergo high frequency mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0172] In certain embodiments, substitution, insertion or deletion may occur within one or more HVRs, as long as such modification does not substantially reduce the ability of antibody to bind to antigen.For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.Such changes may, for example, be outside the antigen contact residues within HVRs.In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two or three amino acid substitutions.

[0173] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, e.g., arg, asp, his, lys, and glu) are identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they have the desired properties.

[0174] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody with an enzyme (e.g., in the case of ADEPT) or a polypeptide which increases the serum half-life of the antibody. b) Glycosylation variants

[0175] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0176] If the antibody contains an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of Fc by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may contain a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to generate antibody variants with specific improved properties.

[0177] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 of the Fc (EU numbering of Fc residues); however, Asn297 may also be located upstream or downstream of position 297, i.e., about ±3 amino acids between positions 294 and 300, due to minor sequence variations of the antibody. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282. US Patent Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 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, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0178] Further provided are antibody variants with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Pat. Appl. Pub. No. 2005 / 0123546 (Umana et al.). Also provided are antibodies with at least one galactose residue in the oligosaccharide attached to the Fc. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). c) Fc variants

[0179] In various embodiments, the antibody comprises a modified Fc as provided herein. In some embodiments, one or more amino acid modifications can be introduced into the Fc of the antibody, thereby creating a modified Fc. The modified Fc can comprise a human Fc sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0180] In certain embodiments, the present invention contemplates antibody variants that have some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important, but where certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, p. 464, of Ravetch and Kinet, Annu. Rev. Immunol., 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, 83:7059-7063 (1986), and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.), and CytoTox 96 (登録商標)Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of interest can be evaluated in vivo, for example in an animal model as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may be performed to confirm that the antibody lacks CDC activity due to its inability to bind C1q. See, for example, C1q and C3c binding ELISA 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 can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0181] Non-limiting examples of antibodies with reduced effector function include those with substitutions at one or more of Fc residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0182] Certain antibody variants have been described with improved or diminished binding to FcRs (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)).

[0183] In certain embodiments, the antibody variant comprises an Fc with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 of Fc (EU numbering of residues).

[0184] In some embodiments, changes are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), 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).

[0185] See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc variants. d) Cysteine ​​Engineered Antibody Variants

[0186] In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues can be replaced 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. Cysteine ​​engineered antibodies can be created, for example, as described in U.S. Pat. Nos. 7,521,541 and 9,000,130. e) Antibody derivative

[0187] In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous 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 antibody can vary, and when multiple polymers are 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 particular property or function of the antibody to be improved and whether the antibody derivative will be used therapeutically under defined conditions.

[0188] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that do not harm normal cells but heat the non-proteinaceous moiety to a temperature that kills cells proximal to the antibody-non-proteinaceous moiety. C. Recombinant Methods and Compositions

[0189] Antibodies, modified Fc and Fc fusions may be produced using recombinant methods and compositions known in the art. See, for example, U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an antibody, modified Fc or Fc fusion described herein. 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., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further 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): (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 a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a method of making an antibody, modified Fc or Fc fusion is provided, comprising culturing a host cell comprising a nucleic acid encoding an antibody, modified Fc or Fc fusion as provided above under conditions suitable for expression of the antibody, modified Fc or Fc fusion, and optionally recovering the antibody, modified Fc or Fc fusion from the host cell (or host cell culture medium).

[0190] For recombinant production of an antibody, modified Fc, or Fc fusion, e.g., nucleic acid encoding the antibody, modified Fc, or Fc fusion, as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. For antibodies, such nucleic acid can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody).

[0191] Suitable host cells for cloning or expressing protein-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, Fc-containing proteins can be produced in bacteria, especially if glycosylation and Fc effector functions are not required. For expression of polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli). After expression, the protein can be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0192] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable as cloning or expression hosts for protein-encoding vectors, including bacterial and yeast strains that have been "humanized" in their glycosylation pathways, resulting in the production of proteins with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0193] Suitable host cells for the expression of glycosylated proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0194] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0195] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with 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 hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, e.g., as described in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR -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 (ed. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). D. Assay

[0196] The modified Fc's provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art. 1. Binding and other assays

[0197] Various techniques are available for determining the binding of modified Fc-containing agents provided herein (such as antibodies or Fc conjugates (including Fc fusions)) to FcRn. One such assay is an enzyme-linked immunosorbent assay (ELISA) to confirm the ability to bind to human FcRn at various pHs, such as pH 7.4 and pH 6. Various techniques are also available for determining the binding of an antibody to its antigen, including 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 modified Fc-containing agent, and the binding of the antibody or modified Fc-containing agent to the antigen or FcRn of interest is determined.

[0198] In one embodiment, the antibodies of the invention are tested for their antigen binding activity by known methods, e.g., ELISA, Western blot, etc. In another embodiment, the modified Fc-containing agents are tested for their binding activity to FcRn by known methods, e.g., ELISA, Western blot, etc.

[0199] In another aspect, competitive assays can be used to identify antibodies that compete with any of the antibodies of the present invention for binding to antigen.In certain embodiments, such competitive antibodies bind to the same epitope (e.g., linear or structural epitope) that is bound by any of the antibodies of the present invention, more specifically, to any of the epitopes that are specifically bound by the class I, class II, class III or class IV antibodies described herein (see, e.g., Example 1 and Table 4).Detailed exemplary methods for mapping the epitope that an antibody binds are provided in Morris (1996), "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0200] In an exemplary competitive assay, the immobilized antigen is incubated in a solution containing a first labeled antibody (e.g., one or more antibodies disclosed herein) that binds to the antigen, and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in a hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, the 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 compared 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). 2. Activity Assay

[0201] In one embodiment, an assay is provided for identifying antibodies and Fc conjugates with biological activity.Biological activity can include, for example, the ability to cross the blood-brain barrier and enter the brain and / or CNS, and the ability to cross the BBB and transport compounds associated with modified Fc to the brain and / or CNS.Antibodies and Fc conjugates with such biological activity in vivo and / or in vitro are provided.

[0202] 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: Human FcRn (e.g., FCGRT (UniProtKB-P55899, FCGRTN_HUMAN) and β separated by the P2A sequence) are used. 2 MDCK II cells (American Type Culture Collection, Manassas, VA) transfected to express IgG1 (UniProtKB-P61769, B2MG_HUMAN) (Kim et al. PLoS one 2011;6(4):e18556)) are seeded in Transwell® permeable support plates, 0.4 μm pore size (Corning Inc., Corning, NY) for 3 days. On day 3, fresh medium containing test antibody and a fluorescent marker dye, e.g., Lucifer Yellow (Molecular Probes, Eugene, OR), is added to the apical compartment. Fresh medium without test antibody and Lucifer Yellow is added to the basolateral compartment. The pH of both chambers is 7.4. Plates are incubated at 37°C and 5% CO 2Incubate overnight in a humidified incubator. On day 4, medium is collected from the apical and basolateral compartments and the antibody concentrations in the two compartments are assayed, 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. Data can be normalized by dividing the transcytosis concentration of each antibody or Fc conjugate by the transcytosis concentration of a reference antibody, typically containing wild-type Fc.

[0203] In some embodiments, an antibody or Fc conjugate provided herein exhibits 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, an antibody or Fc conjugate provided herein exhibits 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. E. Immunoconjugates and Fc conjugates

[0204] The present invention also provides conjugates comprising the antibodies or modified Fc herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes. When such conjugates comprise an antibody, in some embodiments they may be referred to as immunoconjugates.

[0205] In one embodiment, the antibodies or modified Fc herein are coupled to a neuropathic drug, chemotherapeutic agent and / or imaging agent to more efficiently transport the drug, chemotherapeutic agent and / or imaging agent across the BBB.

[0206] 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, a gene encoding a modified Fc and a gene encoding, for example, a neuropathic drug, and expressing them as a single protein). In certain embodiments, direct conjugation is by forming a covalent bond between a reactive group on the modified Fc or antibody and a corresponding group or acceptor on, for example, a neuropathic 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 (non-limiting examples, sulfhydryl or carboxyl groups) that forms a covalent bond with the other molecule to be conjugated under appropriate conditions. As one non-limiting example, a molecule (i.e., an amino acid) with a desired reactive group (i.e., a cysteine ​​residue) can be introduced into an antibody or modified Fc to form a disulfide bond with, for example, a neuropathic drug. Methods for covalent conjugation of nucleic acids and proteins are also known in the art (i.e., photocrosslinking, see, for example, Zatsepin et al. Russ. Chem. Rev. 74:77-95 (2005)).

[0207] Non-covalent conjugation can be by any non-covalent means, including hydrophobic bonds, ionic bonds, electrostatic interactions, and the like, as will be readily appreciated by one of skill in the art.

[0208] Conjugation may also be performed using various linkers. For example, antibodies and neuropharmaceuticals or modified Fc and neuropharmaceuticals may be conjugated using various 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 HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies, modified Fc or Fc conjugates. See WO 94 / 11026. Peptide linkers composed of 1-20 amino acids linked by peptide bonds can also be used. In certain such embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other such embodiments, the one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. The linker can be a "cleavable linker" that facilitates release of the neuropharmaceutical upon delivery to the brain. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); US Pat. No. 5,208,020) may be used.

[0209] The invention herein expressly contemplates, but is not limited to, conjugates prepared with crosslinker 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 (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0210] In one embodiment, the immunoconjugate is an antibody that binds a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0 425 511). 235); auristatins such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (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. Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (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. ... 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; trichothecenes; and CC1065. In some embodiments, an Fc conjugate is provided that comprises the modified Fc herein conjugated to one or more of said drugs.

[0211] In another embodiment, the conjugate comprises an antibody or Fc as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding 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, diansin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0212] 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 are available for the production of 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 a radioactive substance is used for detection, it may contain a radioactive atom, such as tc99m or I123 for scintigraphy studies, or a spin label, such as iodine-123 again, 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).

[0213] In some embodiments, the Fc conjugate comprises a modified Fc as provided herein fused to a protein, such as a therapeutic protein and / or a detectable protein, In such embodiments, such an Fc conjugate may be referred to as an Fc fusion. Non-limiting exemplary therapeutic proteins that can be conjugated to the modified Fc provided herein include TNF-R1, CTLA-4, IL-1R1, alpha-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, acid alpha-glucosidase, glucocerebrosidase, alpha-galactosidase A, hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase, beta-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase, palmitoyl-protein thioesterase 1, and tripeptidyl aminopeptidase 1. In some embodiments, the extracellular domain of a therapeutic protein, such as the extracellular domain of TNF-R1, CTLA-4, or IL-1R1, is conjugated to a modified Fc as provided herein. F. Methods and Compositions for Diagnostics and Detection

[0214] In some embodiments, an antibody or Fc conjugate for use in a diagnostic or detection method is provided.

[0215] Exemplary disorders that may 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 may be used to detect antigens in the CNS (e.g., in the brain), for example, to diagnose a disease or disorder associated with the presence or elevated levels of the antigen.

[0216] In certain embodiments, labeled antibodies and Fc conjugates are provided. Labels include, but are not limited to, directly detectable labels or moieties (fluorescent, chromophore, electron-dense, chemiluminescent and radioactive labels) and indirectly detectable moieties (e.g., enzymes or ligands) through enzymatic reactions or molecular interactions. Exemplary labels include radioisotopes, 32 P, 14 C. 125 I, 3 H and 131 Examples of suitable dye precursors include, but are not limited to, I, rare earth chelates or fluorophores such as 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-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide, such as heterocyclic oxidases such as uricase and xanthine oxidase coupled with HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, and stable free radicals.

[0217] 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. In another aspect, the antibody or Fc conjugate has modified glycosylation, for example, to produce the antibody, Fc conjugate, or modified Fc in a system that lacks normal human glycosylation enzymes. In another aspect, the Ig backbone is modified to have limited or no effector function naturally.

[0218] Various techniques are available for determining the binding of an antibody or Fc conjugate to a target protein. One such assay is the enzyme-linked immunosorbent assay (ELISA) to confirm the ability to bind to a target protein. According to this assay, an antigen-coated plate 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.

[0219] Assays to assess 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 the CNS target protein of interest.

[0220] In one embodiment, an assay is provided for identifying anti-BACE1 antibodies with biological activity.Biological activity can include, for example, inhibition of BACE1 aspartyl protease activity.Also provided are antibodies with such biological activity in vivo and / or in vitro, as assessed, for example, by homogeneous time-resolved fluorescence HTRF assay or microfluidic capillary electrophoresis (MCE) assay, using synthetic substrate peptides or in vivo in cell lines expressing BACE1 substrates such as APP. F. Pharmaceutical Preparations

[0221] Pharmaceutical formulations of the antibodies or Fc conjugates described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or Fc conjugates having the desired degree of purity with any one or more pharma- ceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th ed., Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers, excipients or stabilizers are generally non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; tides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein include soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as rHuPH20 (HYLENEX). (登録商標)Further included are intercalating drug dispersing agents such as human soluble PH-20 hyaluronidase glycoproteins, such as those from the PH-20 family of hyaluronidases (Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0222] Exemplary lyophilized antibody or Fc conjugate formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody or Fc conjugate formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0223] The formulations herein may also contain two or more active ingredients as necessary for the specific indication to be treated, preferably with 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 diseases, seizure disorders, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders or CNS inflammation.Examples of such medicaments are discussed herein.Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

[0224] The active ingredient may also be incorporated into microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences 16th Edition, edited by Osol, A. (1980). One or more active ingredients may be encapsulated in liposomes that are coupled to the antibodies or Fc conjugates described herein (see, for example, US Patent Publication No. 20020025313).

[0225] Slow-release preparations can also be prepared.Suitable examples of slow-release preparations include semipermeable matrix of solid hydrophobic polymers containing antibody or Fc conjugate, the matrix is ​​in the form of shaped article, for example, film, or microcapsule.Non-limiting examples of slow-release matrix include polyester, hydrogel (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (US Pat. No. 3,773,919), copolymer of L-glutamic acid and γ-L-ethyl glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymer such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0226] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes. G. Therapeutic Methods and Compositions

[0227] Any of the antibodies or Fc conjugates provided herein can be used in therapeutic methods. In one aspect, the antibodies or Fc conjugates are provided for use as pharmaceuticals. For example, the present invention provides a method for transporting therapeutic compounds across the blood-brain barrier, comprising exposing the antibodies or Fc conjugates of the present invention to the BBB, such that modified Fc allows the antibody or Fc conjugate to be transported across the BBB, and the antibody or Fc conjugate comprises a therapeutic compound. In another example, the present invention provides a method for transporting neuropathic drugs across the blood-brain barrier, comprising exposing the antibodies or Fc conjugates of the present invention to the BBB, such that modified Fc allows the antibody or Fc conjugate to be transported across the BBB, and the antibody or Fc conjugate comprises a neuropathic drug. In one embodiment, the BBB is in a mammal (e.g., a human), such as one having a neurological disorder, including, but not limited to, Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, traumatic brain injury, and the like.

[0228] In one embodiment, the neurological disorder is selected from neuropathy, amyloidosis, cancer (e.g. involving the CNS or brain), eye disease or disorder, viral or microbial infection, inflammation (e.g. of the CNS or brain), ischemia, neurodegenerative disease, stroke, behavioral disorder, lysosomal storage disease, etc. The antibodies and Fc conjugates of the invention are particularly suitable for the treatment of such neurological disorders, due to their ability to transport one or more relevant active ingredients / coupled therapeutic compounds across the BBB to the CNS / brain, such disorders finding their molecular, cellular or viral / microbial basis.

[0229] Neuropathic disorders are diseases or abnormalities of the nervous system characterized by inappropriate or uncontrolled nerve signaling or the lack thereof, and include, but are not limited to, chronic pain (including nociceptive pain), cancer-related pain, pain caused by injury to body tissues, including neuropathic pain (pain caused by abnormalities in the nerves, spinal cord, or brain) and psychogenic pain (associated wholly or mostly with psychological disorders), headaches, migraines, neuropathy, and symptoms and syndromes that often accompany such neuropathic disorders, such as dizziness or nausea.

[0230] For neuropathic disorders, 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, meloxicam, nabumetone, oxaprozin, piroxicam, sulindac and tolmetin), corticosteroids (i.e., cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone and triamcinolone), antimigraine medications (i.e., sumatriptin, almotriptan, frovatriptan, sumatriptan, rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan and ergotamine), Acetaminophen, salicylates (i.e., aspirin, choline salicylate, magnesium salicylate, diflunisal, and salsalate), anticonvulsants (i.e., carbamazepine, clonazepam, gabapentin, lamotrigine, pregabalin, tiagabine, and topiramate), anesthetics (i.e., isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclomethicine, dimethicone, propoxycaine, benzoquinone, benzophenone, benzocaine ... Neuropathic drugs may be selected that are analgesics, including but not limited to cefotaxime, procaine, novocaine, proparacaine, tetracaine, articaine, bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, saxitoxin and tetrodotoxin, and cox-2 inhibitors (i.e., celecoxib, rofecoxib and valdecoxib). In the case of neuropathic disorders with dizziness, neuropathic drugs may be selected that are antidizziness drugs, including but not limited to meclizine, diphenhydramine, promethazine and diazepam.In the case of neuropathic disorders with nausea complications, neurological agents may be selected that are anti-nausea medications including, but not limited to, promethazine, chlorpromazine, prochlorperazine, trimethobenzamide and metoclopramide.

[0231] 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's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), Guam-Parkinson-dementia complex, cerebral amyloid angiopathy, Huntington's disease, progressive supranuclear palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, transmissible spongiform encephalopathies, HIV-associated dementia, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), and eye diseases associated with beta-amyloid deposits (i.e., macular degeneration, drusen-associated optic neuropathy, and cataracts).

[0232] In the case of amyloidosis, antibodies or other binding molecules (including, but not limited to, small molecules, peptides, aptamers, or other protein binding agents) that specifically bind to a target selected from beta-secretase, tau, presenilin, amyloid precursor protein or portions thereof, amyloid beta peptide or oligomers or fibrils thereof, death receptor 6 (DR6), receptor for advanced glycation end products (RAGE), parkin, and huntingtin; cholinesterase inhibitors (i.e., galantamine, donepezil, rivastigmine, and tacrine); NMDA receptor antagonists (i.e., memantine), monoamine depleting agents (i.e., tetrabenazine); ergoloid mesylates; 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, 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 nicotinamide); dimebolins; homotaurines (i.e., 3-aminopropanesulfonic acid; 3APS); serotonin receptor activity modulators (i.e., xaliproden); interferons, and neurological agents including, but not limited to, glucocorticoids may be selected.

[0233] Cancers of the CNS are characterized by abnormal proliferation of one or more CNS cells (i.e., neuronal cells) and include, but are not limited to, glioma, glioblastoma multiforme, meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma, medulloblastoma, ganglioglioma, schwannoma, neurofibroma, neuroblastoma, and epidural, intramedullary or intradural tumors.

[0234] In the case of cancer, a neurological agent that is a chemotherapeutic agent may be selected. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ethylenimines and methylamelamines, including ylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and and 9-aminocamptothecin;bryostatins;kallistatins;CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs);podophyllotoxins;podophyllic acid;teniposide;cryptophycins (especially cryptophycin 1 and cryptophycin 8);dolastatins;duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1);eleutherobin;pancratistatins;sarcodictiins;spongistatins;chlorambucil, chlorna Nitrogen mustards such as chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin γ1I and calicheamicin ω11 (see, e.g., Agnew, Chem Inti. Ed. Engl., 33:183-186 (1994)); the dynemicins, including dynemicin A; the esperamicins; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazomethanesulfonyl esters, Zo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptomycin, Antibiotics such as zosyn, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidins such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine. Gin analogues;androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenal agents such as aminoglutethimide, mitotane, trilostane;folic acid supplements such as floric acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;elfomitine;elliptinium acetate;epothilone;etoglucide;gallium nitrate;Hydroxyurea; Lentinan; Lonidynin; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Fenameth; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharma- ceutically acceptable salts, acids, or derivatives of any of the above;and combinations of two or more of the above, such as CHOP (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin).

[0235] Also included in this definition of chemotherapeutic agents are anti-hormonal agents, often in the form of systemic or whole body treatments, that act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. Chemotherapeutic agents may be hormones themselves. Examples include, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, keoxyfene, LY117018, onapristone, and FARESTON® toremifene; antiprogesterones; estrogen receptor downregulators (ERDs); agents that function to suppress or shut down the ovaries, such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate, and luteinizing hormone releasing hormones (LHRH) such as tripterelin. agonists; 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, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole.Additionally, such definition of chemotherapeutic agent includes bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®, DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid / zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those directed against, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptors. those that inhibit the expression of genes in signal transduction pathways implicated in aberrant cell proliferation, such as (EGFR); THERATOOPE® vaccines and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; lapatinib ditosylate (also known as GW572016, an ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor); and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0236] Another group of compounds that may be selected as neuropharmaceuticals for treating or preventing cancer are anti-cancer immunoglobulins (including, but not limited to, trastuzumab, pertuzumab, bevacizumab, alemtuzumab, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, panitumumab, and rituximab). In some examples, 131 Antibodies coupled with toxic labels or conjugates, including but not limited to, tositumomab or trastuzumab emtansine bearing an I radiolabel, can be used to target and kill desired cells (i.e., cancer cells).

[0237] Ocular diseases or disorders are diseases or disorders of the eye, which for the purposes of this specification are considered to be CNS organs separated by the BBB. Ocular diseases or disorders include disorders of the sclera, cornea, iris and ciliary body (i.e., scleritis, keratitis, corneal ulcers, corneal abrasions, snow blindness, arc eye, Thygeson's punctate superficial keratitis, 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, retinoschisis, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, age-related macular degeneration, macular degeneration (wet or atrophic), epiretinal membrane, retinitis pigmentosa and macular edema), glaucoma, These include, but are not limited to, floaters, optic nerve and visual pathway disorders (i.e., Leber's hereditary optic neuropathy and optic disc drusen), ocular muscle / binocular control / refractive disorders (i.e., strabismus, ophthalmoplegia, progressive external ophthalmoplegia, esotropia, exotropia, hyperopia, myopia, astigmatism, anisometropia, presbyopia and ophthalmoplegia), visual impairment and blindness (i.e., amblyopia, Leber's congenital amaurosis, scotoma, color blindness, color blindness, night blindness, blindness, river blindness and microphthalmia / coloboma), red eye, Argyll-Robertson pupil, keratomycosis, xerophthalmia and aniridia.

[0238] For ocular diseases or disorders, antiangiogenic ophthalmic drugs (i.e., bevacizumab, ranibizumab, and pegaptanib), ophthalmic glaucoma drugs (i.e., carbachol, epinephrine, demecarium bromide, apraclonidine, brimonidine, brinzolamide, levobunolol, timolol, betaxolol, dorzolamide, bimatoprost, carteolol, metipranolol, dipivefrin, travoprost, and latanoprost), carbonic anhydrase inhibitors (i.e., methazolamide, cyclosporine ... amide and acetazolamide), ophthalmic antihistamines (i.e., naphazoline, phenylephrine, and tetrahydrozoline), ophthalmic lubricants, ophthalmic steroids (i.e., fluorometholone, prednisolone, loteprednol, dexamethasone, difluprednate, rimexolone, fluocinolone, medrysone, and triamcinolone), ophthalmic anesthetics (i.e., lidocaine, proparacaine, and tetracaine), ophthalmic anti-infectives (i.e., levofloxacin, , gatifloxacin, ciprofloxacin, moxifloxacin, chloramphenicol, bacitracin / polymyxin b, sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin, sulfisoxazole, gentamicin, idoxuridine, erythromycin, natamycin, gramicidin, neomycin, ofloxacin, trifluridine, ganciclovir, vidarabine), ophthalmic anti-inflammatory drugs (i.e., nepafenac, keto Neuropathic drugs that are ophthalmic antihistamines or decongestants (i.e., ketotifen, olopatadine, epinastine, naphazoline, cromolyn, tetrahydrozoline, pemirolast, bepotastine, naphazoline, phenylephrine, nedocromil, lodoxamide, phenylephrine, emedastine and azelastine) may be selected.

[0239] Viral or microbial infections of the CNS include any of a variety of pathogenic organisms, including viruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria spp., Streptococcus spp., Pseudomonas spp., Proteus spp., E. coli, S. aureus, Pneumococcus spp., Meningococcus spp., Haemophilus spp., and Mycobacterium tuberculosis), and fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., Toxoplasma gondii), which result in CNS pathophysiology including, but not limited to, meningitis, encephalitis, myelitis, vasculitis, and abscesses, which can be acute or chronic. gondii) or other microorganisms such as amebas.

[0240] For viral or microbial diseases, antiviral compounds (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 inhibitors (i.e., serotinib ... Oxidant reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir, zalcitabine, telbivudine, and didanosine), protease inhibitors (i.e., darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir, amprenavir, indinavir, and saquinavir), purine nucleosides (i.e., valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir, and cidofovir), and species Antivirals (i.e., enfuvirtide, foscarnet, palivizumab, and fomivirsen), antibiotics (aminopenicillins (i.e., amoxicillin, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temocillin, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, and bacampicillin), cephalosporins (i.e., cefazolin, cephalexin, cephalothin, cefamandole, ceftriaxone, cefovir ... Taxime, 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, nocardicin A and tabtoxin-beta-lactams), beta-lactamase inhibitors combined with another beta-lactam antibiotic (i.e., clavulanic acid, tazobactam and sulbactam),Aminoglycosides (i.e., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromomycin), ansamycins (i.e., geldanamycin and herbimycin), carbacephems (i.e., loracarbef), glycopeptides (i.e., teicoplanin and vancomycin), macrolides (i.e., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spemetidine), cutinomycin), monobactams (i.e., aztreonam), quinolones (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamides (i.e., mafenide, sulfonamide chrysoidine, sulfacetamide, sulfadiazine, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimetan, anti-inflammatory or cytotoxic antibiotics (i.e., doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin), and various antibacterial compounds (i.e., bacitracin, colistin, and polymyxin B). , but not limited to), antifungals (i.e., metronidazole, nitazoxanide, tinidazole, chloroquine, iodoquinol, and paromomycin), and antiparasitic drugs (quinine, chloroquine, amodiaquine, pyrimethamine, sulfadoxine, proguanil, mefloquine, atovaquone, primaquine, artemisinin, halofantrine, doxycycline, clindamycin, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin, amphotericin B, melarsoprol,Neuropathic drugs may be selected, including but not limited to, eflornithine and albendazole.

[0241] Inflammation of the CNS includes, but is not limited to, inflammation caused by injury to the CNS, which can be physical injury (i.e., due to an accident, surgery, brain trauma, spinal cord injury, concussion) and injury due to or associated with one or more other diseases or disorders of the CNS (i.e., abscess, cancer, viral or microbial infection).

[0242] In the case of CNS inflammation, neuropharmaceuticals may be chosen that address the inflammation itself (i.e., nonsteroidal anti-inflammatory drugs such as ibuprofen or naproxen) or that treat the underlying cause of the inflammation (i.e., antiviral or anticancer drugs).

[0243] As used herein, ischemia of the CNS refers to a group of disorders associated with abnormal blood flow or vascular behavior in the brain or its causes, including, but not limited to, focal cerebral ischemia, global cerebral ischemia, stroke (i.e., subarachnoid hemorrhage and intracerebral hemorrhage), and aneurysm.

[0244] In the case of ischemia, neurological medications may be selected, including, but not limited to, thrombolytic agents (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 to improve blood flow or vascular flexibility (including, for example, blood pressure medications).

[0245] Neurodegenerative diseases are a group of diseases and disorders associated with loss of function or death of nerve cells in the CNS, including, but not limited to, adrenoleukodystrophy, Alexander disease, Alpers disease, amyotrophic lateral sclerosis, ataxia telangiectasia, Batten disease, Cockayne syndrome, corticobasal degeneration, degeneration caused by or associated with amyloidosis, Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy disease, multiple system atrophy, multiple sclerosis, primary lateral sclerosis, progressive supranuclear palsy, spinal muscular atrophy, transverse myelitis, Refsum's disease, and spinocerebellar ataxia.

[0246] In the case of neurodegenerative diseases, neuropharmaceuticals that are growth hormones or neurotrophic factors may be selected; examples include 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)-alpha, TGF-beta, vascular endothelial growth factor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNF), and the like. NTF), glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin, persephin, interleukin, 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), netrin, saposin, semaphorin, and stem cell factor (SCF).

[0247] Seizure diseases and disorders of the CNS involve inappropriate and / or abnormal electrical conduction in the CNS and include epilepsy (i.e., absence seizures, atonic seizures, benign rolandic epilepsy, childhood absence, clonic seizures, complex partial seizures, frontal lobe epilepsy, febrile seizures, infantile spasms, juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Dravet syndrome, Ohtahara syndrome, West syndrome, myoclonic seizures, mitochondrial disorders, progressive myoclonic epilepsy, psychogenic seizures, reflex epilepsy, Lamella syndrome, and others). including, but not limited to, Smussen's syndrome, simple partial seizures, secondary generalized seizures, temporal lobe epilepsy, tonic-clonic seizures, tonic seizures, psychomotor seizures, limbic epilepsy, partial onset seizures, generalized onset seizures, status epilepticus, abdominal epilepsy, akinetic seizures, autonomic seizures, bilateral generalized myoclonus, catamenial epilepsy, drop attacks, affective seizures, focal seizures, galvanic seizures, Jackson-March, Lafora's disease, motor seizures, multifocal seizures, nocturnal seizures, photosensitive seizures, pseudoseizures, sensory seizures, petit mal seizures, Sylvan seizures, withdrawal seizures and visual reflex seizures).

[0248] For seizure disorders, barbiturate anticonvulsants (i.e., primidone, metharbital, mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal, barbital, bralobarbital, and phenobarbital), benzodiazepine anticonvulsants (i.e., diazepam, clonazepam, and lorazepam), carbamate anticonvulsants (i.e., felbamate), carbonic anhydrase inhibitor anticonvulsants (i.e., acetazolamide, topiramate, and zonisamide), dibenzazepine anticonvulsants (i.e., rufinamide, carbamazepine, and oxcarbazepine), fatty acid derivative anticonvulsants (i.e., divalproex and valproic acid), gamma-aminobutyric acid analogs (i.e., pregabalin, gabapentin, and vigabatrin), gamma-aminobutyric acid reuptake inhibitors (i.e., ribazantine, ribazantine, and ribazantine), and ribazantine-based anticonvulsants (i.e., ribazantine, and ribazantine). Neurologic agents may be selected that are anticonvulsants or antiepileptics, including, but not limited to, uptake inhibitors (i.e., tiagabine), gamma-aminobutyric acid transaminase inhibitors (i.e., vigabatrin), hydantoin anticonvulsants (i.e., phenytoin, ethotoin, fosphenytoin, and mephenytoin), miscellaneous anticonvulsants (i.e., lacosamide and magnesium sulfate), progestins (i.e., progesterone), oxazolidinedione anticonvulsants (i.e., paramethadione and trimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam), succinimide anticonvulsants (i.e., ethosuximide and methsuximide), triazine anticonvulsants (i.e., lamotrigine), and urea anticonvulsants (i.e., phenacemide and feneturide).

[0249] Behavioral disorders are disorders of the CNS characterized by abnormal behavior on the part of an affected subject, and include, but are not limited to, sleep disorders (i.e., insomnia, parasomnias, night terrors, circadian rhythm sleep disorders and narcolepsy), mood disorders (i.e., depression, suicidal depression, anxiety disorders, chronic affective disorders, phobias, panic attacks, obsessive-compulsive disorder, attention deficit hyperactivity disorder (ADHD), attention deficit disorder (ADD), chronic fatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolar disorder), eating disorders (i.e., anorexia or bulimia), psychoses, developmental behavioral disorders (i.e., autism, Rett syndrome, Asperger's syndrome), personality disorders and psychotic disorders (i.e., schizophrenia, delusional disorder, etc.).

[0250] For behavioral disorders, neurological medications include atypical antipsychotics (i.e., risperidone, olanzapine, apripiprazole, quetiapine, paliperidone, asenapine, clozapine, iloperidone, and ziprasidone), phenothiazine antipsychotics (i.e., prochlorperazine, chlorpromazine, fluphenazine, perphenazine, trifluoperazine, thioridazine, and mesoridazine), thioxanthenes (i.e., thiothixene, ), various antipsychotics (i.e., pimozide, lithium, molidone, haloperidol, and loxapine), selective serotonin reuptake inhibitors (i.e., citalopram, escitalopram, paroxetine, fluoxetine, and sertraline), serotonin-norepinephrine reuptake inhibitors (i.e., duloxetine, venlafaxine, desvenlafaxine), tricyclic antidepressants (i.e., doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine, imipramine, protriptyline, and desipramine), tetracyclic antidepressants (i.e., mirtazapine and maprotiline), phenylpiperazine antidepressants (i.e., trazodone and nefazodone), monoamine oxidase inhibitors (i.e., isocarboxazid, phenelzine, selegiline, and tranylcypromine), benzodiazepines (i.e., alprazolam, estazolam, flurazepam, clonazepam, lorazepam, and diazepam), lepinephrine-dopamine reuptake inhibitors (i.e., bupropion), CNS stimulants (i.e., phentermine, diethylpropion, methamphetamine, dextroamphetamine, amphetamine, methylphenidate, dexmethylphenidate, lisdexamfetamine, modafinil, pemoline, phendimetrazine , benzphetamine, phendimetrazine, armodafinil, diethylpropion, caffeine, atomoxetine, doxapram, and mazindol), anxiolytics / sedatives / hypnotics (including, but not limited to, barbiturates (i.e., secobarbital, phenobarbital, and mephobarbital), benzodiazepines (above), and miscellaneous anxiolytics / sedatives / hypnotics (i.e., diphenhydramine, sodium oxybate, zaleplon, hydroxyzine, chloral hydrate, alpidem, buspirone, doxepin, eszopiclone, ramelteon, meprobamate, and ethchlorvynol), secretins (e.g., see Ratliff-Schaub et al. Autism The therapeutic agent may be selected from behavior-modifying compounds, including, but not limited to, agonists, antagonists, and inhibitors of stimulants (see, e.g., Cowen et al., J. Neurochem. 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)).

[0251] Lysosomal storage disorders are metabolic disorders that in some cases involve or have CNS-specific symptoms; such disorders include, but are not limited to, Tay-Sachs disease, Gaucher disease, Fabry disease, mucopolysaccharidoses (types I, II, III, IV, V, VI, and VII), glycogen storage diseases, GM1 gangliosidosis, metachromatic leukodystrophy, Farber disease, Canavan leukodystrophy, and neuronal ceroid lipofuscinosis types 1 and 2, Niemann-Pick disease, Pompe disease, and Krabbe disease.

[0252] In the case of lysosomal storage diseases, neuropharmaceuticals may be selected that mimic the activity of the enzyme itself or that is otherwise impaired in the disease. Exemplary recombinant enzymes for treating lysosomal storage disorders include, but are not limited to, those set forth, for example, in U.S. Patent Application Publication No. 2005 / 0142141 (i.e., alpha-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, acid alpha-glucosidase, glucocerebrosidase, alpha-galactosidase A, hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase, beta-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase, palmitoyl-protein thioesterase 1, and tripeptidyl aminopeptidase 1).

[0253] In one embodiment, the antibodies or Fc conjugates of the invention are used to detect neuropathy prior to the onset of symptoms and / or to assess the severity or duration of a disease or disorder, hi one embodiment, the antibodies or Fc conjugates allow for detection and / or imaging of neuropathy, including imaging by radiography, tomography, or magnetic resonance imaging (MRI).

[0254] In one aspect, an antibody or Fc conjugate of the invention is provided for use as a medicament. In a further aspect, an antibody or Fc conjugate is provided for use in the treatment of a neurological disease or disorder (e.g., Alzheimer's disease). In certain embodiments, an antibody or Fc conjugate is provided for use in the treatment method described herein. In certain embodiments, the invention provides an antibody or Fc conjugate for use in a method of treating an individual with a neurological disease or disorder, comprising administering to the individual an effective amount of the antibody or Fc conjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In a further embodiment, the invention provides an antibody or Fc conjugate for use in reducing or inhibiting amyloid plaque formation in a patient at risk for 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 invention for use in the method of the invention improves the uptake of neuropathic drugs compared to an antibody or Fc conjugate comprising a wild-type Fc.

[0255] In a further aspect, the present invention provides the use of the antibody or Fc conjugate of the present invention in the manufacture or preparation of a medicament.In one embodiment, the medicament is for treating a neurological disease or disorder.In a further embodiment, the medicament is for use in a method for treating a neurological disease or disorder, comprising administering an effective amount of the medicament to an individual having a neurological disease or disorder.In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0256] In a further aspect, the present invention provides a method for treating Alzheimer's disease. In one embodiment, the method comprises administering an effective amount of an antibody of the present invention that binds to BACE1 or Abeta to an individual with Alzheimer's disease. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. An "individual" according to any of the above embodiments may be a human.

[0257] The antibody and Fc conjugate of the present invention can be used alone or in combination with other drugs in treatment.For example, the antibody or Fc conjugate of the present invention can be co-administered with at least one additional therapeutic agent.In certain embodiments, the additional therapeutic agent is an effective therapeutic agent for treating the same or different neurological disorder that the antibody or Fc conjugate is used for treatment. Exemplary additional therapeutic agents include, but are not limited to, the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, lovastigmine and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, gamma-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid 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 neurological drugs.

[0258] The various combination therapies described above and herein include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) as well as separate administration, where administration of the antibody or Fc conjugate of the invention may occur prior to, concurrently with, and / or after administration of the additional therapeutic agent and / or adjuvant. In one embodiment, administration of the antibody or Fc conjugate and administration of the additional therapeutic agent occur within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. The antibodies and Fc conjugates of the invention can also be used in combination with other interventional therapies, such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and appropriate for the neurological disorder being treated or prevented.

[0259] 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 administration, and, if desired for localized treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, by injection, for example, intravenous or subcutaneous injection, depending in part on whether administration is temporary or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0260] The antibodies and Fc conjugates of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to the medical profession. The antibodies or Fc conjugates need not be, but are optionally, formulated with one or more agents currently used to prevent or treat the disorder in question, or to prevent, alleviate, or ameliorate one or more side effects of antibody or Fc conjugate administration. The effective amount of such other agents depends on the amount of antibody or Fc conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and by the same routes of administration as described herein, or about 1-99% of the dosages described herein, or in any dosage and by any route empirically / clinically determined to be appropriate.

[0261] For the prevention or treatment of 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 being treated, the type of antibody or Fc conjugate, the severity and course of the disease, whether the antibody or Fc conjugate is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history, and response to the antibody or Fc conjugate, and the discretion of the attending physician. The antibody or Fc conjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the antibody or Fc conjugate may be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administration over several days or longer, depending on symptoms, treatment is generally maintained until a desired suppression of disease symptoms occurs. One exemplary dosage of antibody or Fc conjugate would be in the range of about 0.05 mg / kg to about 40 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, or 40 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives about two to about twenty, or, for example, about six doses of the antibody or Fc conjugate). An initial larger loading dose, followed by one or more smaller doses, may be administered. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays described herein and known in the art. H. Manufactured articles

[0262] In another aspect of the present invention, an article of manufacture is provided that includes materials useful for treating, preventing, and / or diagnosing the above disorders. The article of manufacture includes a container and a label or package insert that is inserted into or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition alone or in combination with another composition that is effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced 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 condition of choice. Furthermore, the article of manufacture may include (a) a first container in which a composition comprising an antibody comprising a modified Fc of the present invention or an Fc conjugate of the present invention is contained, and (b) a second container in which a composition comprising an additional cytotoxic or otherwise therapeutic agent is contained. In this embodiment of the present invention, the article of manufacture may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharma- ceutically 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. EXAMPLES

[0263] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the invention. Example 1 - Method

[0264] Plasmid construction and antibody and FcRn production - Antibodies, antibody Fc, and human and mouse FcRn complexes, including the antibody heavy and light chains or Fc, or in the case of FcRn, the cloned sequences encoding FCGRT or Fcgrt (encoding human FcRn alpha chain and mouse FcRn alpha chain, respectively) and beta2 microglobulin (β2 M), were expressed in mammalian expression vectors by standard molecular biology techniques as previously described (Eaton, Wood et al. 1986) using standard techniques. FcRn is expressed with a His tag and purified by immobilized metal ion chromatography (IMAC) or purification on an immobilized IgG column (e.g., Sigma A0919, A2909 for mouse IgG agarose or rabbit IgG agarose). Antibodies were expressed in CHO cells as transient transfection cultures (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).

[0265] Antibody-FcRn affinity measurements - The affinity of antibodies containing 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. Modified Fc antibodies were immobilized by protein L binding at a surface density of 1000 RU. Neutral pH binding was determined in HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% v / v surfactant P20). Acidic pH binding was determined in MBS-P (0.01 M MES pH 6.0, 0.15 M NaCl, 0.005% v / v surfactant P20). Association rates (ka) and dissociation rates (kd) were calculated using a one-to-one Langmuir binding model by simultaneously fitting the association and dissociation sensorgrams (BIAevaluations version 4.1). All fits had a chi-square / Rmax value less than 10% and met the acceptable goodness of fit criteria. D ) was calculated as the ratio kd / ka. Alternatively, the equilibrium binding K D Values ​​were determined from the dependence of steady-state binding levels on analyte concentration. The so-called steady-state analysis is suitable for measuring weak to moderate interactions. Undetectable binding or binding that is too weak to allow accurate affinity analysis is set to above 10 μM in the tables herein.

[0266] Transcytosis assay - an in vitro assay was used to measure the transcytosis activity of modified Fc antibodies. In this two-chamber transwell assay, a layer of epithelial cells expressing hFcRn or mFcRn is established on a membrane separating two chambers. Since tight junctions formed between the cells preclude antibody diffusion, antibody passage from one chamber to the other is only possible by intracellular transport. Therefore, transport of antibodies through this cell layer from one chamber to the other is used as a model for transcytosis. A similar assay is described, for example, in Claypool et al. Journal of Biological Chemistry 2002 Aug. 2;277(31):28038-50. MDCK II cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified minimal 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 (Thermo Fisher Scientific, Waltham, MA) at 37°C and 5% CO . 2 MDCK II cells were grown in a humidified incubator at 37 °C. MDCK II cells were first transfected with the gene encoding hFcRn (FCGRT (UniProtKB-P55899, FCGRTN HUMAN) separated by the P2A sequence) and β 2 m (UniProtKB-P61769, B2MG_HUMAN) (Kim et al. PLoS one 2011;6(4):e 18556)) and then 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 in constant antibiotic selection (5 μg / mL puromycin). FITC anti-human β2 Its FCGRT and β were assessed by flow cytometry using M (BioLegend). 2 Final clones were selected based on M cell surface expression.

[0267] Transcytosis assays were performed as follows: FcRn-expressing cells were seeded in Transwell® permeable support plates, 0.4 μm pore size (Corning Inc., Corning, NY) for 3 days. On day 3, fresh medium containing test antibodies and the fluorescent marker dye Lucifer Yellow (Molecular Probes, Eugene, OR) was added to the apical compartment. Fresh medium without test antibodies and Lucifer Yellow was added to the basolateral compartment. The pH of both chambers was 7.4. Plates were incubated at 37°C and 5% CO 2 The cells were incubated overnight in a humidified incubator. On the fourth day, the medium was collected from the apical and basolateral compartments, and the antibody concentrations in the two compartments were assayed 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 a reference antibody, typically a 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 levels of Lucifer Yellow above the control level were discarded, as this indicated that the epithelial barrier was not intact.

[0268] Wild-type and transgenic mouse PK and PD studies - Wild-type C57B / 6 mice aged 6 to 8 weeks were used for mFcRn studies. For mouse studies modeling hFcRn, transgenic Tg32 mice expressing the human FcRn alpha chain (FCGRT) transgene under the control of a human promoter and carrying a knockout allele of the mouse FcRn alpha chain (Fcgrt tmIDcr )-B6.Cg-Fcgrt tmIDcrTg(FCGRT)32Dcr / DcrJ, (JAX stock number 014565) was used (Petkova SB2006 Int. Immunol 18(2):1759-69; Roopenian, DC, 2010, Methods Mol. Biol. 602:93-104).

[0269] Dosing, Sample Collection for Mouse Studies - Mice were injected intravenously with the indicated doses of antibody as outlined below. After various times post-dose, blood was collected and either plasma or serum was isolated for measurement of antibody concentrations. For plasma collection, whole blood was collected into EDTA microcontainer tubes (BD Diagnostics) prior to perfusion, centrifuged at 5,000 x g for 15 minutes, and the supernatant was isolated for measurement of plasma antibody concentrations. For serum collection, whole blood was collected into serum separator microcontainer tubes (BD Diagnostics), allowed to clot for at least 30 minutes, and spun down at 5,000 x g for 90 seconds. The supernatant was isolated for serum antibody measurements. At the indicated times, mice were perfused with D-PBS and brains were collected for measurement of antibody concentrations and / or Abeta. For brain antibody concentration measurements, a half brain from each mouse was homogenized in 1% NP-40 (Cal-Biochem) in PBS containing Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics). 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 measurements. 1-40 For the measurements, the hemibrain was homogenized in 5 M guanidine hydrochloride buffer, the samples were rotated at room temperature for 3 h, and then 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 to determine Abeta. 1-40 The supernatant was isolated for measurement.

[0270] Measurement of antibody concentrations in mouse plasma or serum (pharmacokinetics) - Total antibody concentrations in mouse plasma or serum were measured by a common human Fc ELISA. Nunc 384-well MaxiSorp immunoplates were loaded with donkey anti-human IgG F(ab') 2 Plates were coated with Fc fragment- and Fc fragment-specific polyclonal antibodies (Jackson ImmunoResearch) overnight at 4°C. 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. Plates were washed with PBS and 0.05% Tween 20 using a microplate washer (Bio-Tek Instruments Inc.) and standards and samples diluted in PBS containing 0.5% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.05% Tween 20 and 15 ppm (parts per million) Proclin were added for 2 hours at 25°C. Bound antibodies were analyzed by HRP-conjugated F(ab') 2 Detection was with goat anti-human IgG and Fc-specific polyclonal antibodies (Jackson ImmunoResearch), development with TMB (KPL Inc.), and absorbance (A) was measured at 450 nm on a Multiskan Ascent reader (Thermo Scientific). Concentrations were determined from a standard curve using a four-parameter nonlinear regression program.

[0271] 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 recombinant human BACE1 ECD as a coat 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 concentration 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), standards and samples diluted in PBS containing 0.5% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.05% Tween 20 and 15 ppm Proclin were incubated on the plates with gentle agitation for 2 hours at room temperature. Bound antibodies were analyzed by HRP-conjugated F(ab') 2 Detection was with goat anti-human IgG Fc-specific polyclonal antibody (Jackson ImmunoResearch). Finally, the plates were developed using the substrate 3,3',5,5'-tetramethylbenzidine (TMB) (KPL, Inc., Gaithersburg, MD). Absorbance was measured at a wavelength of 450 nm with a reference at 630 nm in a Multiskan Ascent reader (Thermo Scientific, Hudson, NH). Concentrations were determined from a standard curve using a four-parameter nonlinear regression program. The lower limit of detection (LLOD) of the free anti-BACE1 mouse ELISA was 0.06 ng / ml.

[0272] PD assay-A beta in mouse brain samples 1-40 Concentrations were measured using an ELISA similar to the method described above for PK analysis. 1-40A rabbit polyclonal antibody specific for the C-terminus of (Millipore, Bedford, MA) was coated onto the plate, and biotinylated anti-mouse Abeta 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.

[0273] Radiolabeling studies - Antibodies were radiolabeled using a modified Chizzonite radioiodination protocol. 125 The nucleosomes were labeled with I (Chizzonite, Truitt et al., 1991). Wild-type or human FCGRT homozygous transgenic mice (Tg32) were transfected with 125 I]-labeled antibody (5 mCi iv) was administered. Blood, brain and other tissues were collected at the indicated time points after injection. Samples were then analyzed for total radioactivity per gram of tissue. Tissue radioactivity was corrected for contributions from vascular blood concentrations (n=2 / group). Example 2 - Pharmacokinetics and Pharmacodynamics of hIgG1 Wild-Type and Fc-Modified Antibodies in Wild-Type and Human FCGRT Transgenic Mice

[0274] Two antibodies containing modified Fc, M428L / N434A (LA) and M252Y / S254T / T256E (YTE), previously identified as having improved binding to hFcRn at pH 6, and two antibodies containing wild-type hIgG1 and hIgG4, were expressed and purified and their affinity for hFcRn was measured at pH 7.4 and pH 6. In addition, the transcytosis of each antibody containing 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 for these antibodies. Table 2 TIFF2025037985000005.tif254170

[0275] As shown in Table 2, antibodies containing the LA or YTE Fc modifications had improved FcRn binding at pH 6, no detectable difference in binding at pH 7.4, and showed little improvement in transcytosis compared to the wild-type control (above 10 μM indicates affinity at pH 7.4 was undetectable or too weak to measure). The LA and YTE variants increased transcytosis by 5.1-fold and 1.9-fold, respectively.

[0276] To evaluate whether antibodies with enhanced FcRn affinity at pH 6 could improve brain uptake, we investigated the effect of wild-type (Fcgrt + / + We determined antibody pharmacokinetics and Abeta pharmacodynamics in mice. As shown in Figure 1, anti-BACE1-hIgG1-YTE exhibited faster plasma clearance (Figure 1A) and improved brain antibody concentrations (Figure 1C) compared to anti-BACE1 hIgG1 with wild-type Fc. Consistent with the improved brain concentrations of the antibody, anti-BACE1-hIgG1-YTE administration significantly reduced brain Abeta 1-40 In contrast, anti-gD-hIgG1 (control) and anti-BACE1-hIgG1 antibodies significantly reduced brain Abeta levels, consistent with the low levels of these antibodies detected in the brain. 1-40 The levels of BACE1 were not significantly affected by anti-BACE1-hIgG1 (Figure 1B, C). Compared with anti-BACE1-hIgG1, anti-BACE1-hIgG1-YTE inhibited the wild-type (Fcgrt + / + ) increased brain exposure in mice in terms of both maximum concentration (Cmax) and area under the curve (AUC) (Figure 1D).

[0277] Surprisingly, the transgenic Tg32 (FCGRT + / + Fcgrt - / -When anti-BACE1-hIgG1-YTE was tested in mice, it also showed improved brain uptake and reduced brain A beta-blockers compared to anti-BACE1-hIgG1. 1-40 There was also no decrease in the levels of IgG1 and hFcRn (Figures 2A, 2B, and 2C). To investigate the reason for this, we tested the binding of wild-type hIgG1 antibody and hIgG1 antibody containing the YTE modification to mFcRn and hFcRn at pH 7.4 and pH 6. The data showed that the YTE modification improved binding to mFcRn by approximately 10-fold at both pH 7.4 and pH 6.0, but binding to hFcRn was improved only at pH 6.0 (Figure 2D; K in Figure 2D). D (Data slightly differ from those in Table 2 because they were measured in different experiments.) These results suggested that a stronger affinity for FcRn at neutral pH but not at pH 6 allows for increased transport to the brain. Example 3 - Evaluation of hIgG1 and hIgG4 Fc modifications

[0278] 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.

[0279] Antibodies containing the 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.

[0280] Table 3 shows Fc-modified antibodies with improved binding to human FcRn at pH 6.0 that did not substantially improve in vitro transcytosis. TIFF2025037985000006.tif254170

[0281] As shown in Table 3, for modified Fc antibodies with enhanced affinity for hFcRn at pH 6 but no measurable affinity at pH 7.4, no or only slight improvements in transcytosis were observed (1.7-7.8), consistent with the YTE results described in Example 2. In contrast, the antibody containing the N434W Fc modification showed measurable but weak pH 7.4 affinity (5.5 μM) and increased transcytosis (26-fold) compared to the wild-type antibody.

[0282] Single Fc modifications were combined into double, triple and quadruple Fc modifications, and anti-BACE1 antibodies containing 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 containing double, triple and quadruple Fc modifications, respectively, with improved binding to human FcRn at pH 7.4 and improved transcytosis activity. Figure 3 shows the correlation between pH 7.4 FcRn affinity and pH 6 FcRn affinity of modified Fc antibodies. Each point represents a single antibody. Open triangles show an exemplary antibody of the present disclosure containing quadruple Fc modifications M252Y / T307Q / Q311A / N434Y, which was further tested in vivo, and designated as Q95(YQAY). Figure 4 shows the normalized transcytosis activity of the Fc modified antibodies shown in Tables 3, 4, 5 and 6. TIFF2025037985000007.tif254170TIFF2025037985000008.tif254170TIFF2025037985000009.tif254170TIFF2025037985000010.tif254170

[0283] As shown in the table above, a series of Fc modified antibodies were generated with substantially improved transcytosis activity, with some Fc modifications promoting transcytosis over 100-fold compared to the wild-type IgG1 antibody.

[0284] Tables 7 and 8 show the steady state affinities of certain anti-BACE1 and certain anti-Abeta antibodies with modified Fc as determined by BIACORE using an anti-human Fab capture chip. TIFF2025037985000011.tif254170TIFF2025037985000012.tif254170Example 4 - Pharmacokinetics of antibodies containing modified Fc in Tg32 mice

[0285] A single 25 mg / kg intravenous (IV) dose of anti-gD antibody comprising wild-type human IgG1 (anti-gD-hIgG1), anti-gD antibody comprising hIgG1 with the YY (D1, M252Y / N434Y) Fc modification (anti-gD-hIgG1-YY), anti-gD antibody comprising hIgG1 with the YQAY (Q95, M252Y / T307Q / Q311A / N434Y) Fc modification (anti-gD-hIgG1-YQAY), or anti-gD antibody comprising hIgG1 with the LA (M428L / N434A) Fc modification (anti-gD-IgG1-LA) was administered to transgenic mice expressing hFCGRT and lacking mFCGRT (Tg32 (FCGRT + / + Fcgrt - / - ) mice. As shown in Figure 5A and Figure 5B, serum levels of anti-gD antibodies containing different hIgG1 Fc were similar, but brain levels of anti-gD-hIgG1-YY, anti-gD-hIgG1-YQAY, and anti-gD-hIgG1-LA were higher than that of anti-gD-hIgG1.

[0286] The binding affinity of the anti-gD antibodies to hFcRn was measured at pH 6 and pH 7.4 as described above. The YY and YQAY Fc modified antibodies resulted in a similar hFcRn affinity enhancement in the anti-gD antibodies as observed in the anti-BACE1 context (Figure 5C) (Tables 4 and 6). The PK results and hFcRn affinity are summarized together in Figure 5C, which shows that anti-gD-hIgG1-YY and anti-gD-hIgG1-YQAY had a greater brain / serum ratio (measured as %AUC / AUC for brain / serum). Both Fc modifications significantly improved binding to hFcRn at pH 7.4 and pH 6, resulting in high transcytosis scores (79 for YY and 100 for YQAY in the context of anti-BACE1 - Tables 4 and 6). Example 5 - Pharmacodynamics of antibodies containing modified Fc in Tg32 mice

[0287] Pharmacodynamics (PD) assays were performed to compare the efficacy of anti-BACE1 antibodies containing wild-type hIgG1 (anti-BACE1-hIgG1) and hIgG1 with the YQAY Fc modification (anti-BACE1-hIgG1-YQAY) in transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT (FCGRT + / + Fcgrt - / - ) Mice were assessed for Abeta levels after a single 50 mg / kg IV dose. Brain antibody levels and Abeta 1-40 Levels were assayed as described in Example 1. Consistent with previous results, Fc-modified anti-BACE1-hIgG1-YQAY showed greater brain uptake than anti-BACE1-hIgG1 wild type (Figure 6B). Furthermore, brain Abeta levels were significantly higher after administration of Fc-modified anti-BACE1-hIgG1-YQAY. 1-40 The levels were reduced, whereas anti-BACE1-hIgG1 showed little or no reduction (FIG. 6C). As shown in FIG. 6D and FIG. 6E, anti-BACE1-hIgG1-YQAY had a larger Cmax and AUC last , as well as a greater reduction in Abeta.

[0288] Similar experiments were performed using different anti-BACE1 antibodies formatted as anti-BACE1-hIgG1, anti-BACE1-hIgG1-YY and anti-BACE1-hIgG1-YQAY. + / + Fcgrt - / - ) Mice were given a single 50 mg / kg IV dose of anti-BACE1-hIgG1, anti-BACE1-hIgG1-YY, or anti-BACE1-hIgG1-YQAY. Brain antibody levels and Abeta 1-40 Levels were assayed as described in Example 1. Although all three antibodies had similar serum concentrations (Figure 13A), anti-BACE1-hIgG1-YQAY showed the greatest brain uptake, with anti-BACE1-hIgG1-YY trending towards enhanced brain uptake compared to anti-BACE1-hIgG1 (Figure 13B). Consistent with greater brain uptake, brain Abeta 1-40 The levels were most significantly decreased after administration of Fc-modified anti-BACE1-hIgG1-YQAY, and a trend toward decreased levels was observed after administration of Fc-modified anti-BACE1-hIgG1-YY (FIG. 13C). As shown in FIG. 13D, anti-BACE1-hIgG1-YQAY had a larger brain Cmax and AUC than anti-BACE1-hIgG1, and anti-BACE1-hIgG1-YY was associated with a larger brain Cmax and AUC than anti-BACE1-hIgG1, as well as a larger reduction in A beta. Example 6 - FCGRT + / + , F.C.G.R.T. + / - and Fcgrt + / + Pharmacokinetics of a highly transcytotic IgG1 variant in mice

[0289] A single intravenous (IV) dose of 5 mg / kg of anti-gD-hIgG1 antibody, anti-gD-hIgG1-YY, anti-gD-hIgG1-YQAY, or anti-gD antibodies including hIgG1 with the YTE (M252Y / S254T / T256E) Fc modification (anti-gD-hIgG1-YTE) were administered to homozygous transgenic Tg32 mice expressing hFCGRT and lacking mFCGRT (FCGRT + / + Fcgrt - / -) mice; hemizygous (FCGRT + / - Fcgrt + / - ) mice; and wild-type (Fcgrt + / + ) mice. 5 μCi of each antibody was administered. 125 All antibodies were generated from homozygous Tg32 mice and hemizygous (FCGRT + / - Fcgrt + / - ) mice. 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 (Figures 7A, 7B, 7C). Figure 7D shows the affinity of anti-gD-hIgG1 and each Fc-modified antibody for hFcRn and mFcRn at pH 7.4 and pH 6, along with the plasma AUC relative to wild type for each antibody in each mouse strain.

[0290] Brain PK of anti-gD-hIgG1 and the three Fc modified antibodies in homozygous hFCGRT mice is shown in Figure 8. Anti-gD-hIgG1-YY and anti-gD-hIgG1-YQAY showed greater brain uptake than anti-gD-hIgG1 or anti-gD-hIgG1-YTE (Figure 8A). The increased brain uptake of anti-gD-hIgG1-YY and anti-gD-hIgG1-YQAY was specific to homozygous hFCGRT Tg32 mice (Figure 8B). Anti-gD-hIgG1-YY resulted in a 2.2-fold increased brain exposure compared to anti-gD-hIgG1, and anti-gD-hIgG1-YQAY resulted in an even greater increase in brain exposure, a 3.4-fold increase compared to anti-gD-hIgG1, as measured by AUC in mice homozygous for hFCGRT (Figure 8C).

[0291] The pharmacokinetics of anti-gD-hIgG1 and the three Fc-modified antibodies were also determined in other tissues, including liver, colon, and lung (Figures 9-11). These tissues also showed a moderate improvement in tissue penetration with the Fc-modified antibodies, although the improvement was less than that seen in the brain. Without intending to be bound by any particular theory, these tissues may have lower levels of FcRn or contain vasculature with fenestrated capillaries, which may allow some level of antibody penetration by diffusion. Example 7 - Pharmacokinetics and pharmacodynamics of highly transcytotic IgG1 variants in cynomolgus monkeys

[0292] Cynomolgus Monkey PK / PD Studies. For both studies, four male cynomolgus monkeys aged 3-5 years were used per experimental group.

[0293] In the first study, anti-BACE1 hIgG1 wild type antibody and anti-BACE1 hIgG1 antibody with modified Fc (anti-BACE1-YQAY, YEY, YPY) were administered at 50 mg / kg by intravenous bolus injection into the saphenous vein on day 0. CSF and blood samples were collected at various time points from 7 days before administration to 29 days after administration. Samples were collected at the same time points.

[0294] In the second study, anti-BACE1 hIgG1 wild-type and anti-BACE1 hIgG1 antibodies with modified Fc (anti-BACE1-YQAY, YY, YLYI, YIY) were administered at 50 mg / kg by intravenous bolus injection into the saphenous vein. CSF and blood samples were collected at various time points from 7 days before to 7 days after administration. At 2 and 7 days after administration, the brains of two animals were harvested after total body perfusion. Brain regions were partially dissected and immediately frozen. Different brain regions were homogenized in 1% NP-40 (Cal-Biochem) in PBS containing Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics). Homogenized brain samples were rotated at 4°C for 1 hour and then spun at 14,000 rpm for 20 minutes. The supernatant was isolated for brain pharmacokinetic and pharmacodynamic (sAPPβ / α) analysis.

[0295] Pharmacokinetic assay. Total antibody concentrations in monkey serum were measured using an LCMS method using affinity capture with Pure Proteome Protein A Magnetic Beads (Millipore) followed by denaturation, reduction, alkylation and trypsin digestion. A signature peptide from the Fc region was selected as a surrogate for quantification of total antibody concentration. The MQC of the assay 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 coat and monkey-adsorbed goat anti-human IgG antibody conjugated to HRP (Bethyl) as detection. The assay had an MQC value of 1.6 ng / mL in CSF and brain.

[0296] Pharmacodynamic assays.sAPPβ / α ratio.CSF and brain concentrations of sAPPα and sAPPβ were determined using the sAPPα / sAPPβ multiplex ECL assay. sAPPα was captured using anti-A beta monoclonal antibody 6E10 and APPβ was captured using an antibody against amino acids 591-596 of APP. Both analytes were detected using an antibody against the N-terminus of APP. CSF was thawed on ice and then diluted 1:10 in 1% BSA in TBS-Tween 20. The assay had LLOQ values ​​of 0.05ng / ml and 0.03ng / ml for sAPPα and sAPPβ, respectively.

[0297] Results: As shown in Figure 14A, in the first study, anti-BACE1 antibodies with modified Fc showed a significant decrease in cerebrospinal fluid (CSF) sAPPβ / α ratio compared to anti-BACE1 hIgG1 wild-type antibody. As shown in Figure 14B and Figure 14C, FcRn affinity at neutral pH appeared to correlate with increased clearance of the antibody from serum, with anti-BACE1 hIgG1 wild-type antibody being cleared more slowly than either of the antibodies with modified Fc.

[0298] As shown in Figure 15A, in the second test, the anti-BACE1 antibody with modified Fc showed a significant decrease in CSF sAPPβ / α ratio compared to the anti-BACE1 hIgG1 wild-type antibody. As shown in Figure 15B, the anti-BACE1 antibody with modified Fc showed a substantial decrease in brain sAPPβ / α ratio compared to the anti-BACE1 hIgG1 wild-type antibody. Figure 15C shows a comparison of the CSF sAPPβ / α ratio and the brain sAPPβ / α ratio for each antibody. CSF and brain showed similar PD effects with various antibodies.

[0299] As shown in Figure 15D, all of the anti-BACE1 antibodies with modified Fc were present in higher concentrations in the brain (average of cortex and hippocampus) on day 2 than the anti-BACE1 hIgG1 wild-type antibody, and the anti-BACE1 antibodies with YQAY, YY, and YIY Fc modifications had higher brain concentrations on day 7 than the anti-BACE1 hIgG1 wild-type antibody. The fold improvement in antibody concentration on day 2 was 7.4 and on day 7 was 4.7 for anti-BACE1 hIgG1 YY. The fold improvement in antibody concentration on day 2 was 7.4 and on day 7 was 8.1 for anti-BACE1 hIgG1 YQAY (Figure 15E).

[0300] FIG. 15F shows the correlation between brain sAPPβ / α ratio and brain antibody concentration, demonstrating that higher levels of anti-BACE1 antibodies result in a lower sAPPβ / α ratio and a stronger PD response in the brain.

[0301] FIG. 15G shows the average CSF concentration of anti-BACE1 antibodies with wild-type or modified hIgG1 Fc. Anti-BACE1 antibodies with hIgG1 Fc, including YY and YQAY modifications, showed higher CSF concentrations compared to anti-BACE1 antibodies with hIgG1 wild-type Fc at various time points. FIG. 15H shows the ratio of the concentration of anti-BACE1 antibodies in CSF to the concentration of anti-BACE1 antibodies in serum. Modified Fc, YY, YQAY, YLYI and YIY, all resulted in an increased proportion of anti-BACE1 antibodies in CSF. As shown in FIG. 151 and FIG. 15J, anti-BACE1 antibodies with modified Fc were cleared from serum more quickly than anti-BACE1 hIgG1 wild-type antibodies. Example 8 - Evaluation of anti-Abeta antibody target binding in PS2APP transgenic mice

[0302] As discussed in Example 2, modified Fc containing M252Y / S254T / T256E (YTE) improved binding to mFcRn at both pH 7.4 and pH 6.0, but hFcRn only at pH 6.0. In wild-type mice, anti-BACE1-hIgG1-YTE demonstrates improved brain uptake compared to anti-BACE1-hIgG1. To determine whether improved brain uptake is observed with antibodies that bind to other brain antigens, anti-Abeta-hIgG4-YTE antibodies were administered to PS2APP mice co-expressing human APP (hAPP) with the Swedish mutation K670N / M671L and human presenilin 2 with the N141I mutation, driven by the Thy1 and PrP promoters, respectively. 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 therefore target binding of anti-Abeta antibodies can be assessed. In general, after administration of anti-Abeta antibodies, antibody binding is observed along the periphery of amyloid plaques and within the mossy fiber hippocampal tract. This specific staining pattern is not observed with an isotype-matched control antibody (data not shown; see, for example, 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).

[0303] In vivo dosing. Transgenic PS2APP or non-transgenic (Ntg) littermates were randomized into treatment groups and received a single intravenous (iv) dose of anti-Abeta hIgG4 or anti-Abeta hIgG4-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 dosing, animals were sacrificed and terminal plasma was collected by cardiac puncture followed by perfusion with phosphate-buffered saline (PBS); the right hemibrain was removed and dropwise fixed in 4% paraformaldehyde. The hippocampus, cortex and cerebellum were dissected from the left hemibrain, weighed and stored at -80°C.

[0304] Immunohistochemistry. Right hemibrains were drop-fixed in 4% paraformaldehyde for 48 h and then transferred to 30% sucrose in PBS. Free-floating sagittal cryosections (35 μm) of mouse brains were washed in PBS, then PBS-Triton X100 (PBST, 0.1%), then blocked with PBST (0.3%) containing 5% bovine serum albumin (BSA), and incubated overnight at 4 °C with primary antibodies diluted in 1% BSA in PBST (0.3%). Localization of administered human antibodies was performed using goat anti-human IgG-Alexa594 (or Alexa555, 1:100–1:500; Thermo-Fisher, Waltham, MA). Plaques were detected using the Ap fluorescent marker methoxy-X04.

[0305] Fluorescence microscopy. Whole slide images are captured at 20x using a Pannoramic 250 (3D Histech, Hungary) equipped with a PCO. Edge camera (Kelheim, Germany), Lumencor Spectra X (Beaverton, OR) and Semrock filters (Rochester, NY). The ideal exposure for each channel is determined based on the brightest intensity specimen and set to run the entire set of slides as a batch. Images were also captured at 20x using a Leica DM5500B light microscope using Leica Application Suite Advanced Florescence software (LAS AF4.0). Confocal images were taken using a 20x or 40x oil immersion objective on a Zeiss LSM800 confocal laser scanning microscope using Zen2.3 software. Quantification of mossy fiber staining was performed by measuring integrated density from 2–4 sections per animal using ImageJ (NIH).

[0306] In vivo antibody pharmacokinetic (PK) measurements. Cerebellar samples were weighed and homogenized in 300 μl of 1% NP-40 (containing Roche Complete ETDA-free protease inhibitor cocktail) using a Qiagen TissueLyser II (2 × 3 min at 30 Hz). Samples were then placed on ice for 20 min and then centrifuged at 20,000 × g for 20 min. Supernatants were 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 loaded with sheep anti-human IgG F(ab') 2The plates were coated overnight at 4°C with Fc fragment-specific polyclonal antibodies (Jackson ImmunoResearch, West Grove, PA, USA). The plates were then blocked with PBS containing 0.5% BSA for 1 hour at room temperature. Each antibody (anti-Abeta hIgG4 or anti-Abeta hIgG4-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, 0.35 M sodium chloride (NaCl), 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 5 mM EDTA, 0.05% Tween 20 and 15 ppm Proclin were incubated on the plates with gentle agitation for 2 hours at room temperature. Bound antibodies were detected with horseradish peroxidase-conjugated F(ab')2 goat anti-human IgG, Fc fragment-specific polyclonal antibody (Jackson ImmunoResearch). Finally, the plates were developed using the substrate 3,3',5,5'-tetramethylbenzidine (KPL, Inc., Gaithersburg, MD, USA). Absorbance was measured at a wavelength of 450 nm with a reference at 630 nm in a Multiskan Ascent reader (Thermo Scientific, Hudson, NH, USA). Concentrations were determined from a 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.

[0307] Results. As shown in Figures 16A-B, a dose-dependent increase in plasma and brain PK was observed for anti-Abeta hIgG4, while anti-Abeta hIgG4-YTE showed lower serum exposure and enhanced brain uptake at all doses. The brain:plasma ratio for anti-Abeta hIgG4 was approximately 0.15% and for anti-Abeta hIgG4 YTE was approximately 0.75%.

[0308] Figure 16C shows in vivo target binding as measured by antibody binding to the mossy fiber hippocampal tract. Anti-Abeta hIgG4 YTE shows significant binding after administration of 20 mg / kg, and increases in binding as dose increases. In contrast, anti-Abeta hIgG4 shows much less staining compared to anti-Abeta hIgG4 YTE after administration at 20 mg / kg and 40 mg / kg. Figure 16D is a bar graph showing staining levels from Figure 16C.

[0309] Figures 16E and 16F show binding of anti-Abeta hIgG4 and anti-Abeta hIgG4 YTE to the periphery of amyloid plaques in the subiculum (16E) and prefrontal cortex (16F) following administration at 20 mg / kg. The level of binding of anti-Abeta hIgG4 YTE to amyloid plaques in these brain regions is much greater than that observed for anti-Abeta hIgG4. Example 9 - Pharmacokinetics of highly transcytotic IgG4 variants in cynomolgus monkeys

[0310] Cynomolgus monkey PK study. Four 3- to 5-year-old male cynomolgus monkeys were used per experimental group. Anti-Abeta hIgG4 wild-type antibody, and anti-Abeta hIgG4 antibody with modified Fc (anti-Abeta-YQAY, YEY, YY) were administered at 50 mg / kg by intravenous bolus injection into the saphenous vein on day 0. The binding affinity of each antibody 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). TIFF2025037985000013.tif37170

[0311] CSF and blood samples were collected at various time points from 7 days pre-dose to 7 days post-dose. Samples were collected at the same time. At 2 and 7 days post-dose, the brains of two animals were harvested after total body perfusion. Brain regions were partially dissected and immediately frozen. Different brain regions were homogenized in 1% NP-40 (Cal-Biochem) in PBS containing Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics). Homogenized brain samples were rotated at 4°C for 1 hour and then spun at 14,000 rpm for 20 minutes. The supernatant was isolated for brain pharmacokinetic analysis.

[0312] Pharmacokinetic assay. Total antibody concentrations in monkey serum, CSF and brain samples were measured using an ELISA method using monkey-adsorbed sheep anti-human IgG polyclonal antibody (Binding Site) as coat and monkey-adsorbed goat anti-human IgG antibody conjugated to HRP (Bethyl) as detection. The assay had an MQC value of 1.6 ng / ml in CSF and brain.

[0313] Results. Figure 17A shows the mean brain concentrations of anti-Abeta antibodies on days 2 and 7. Figure 17B summarizes the fold improvement in brain concentration observed with each modified hIgG4 Fc. Anti-Abeta antibodies with modified Fc showed a 2.7-4.7-fold improvement in brain uptake compared to wild-type Fc on day 2, and a 3.7-4.2-fold improvement on day 7.

[0314] FIG. 17C shows the average CSF concentration of anti-Abeta antibodies with wild-type or modified hIgG4 Fc. Anti-Abeta antibodies with hIgG4 Fc containing YEY and YQAY modifications showed higher CSF concentrations compared to anti-Abeta antibodies with hIgG4 wild-type Fc. A slight improvement was also observed with Fc containing YY modification. FIG. 17D shows the ratio of the concentration of anti-Abeta antibodies in CSF to the concentration of anti-Abeta antibodies in serum. Modified Fc, YY, YEY and YQAY, all resulted in an increase in the percentage of anti-Abeta antibodies in CSF. As shown in FIG. 17E and FIG. 17F, anti-Abeta antibodies with modified Fc were cleared from serum more quickly than anti-Abeta hIgG4 wild-type antibodies.

[0315] FIG. 18A shows serum exposure and affinity of Fc for hFcRn at pH 7.4 in cynomolgus monkeys (K D (7.4)) are shown. Both anti-BACE1 hIgG1 (circles) and anti-Abeta IgG4 (squares) Fc engineered variants are shown in the plot. WT Fc is circled and labeled.

[0316] FIG. 18B shows antibody distribution to the brain of cynomolgus monkeys (% [mAb 脳 ] / [mAb 血清 ]) and the affinity of Fc for hFcRn at pH 7.4 (K D( 7.4) Correlation between Fc-engineered variants of both anti-BACE1 hIgG1 (circles) and anti-Abeta IgG4 (squares) are shown in the plot. WT Fc is circled and labeled.

[0317] 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 contexts. Improved CSF exposure was also observed, especially compared to serum exposure. Without intending to be bound by any particular theory, increased affinity for FcRn at pH 7.4 may increase the proportion of IgG that is transcytosed rather than recycled or degraded, resulting in improved 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 antibody that is internalized and transcytosed into the cell, resulting in improved brain and CSF exposure.

[0318] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties. References Abbott, N.J., L. Ronnback, and E. Hansson (2006). "Astrocyte-endothelial interactions at the blood-brain barrier." Nat Rev Neurosci 7(1):41-53. Abuqayyas, L. and JPBalthasar (2013). “Investigation of the role of FcgammaR and FcRn in mAb distribution to the brain.” Mol Pharm 10(5):1505~1513. Atwal, JK, Y. Chen, C. Chiu, DLMortensen, WJ Meilandt, Y. Liu, C. Heise, K. Hoyte, W. Luk, Y. Lu, K. Peng, and P. Wu 、L. Rouge, Y. Zhang, RALazarus, K. Scearce-Levie, W. Wang, Y. Wu, M. Tessier-Lavigne, and RJWatts(2011).A therapeutic antibody targeting BACE1 inhibits amyloid-beta production in vivo.」Sci Transl Med 3(84):84ra43. Borrok, MJ, Y. Wu, N. Beyaz, XQYu, V. Oganesyan, WF Dall'Acqua, P. Tsui(2015).pH-dependent binding engineering reveals an FcRn affinity threshold that governs IgG recycling.J Biol Chem 290(7):4282~4290. Challa,DK、R.Velmurugan、RJOberおよびE.Sally Ward(2014).「FcRn:from molecular interactions to regulation of IgG pharmacokinetics and functions.」Curr Top Microbiol Immunol 382:249~272. Chizzonite,R.、T.Truitt、FJPodlaski、AGWolitzky、PMQuinn、P.Nunes、ASSternおよびMKGately(1991).「IL-12:monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts.」J Immunol 147(5):1548~1556. Cooper, PR, GJCiambrone, CMKliwinski, E. Maze, L. Johnson, Q. Li, Y. Feng, and PJ Hornby (2013). 1534:13~2 Couch, JA, YJYu, Y. Zhang, JMTarrant, RNFuji, WJ Meilandt, H. Solanoy, RKTong, K. Hoyte, W. Luk, Y. Lu, K. Gadkar, S. Prabh u、BAOrdonia、Q.Nguyen、Y.Lin、Z.Lin、M.Balazs、K.Scearce-Levie、JAErnst、MSDennisおよびRJWatts(2013).「Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.」Sci Transl Med 5(183):183ral57、181~112. Dall'Acqua, WF, PAKiener, H. Wu (2006).J Biol Chem 281(33):23514~23524. Dall'Acqua,WF、RMWoods、ESWard、SRPalaszynski、NKPatel、YABrewah、H.Wu、PAKienerおよびS.Langermann(2002).「Increasing the affinity of a human IgG1 for the neonatal Fc receptor:biological consequences.」J Immunol 169(9):5171~5180. Datta-Mannan,A.、DRWitcher、Y.Tang、J.WatkinsおよびVJWroblewski(2007).「Impact of modulating the interaction of IgG with the neonatal Fc receptor.」J Biol Chem 282(3):1709~1717. Eaton, DL, WIWood, D. Eaton, PEHass, P. Hollingshead, K. Wion, J. Mather, RMLawn, GVehar, and C. Gorman (1986). molecule.」Biochemistry 25(26):8343~8347. Fishman,JB、JBRubin、JVHandrahan、JRConnorおよびREFine(1987).「Receptor-mediated transcytosis of transferrin across the blood-brain barrier.」J Neurosci Res 18(2):299~304. Fowler,DM, CLAraya, SJFleishman, EHKellogg, JJStephany, D.Baker, and S.Fields(2010).High-resolution mapping of protein sequence-function relationships.Nat Methods 7(9):741~746 Ghetie,V.およびESWard(2002).「Transcytosis and catabolism of antibody.」Immunol Res 25(2):97~113. Ghosh, AK, S. Gemma, J. Tang (2008). Neurotherapeutics 5(3):399~408. Hardy,J.およびDJSelkoe(2002).. Science 297(5580):353~356. Hinton,PR, MGJohlfs, JMXiong, K. Hanestad, KCOng, C. Bullock, S. Keller, MTTang, JYTso, M. Vasquez, N. Tsurushita (2004). Engineered human IgG antibodies with longer serum half-lives in primates.J Biol Chem 279(8):6213~6216. Hinton,PR、JMXiong、MGJohlfs、MTTang、S.KellerおよびN.Tsurushita(2006).「An engineered human IgG1 antibody with longer serum half-life.」J Immunol 176(1):346~356. Igawa, T., A. Maeda, K. Haraya, T. Tachibana, Y. Iwayanagi, F. Mimoto, Y. Higuchi, S. Ishii, and S. Igawa. Tamba、N.Hironiwa、K.Nagano、T.Wakabayashi、H.TsunodaおよびK.Hattori(2013).「Engineered monoclonal antibody with novel antigen-sweeping activity in vivo.」PLoS One 8(5):e63236. Kuo,T.T.およびV.G.Aveson(2011).「Neonatal Fc receptor and IgG-based therapeutics.」MAbs 3(5):422~430. Lo,M.、H.S.Kim、R.K.Tong、T.W.Bainbridge、J.M.Vernes、Y.Zhang、Y.L.Lin、S.Chung、M.S.Dennis、Y.J.Zuchero、R.J.Watts、J.A.Couch、Y.G.Meng、J.K.Atwal、R.J.Brezski、C.SpiessおよびJ.A.Ernst(2017).「Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice.」J Biol Chem 292(9):3900~3908. Mandelkow,E.M.およびE.Mandelkow(2012).「Biochemistry and cell biology of tau protein in neurofibrillary degeneration.」Cold Spring Harb Perspect Med 2(7):a006247. Morgan,M.、S.Anders、M.Lawrence、P.Aboyoun、H.PagesおよびR.Gentleman(2009).「ShortRead:a bioconductor package for input,quality assessment and exploration of high-throughput sequence data.」Bioinformatics 25(19):2607~2608. Pardridge,W.M.(2016).「CSF,blood-brain barrier,and brain drug delivery.」Expert Opinion on Drug Delivery 13(7):963~975. Pardridge,W.M.(2016).「Re-engineering therapeutic antibodies for Alzheimer’s disease as blood-brain barrier penetrating bi-specific antibodies.」Expert Opinion on Biological Therapy 16(12):1455~1468. Petkova,S.B.、S.Akilesh、T.J.Sproule、G.J.Christianson、H.Al Khabbaz、A.C.Brown、L.G.Presta、Y.G.MengおよびD.C.Roopenian(2006).「Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model:potential application in hum orally mediated autoimmune disease.」Int Immunol 18(12):1759~1769. Ponka,P.およびC.N.Lok(1999).「The transferrin receptor:role in health and disease.」Int J Biochem Cell Biol 31(10):1111~1137. Roopenian,D.C.およびS.Akilesh(2007).「FcRn:the neonatal Fc receptor comes of age.」Nat Rev Immunol 7(9):715~725. Spillantini,M.G.、M.L.Schmidt、V.M.Lee、J.Q.Trojanowski、R.JakesおよびM.Goedert(1997).「Alpha-synuclein in Lewy bodies.」Nature 388(6645):839~840. Thinakaran,G.およびEHKoo(2008).「Amyloid precursor protein trafficking,processing,and function.」J Biol Chem 283(44):29615~29619. Vaccaro,C.、J.Zhou、RJOberおよびESWard(2005).「Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels.」Nat Biotechnol 23(10):1283~1288. Wong,AW、TKBaginskiおよびDEReilly(2010).「Enhancement of DNA uptake in FUT8-deleted CHO cells for transient production of afucosylated antibodies.」Biotechnol Bioeng 106(5):751~763. Yeung, YA, MKLeabman, JSMarvin, J. Qiu, CWAdams, S. Lien, MAStarovasnik, HBLowman(2009). primates.」J Immunol 182(12):7663~7671. Yu, YJ, JKAtwal, Y. Zhang, R. Tong, K. R. Wildsmith, C. Tan, N. Bien-Ly, M. Hersom, J. Maloney, W. J. Meilandt, and D. Bumbaca. K. Gadkar, K. Hoyte, W. Luk, Y. Lu, JAErnst, K. Scearce-Levie, JACouch, MSDennis, and RJWatts(2014).Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.」Sci Transl Med 6(261):261ra154. Yu, YJ, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, JMElliott, S. Prabhu, RJ Watts, and MSDennis(2011). reducing its affinity for a transcytosis target.」Sci Transl Med 3(84):84ra44. Zalevsky,J.、AKChamberlain、HMHorton、S.Karki、IWLeung、TJSproule、GALazar、DCRoopenianおよびJRDesjarlais(2010).「Enhanced antibody half-life improves in vivo activity.」Nat Biotechnol 28(2):157~159. [English language] TIFF2025037985000014.tif118170

Claims

1. 11. A method of treating a neurological disorder, 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.

2. 2. The method of claim 1, wherein the neurological disorder is selected from a neuropathy disorder, a neurodegenerative disease, a brain disorder, a cancer, an eye disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and a CNS inflammation.

3. The method of claim 1 , wherein the neurological disorder is a neurodegenerative disease.

4. 4. The method of claim 3, wherein the neurodegenerative disease is selected from Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, amyloidosis, Parkinson's disease, multiple system atrophy, striatonigral degeneration, amyloidosis, tauopathy, Alzheimer's disease, supranuclear palsy, prion disease, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia.

5. 11. A method of delivering an antibody to the brain of a subject, comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein said antibody is active in an in vitro transcytosis assay.

6. 11. A method of increasing brain exposure to an antibody, comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein said antibody is active in an in vitro transcytosis assay.

7. 1. A method for increasing transport of an antibody across the blood-brain barrier (BBB), 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.

8. 8. The method of claim 1, wherein the antibody exhibits a transcytosis activity in an in vitro transcytosis assay of at least 50 when normalized to the same antibody containing wild-type IgG Fc.

9. The method of claim 8, wherein the antibody exhibits 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.

10. The method of claim 1 , wherein the in vitro transcytosis assay comprises cells expressing FcRn.

11. The method of claim 10, wherein the cell is an MDCK II cell.

12. 12. The method of any one of claims 1 to 11, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference antibody having an unmodified IgG Fc of the same species and isotype.

13. 13. The method of any one of claims 1 to 12, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference antibody having an unmodified IgG Fc of the same species and isotype.

14. 14. The method of any one of claims 1 to 13, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 7.4 of 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less.

15. 15. The method of any one of claims 1 to 14, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less.

16. 16. The method of any one of claims 1 to 15, wherein the ratio of the affinity of the antibody comprising the modified IgG Fc for FcRn at pH 7.4 to the affinity of the antibody comprising the 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 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

17. 17. The method of any one of claims 1 to 16, wherein the antibody comprising the modified IgG Fc comprises one or more mutations selected from 252W, 252Y, 286E, 286Q, 307Q, 308P, 310A, 311A, 311I, 428L, 433K, 434F, 434W, 434Y and 436I according to EU numbering.

18. The method of claim 17, wherein the modified IgG Fc comprises 252Y and 434Y.

19. 19. The method of claim 18, wherein the modified IgG Fc comprises 252Y and 434Y, and one or two additional mutations selected from 286E, 286Q, 307Q, 308P, 311A, 311I, 428L, 433K and 436I.

20. 19. The method of claim 18, wherein the modified IgG Fc further comprises 307Q and 311A, or further comprises 286E.

21. 21. The method of any one of claims 1 to 20, wherein the modified IgG Fc comprises a set of mutations selected from the sets of mutations in Tables 4, 5 and 6.

22. 22. The method of any one of claims 1 to 21, wherein the IgG Fc is an IgG1 Fc.

23. 22. The method of any one of claims 1 to 21, wherein the IgG Fc is an IgG4 Fc.

24. The method of any one of claims 1 to 23, wherein the antibody binds to a brain antigen.

25. The antibody is selected from the group consisting of beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-11), interleukin-11 (IL-12), interleukin-12 (IL-13), interleukin-13 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-18), interleukin-18 (IL-19), interleukin-20 (IL-19), interleukin-21 (IL-19), interleukin-22 (IL-19), interleukin-23 (IL-19), interleukin-24 (IL-19), interleukin-25 (IL-19), interleukin-36 (IL-19), interleukin-17 (IL-19), interleukin-18 (IL-19), interleukin-19 (IL-19), interleukin-19 (IL-19), interleukin-2 25. The method of claim 24, wherein the antigen binds to a brain antigen selected from leukin 6 receptor (IL6R), interleukin 1 beta (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).

26. 26. The method of any one of claims 1 to 25, wherein the antibody is a monoclonal antibody.

27. 27. The method of any one of claims 1 to 26, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

28. 28. The method of any one of claims 1 to 27, wherein the antibody is a bispecific antibody.

29. 29. The method of any one of claims 1 to 28, wherein the antibody is an antibody fragment.

30. 30. The method of any one of claims 1 to 29, wherein the modified IgG Fc comprises one or more modifications of a sequence selected from SEQ ID NOs: 1-4.

31. 31. The method of any one of claims 1 to 30, wherein the antibody is conjugated to an imaging agent.

32. 32. The method of any one of claims 1 to 31, wherein the antibody is conjugated to a neuropathic drug.

33. 33. The method of claim 32, wherein the neuropathic drug is selected from an aptamer, an inhibitory nucleic acid, a ribozyme, and a small molecule.

34. 1. A method of treating a neurological disorder, comprising administering to a subject in need thereof an Fc conjugate comprising a modified IgG Fc, wherein said Fc conjugate is active in an in vitro transcytosis assay.

35. 35. The method of claim 34, wherein the neurological disorder is selected from a neuropathy disorder, a neurodegenerative disease, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and a CNS inflammation.

36. 36. The method of claim 35, wherein the neurological disorder is a neurodegenerative disease.

37. 37. The method of claim 36, wherein 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, tauopathy, Alzheimer's disease, supranuclear palsy, prion disease, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia.

38. 16. A method for delivering an Fc conjugate to the brain of a subject, comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein said Fc conjugate is active in an in vitro transcytosis assay.

39. 1. A method for increasing brain exposure to an Fc conjugate, comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein said Fc conjugate is active in an in vitro transcytosis assay.

40. 1. A method for increasing transport of an Fc conjugate across the blood-brain barrier (BBB), comprising administering to a subject in need thereof an antibody comprising a modified IgG Fc, wherein said Fc conjugate is active in an in vitro transcytosis assay.

41. 41. The method of any one of claims 34 to 40, wherein the Fc conjugate exhibits a transcytosis activity in an in vitro transcytosis assay of at least 50 when normalized to the same Fc conjugate comprising a wild-type IgG Fc.

42. 42. The method of claim 41, wherein the Fc conjugate exhibits 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.

43. 43. The method of any one of claims 34 to 42, wherein the in vitro transcytosis assay comprises cells expressing FcRn.

44. 44. The method of claim 43, wherein the cell is an MDCK II cell.

45. 45. The method of any one of claims 34 to 44, wherein the Fc conjugate comprising the 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 having an unmodified IgG Fc of the same species and isotype.

46. 46. ​​The method of any one of claims 34 to 45, wherein the Fc conjugate comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference Fc conjugate having an unmodified IgG Fc of the same species and isotype.

47. 47. The method of any one of claims 34 to 46, wherein the Fc conjugate comprising the modified IgG Fc has a binding affinity for FcRn at pH 7.4 of 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.

48. 48. The method of any one of claims 34 to 47, wherein the Fc conjugate comprising the 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, or between 10 nM and 100 nM.

49. 49. The method of any one of claims 34 to 48, wherein the ratio of the affinity of the Fc conjugate comprising the modified IgG Fc for FcRn at pH 7.4 to the affinity of the Fc conjugate comprising the 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 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

50. 50. The method of any one of claims 45 to 49, wherein the modified IgG Fc comprises one or more mutations selected from 252W, 252Y, 286E, 286Q, 307Q, 308P, 310A, 311A, 311I, 428L, 433K, 434F, 434W, 434Y and 436I according to EU numbering.

51. 50. The method of claim 49, wherein the modified IgG Fc comprises 252Y and 434Y.

52. 52. The method of claim 51 , wherein the modified IgG Fc comprises 252Y and 434Y, and one or two additional mutations selected from 286E, 286Q, 307Q, 308P, 311A, 311I, 428L, 433K and 436I.

53. 52. The method of claim 51 , wherein the modified IgG Fc further comprises 307Q and 311A, or further comprises 286E.

54. 54. The method of any one of claims 34 to 53, wherein the modified IgG Fc comprises a set of mutations selected from the sets of mutations in Tables 4, 5 and 6.

55. 55. The method of any one of claims 34 to 54, wherein the IgG Fc is an IgG1 Fc.

56. 55. The method of any one of claims 34 to 54, wherein the IgG Fc is an IgG4 Fc.

57. 57. The method of any one of claims 34 to 56, wherein the Fc conjugate comprises the modified IgG Fc fused to a therapeutic protein.

58. 58. The method of claim 57, wherein the therapeutic protein is selected from a receptor extracellular domain and an enzyme.

59. 58. The method of claim 57, wherein the receptor extracellular domain is selected from a TNF-R1 extracellular domain (ECD), a CTLA-4 ECD, and an IL-1R1 ECD.

60. 58. The method of claim 57, wherein the enzyme is selected from alpha-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, acid alpha-glucosidase, glucocerebrosidase, alpha-galactosidase A, hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase, beta-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase, palmitoyl-protein thioesterase 1, and tripeptidyl aminopeptidase 1.

61. 57. The method of any one of claims 34 to 56, wherein the Fc conjugate comprises the modified IgG Fc conjugated to a neuropathic drug.

62. 60. The method of claim 59, wherein the neuropathic drug is selected from an aptamer, an inhibitory nucleic acid, a ribozyme, and a small molecule.

63. 57. The method of any one of claims 34 to 56, wherein the Fc conjugate comprises the modified IgG Fc conjugated to an imaging agent.

64. An isolated antibody that binds to a brain antigen, the antibody comprising a modified IgG Fc and being active in an in vitro transcytosis assay.

65. 65. The isolated antibody of claim 64, which exhibits a transcytosis activity in an in vitro transcytosis assay of at least 50 when normalized to the same antibody comprising a wild-type IgG Fc.

66. 66. The isolated antibody of claim 65, which exhibits 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.

67. 67. The isolated antibody of any one of claims 64 to 66, wherein the in vitro transcytosis assay comprises cells expressing FcRn.

68. 68. The isolated antibody of claim 67, wherein the cell is a MDCK II cell.

69. 69. The isolated antibody of any one of claims 64-68, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 7.4 that is greater than the binding affinity of a reference antibody having an unmodified IgG Fc of the same species and isotype.

70. 70. The isolated antibody of any one of claims 64-69, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference antibody having an unmodified IgG Fc of the same species and isotype.

71. 71. The isolated antibody of any one of claims 64-70, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 7.4 of 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less.

72. 72. The isolated antibody of any one of claims 64-71, wherein the antibody comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less.

73. 73. The isolated antibody of any one of claims 64-72, wherein the ratio of the affinity of the antibody comprising said modified IgG Fc for FcRn at pH 7.4 to the affinity of the antibody comprising said 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 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

74. 74. The isolated antibody of any one of claims 64-73, wherein the antibody comprising the modified IgG Fc comprises one or more mutations selected from 252W, 252Y, 286E, 286Q, 307Q, 308P, 310A, 311A, 311I, 428L, 433K, 434F, 434W, 434Y and 436I, according to EU numbering.

75. 75. The isolated antibody of claim 74, wherein the modified IgG Fc comprises 252Y and 434Y.

76. 76. The isolated antibody of claim 75, wherein the modified IgG Fc comprises 252Y and 434Y, and one or two additional mutations selected from 286E, 286Q, 307Q, 308P, 311A, 311I, 428L, 433K and 436I.

77. 76. The isolated antibody of claim 75, wherein the modified IgG Fc further comprises 307Q and 311A, or further comprises 286E.

78. 78. The isolated antibody of any one of claims 64 to 77, wherein the modified IgG Fc comprises a set of mutations selected from the sets of mutations in Tables 4, 5 and 6.

79. 79. The isolated antibody of any one of claims 64 to 78, wherein the IgG Fc is an IgG1 Fc.

80. 79. The isolated antibody of any one of claims 64 to 78, wherein the IgG Fc is an IgG4 Fc.

81. The brain antigen is beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-9), interleukin-11 (IL-9), interleukin-12 (IL-9), interleukin-13 (IL-9), interleukin-14 (IL-9), interleukin-15 (IL-9), interleukin-16 (IL-9), interleukin-17 (IL-9), interleukin-18 (IL-9), interleukin-19 (IL-19), interleukin-20 (IL-19), interleukin-21 (IL-19), interleukin-22 (IL-19), interleukin-23 (IL-19), interleukin-24 (IL-19), interleukin-25 (IL-19), interleukin-26 (IL-19), interleukin-37 (IL-19), interleukin-38 (IL-19), interleukin-39 (IL-19), interleukin-39 81. The isolated antibody of any one of claims 64 to 80, wherein the antibody is selected from leukin 6 receptor (IL6R), interleukin 1 beta (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).

82. 82. The isolated antibody of any one of claims 64 to 81, which is a monoclonal antibody.

83. 83. The isolated antibody of any one of claims 65 to 82, which is a human antibody, a humanized antibody, or a chimeric antibody.

84. 84. The isolated antibody of any one of claims 64 to 83, which is a bispecific antibody.

85. 85. The isolated antibody of any one of claims 64 to 84, which is an antibody fragment.

86. 86. The isolated antibody of any one of claims 64-85, wherein the modified IgG Fc comprises one or more modifications of a sequence selected from SEQ ID NOs: 1-4.

87. 87. The isolated antibody of any one of claims 64 to 86, conjugated to an imaging agent.

88. 88. The isolated antibody of any one of claims 64 to 87, conjugated to a neuropathic drug.

89. 89. The isolated antibody of claim 88, wherein the neuropathic drug is selected from an aptamer, an inhibitory nucleic acid, a ribozyme, and a small molecule.

90. An Fc conjugate comprising a modified IgG Fc, the Fc conjugate being active in an in vitro transcytosis assay.

91. 91. The Fc conjugate of claim 90, which exhibits a transcytosis activity in an in vitro transcytosis assay of at least 50 when normalized to the same Fc conjugate comprising a wild-type IgG Fc.

92. 92. The Fc conjugate of claim 91, exhibiting 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.

93. 93. The Fc conjugate of any one of claims 90 to 92, wherein the in vitro transcytosis assay comprises cells expressing FcRn.

94. 94. The Fc conjugate of claim 93, wherein the cell is an MDCK II cell.

95. 95. The Fc conjugate of any one of claims 90 to 94, wherein the Fc conjugate comprising the 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 having an unmodified IgG Fc of the same species and isotype.

96. 96. The Fc conjugate of any one of claims 90 to 95, wherein the Fc conjugate comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 that is greater than the binding affinity of a reference Fc conjugate having an unmodified IgG Fc of the same species and isotype.

97. 97. The Fc conjugate of any one of claims 90 to 96, wherein the Fc conjugate comprising the modified IgG Fc has a binding affinity for FcRn at pH 7.4 of 10 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less.

98. 98. The Fc conjugate of any one of claims 90 to 97, wherein the Fc conjugate comprising the modified IgG Fc has a binding affinity for FcRn at pH 6 of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less.

99. 99. The Fc conjugate of any one of claims 90 to 98, wherein the ratio of the affinity of said Fc conjugate comprising said modified IgG Fc for FcRn at pH 7.4 to the affinity of said Fc conjugate comprising said 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 5-200, 5-100, 10-200, 10-100, 20-100, or 20-200.

100. 100. The Fc conjugate of any one of claims 90 to 99, wherein the Fc conjugate comprising the modified IgG Fc comprises one or more mutations selected from 252W, 252Y, 286E, 286Q, 307Q, 308P, 310A, 311A, 311I, 428L, 433K, 434F, 434W, 434Y and 436I according to EU numbering.

101. The Fc conjugate of claim 100, wherein the modified IgG Fc comprises 252Y and 434Y.

102. 102. The Fc conjugate of claim 101, wherein said modified IgG Fc comprises 252Y and 434Y, and one or two additional mutations selected from 286E, 286Q, 307Q, 308P, 311A, 311I, 428L, 433K and 436I.

103. 102. The Fc conjugate of claim 101, wherein said modified IgG Fc further comprises 307Q and 311A, or further comprises 286E.

104. 104. The Fc conjugate of any one of claims 90 to 103, wherein the modified IgG Fc comprises a set of mutations selected from the sets of mutations in Tables 4, 5 and 6.

105. 105. The Fc conjugate of any one of claims 90 to 104, wherein the IgG Fc is an IgG1 Fc.

106. 105. The Fc conjugate of any one of claims 90 to 104, wherein the IgG Fc is an IgG4 Fc.

107. 107. The Fc conjugate of any one of claims 90 to 106, comprising the modified IgG Fc fused to a therapeutic protein.

108. The Fc conjugate of claim 107, wherein the therapeutic protein is selected from a receptor extracellular domain and an enzyme.

109. 108. The Fc conjugate of claim 107, wherein said receptor extracellular domain is selected from the TNF-R1 extracellular domain (ECD), CTLA-4 ECD, and IL-1R1 ECD.

110. 108. The Fc conjugate of claim 107, wherein the enzyme is selected from alpha-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, acid alpha-glucosidase, glucocerebrosidase, alpha-galactosidase A, hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase, beta-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase, palmitoyl-protein thioesterase 1 and tripeptidyl aminopeptidase 1.

111. 111. The Fc conjugate of any one of claims 90 to 110, wherein said modified IgG Fc comprises one or more modifications of a sequence selected from SEQ ID NOs: 1 to 4.

112. 112. The Fc conjugate of any one of claims 90 to 111, conjugated to an imaging agent.

113. 112. The Fc conjugate of any one of claims 90 to 111, conjugated to a neuropathic drug.

114. The Fc conjugate of claim 113, wherein the neuropathic drug is selected from an aptamer, an inhibitory nucleic acid, a ribozyme, and a small molecule.

Images