Dosing regimens for treatment of synucleinopathies

Abstract

To provide dosage regimens for treatment of synucleinopathies.SOLUTION: In one regimen, a subject receives 3000-5000 mg of an antibody intravenously every 3-5 weeks. In another regimen, a subject receives 1300-1700 mg of an antibody intravenously every 3-5 weeks.SELECTED DRAWING: None

Description

Technical Field

[0001] Sequence Listing A Sequence Listing containing SEQ ID NOs: 1 to 151 is attached and is hereby incorporated by reference in its entirety. The list, named 503725SEQLIST.txt, was created on Sep. 27, 2017 in ASCII format and is 127,151 bytes in size.

Background Art

[0002] Lewy body disease (LBD) is characterized by degeneration of the dopaminergic system, movement changes, cognitive impairment, and the formation of Lewy bodies (LBs) and / or Lewy neurites [McKeith et al., Neurology (1996) 47:1113-24]. Lewy body disease includes Parkinson's disease (including idiopathic Parkinson's disease), and dementia with Lewy bodies (DLB). Lewy body disease is a common cause of movement disorders and cognitive decline in the aging population [Galasko et al., Arch. Neurol. (1994) 51:888-95]. Constipation is another common symptom in subjects with Lewy body disease [Ondo et al., Neurology 2012;78;1650-1654; Ashraf et al., Movement Disorders 12, 946-951 (1997)].

[0003] Alpha-synuclein is a protein normally associated with synapses and is thought to play a role in neuroplasticity, learning, and memory. Alpha-synuclein is also associated with a central role in the pathogenesis of Lewy body disease. The protein can aggregate in the diseased state to form insoluble fibrils. For example, alpha-synuclein ​​​​​​​​​​​​ Accumulation in LB [Spillantini et al., Nature (1997) 388:839-40; Takeda et al ., J. Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett. (1997) 239: 45-8]. Mutations in the alpha-synuclein gene co-segregate with rare familial forms of parkinsonism [Kruger et al., Nature Gen. (1998) 18:106-8; Polymeropo ulos, et al., Science (1997) 276:2045-7]. Overexpression of alpha-synuclein in transgenic mice [Masliah et al., Science (2000) 287:1265-9] and Drosophila [Feany et al., Nature (20 00) 404:394-8] mimics several pathological aspects of Lewy body disease. Soluble oligomers of alpha-synuclein can be neurotoxic [Conway et al., Proc Natl Acad Sci USA (2000) 97:571-576]. Similar morphological and neuro logical changes in alpha-synuclein accumulation in diverse species and animal models such as humans, mice, and flies suggest that this molecule contributes to the development of Lewy body disease.

[0004] Immunotherapy targeting alpha-synuclein has been reported to reduce alpha-synuclein deposition and behavioral symptoms in a mouse model of Lewy body disease [Ma sliah et al., PLoS ONE 6(4): e19338. doi:10.1371 / journal.pone.0019338]. ​

Summary of the Invention

[0005] In one aspect, the present invention is a method of performing treatment or prevention of a subject having or at risk of having synucleinopathy, comprising intravenously administering to the subject an antibody against alpha-synuclein at a dose of 3000-5000 mg at intervals of 3-5 weeks, or administering to the subject another regimen that delivers the antibody with substantially the same area under the curve. Some such methods have a dose of 3500-4500 mg. In some such methods, the antibody is administered to a plurality of subjects having or at risk of having synucleinopathy, and the subjects receive one or two fixed doses. In some such methods, subjects having a body weight of less than 65 kg receive a dose of 3500 mg of the antibody, and subjects having a body weight of 65 kg or more receive a dose of 4500 mg. In some such methods, the dose is 45-75 mg / kg. In some such methods, the dose is 50-70 mg / kg. In some such methods, the interval is 28 days. In some such methods, the subject receives at least 6 doses of the antibody at intervals of 3-5 weeks. In some such methods, the subject receives at least 12 doses of the antibody at intervals of 3-5 weeks. In some such methods, the subject receives at least 18 doses of the antibody at intervals of 3-5 weeks. In some such methods, the interval is 4 weeks. In some such methods, the subject receives the antibody every 4 weeks for at least 52 weeks.

[0006]

[0007]

[0008]

[0009] ​​​​​​​​​​​​ Some such methods further include monitoring the subject for changes in movement, cognitive impairment, autonomic dysfunction, gastrointestinal dysfunction, visual hallucinations or psychological symptoms.

[0010] In some such methods, monitoring comprises (a) providing the subject with a mobile device programmed to receive and transmit data from internal and / or external sensors of the mobile device regarding movement disorders of the subject having or suspected of having synucleinopathy such that the subject then performs a series of movements to reveal a movement disorder, if any, and internal or external sensors of the device obtain data regarding the movement (b) collecting data transmitted from the mobile device, and (c) comparing the data obtained from the subject to control data to assess the presence or degree of movement disorder in the subject. (b) collecting data transmitted from the mobile device, and (c) comparing the data obtained from the subject to control data to assess the presence or degree of movement disorder in the subject. In some such methods, the mobile device is programmed to receive and transmit data from at least two external sensors attached to the upper and lower limbs of the subject.

[0011] In some such methods, the mobile device obtains data from sensors on the upper and lower limbs of the subject. In some such methods, the mobile device is carried by the subject and obtains data from internal sensors. In some such methods, the series of movements includes tapping, sitting, and standing of the device. In some such methods, monitoring indicates a reduction in movement disorder or cognitive impairment in response to administration of an antibody. In some such methods, the series of movements includes tapping, sitting, and standing of the device. In some such methods, monitoring indicates a reduction in movement disorder or cognitive impairment in response to administration of an antibody.

[0012] In some such methods, the antibody binds within residues 115 - 130 of alpha-synuclein. It binds.

[0013] In some such methods, the antibody binds within residues 118 - 126 of alpha-synuclein. It binds.

[0014] In some such methods, the antibody binds within residues 117 - 123 of alpha-synuclein. It binds. In some such methods, the antibody is NI-202.21D11. In some such methods, the antibody comprises three heavy chain CDRs specified by SEQ ID NOs: 139 - 141 and three light chain CDRs specified by SEQ ID NOs: 143 - 145 respectively. In one such method, the antibody comprises a heavy chain variable region specified by SEQ ID NO: 138 and a light chain variable region specified by SEQ ID NO: 142.

[0015] In some such methods, the antibody comprises three light chain Kabat CDRs of SEQ ID NO: 5 and three heavy chain Kabat CDRs of SEQ ID NO: 10. In some such methods, the antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 8 - 11 and a light chain variable region of any one of SEQ ID NOs: 3 - 5, preferably the heavy chain variable region is the heavy chain variable region of SEQ ID NO: 10 and the light chain variable region is the light chain variable region of SEQ ID NO: 5.

[0016] In some such methods, the antibody is an antibody of human IgG1 isotype. In some such methods, the antibody comprises a heavy chain constant region of SEQ ID NO: 35 where the C-terminal lysine may be absent and a light chain constant region of SEQ ID NO: 30. In some such methods ​​​​The antibody may not have the C-terminal lysine of SEQ ID NO: 35 and comprises the heavy chain constant region of SEQ ID NO: 35 and the light chain constant region of SEQ ID NO: 13.

[0017] In some such methods, the antibody is 9E4. In some such methods, the antibody comprises three heavy chain CDRs specified by SEQ ID NOs: 146-148 respectively and three light chain CDRs specified by SEQ ID NOs: 149-151 respectively. In some such methods, the anti- body comprises the heavy chain specified by SEQ ID NO: 37 and the light chain specified by SEQ ID NO: 32, and the C-terminal lysine of SEQ ID NO: 37 may not be present.

[0018] In some such methods, the antibody binds within residues 1-20 of alpha-synuclein. In some such methods, the antibody binds within residues 4-15 of alpha-synuclein.

[0019] In some such methods, the antibody is NI-202.12F4. In some such methods, the antibody comprises three heavy chain CDRs specified by SEQ ID NOs: 131-133 respectively and three light chain CDRs specified by SEQ ID NOs: 135-137 respectively. In some such methods, the antibody comprises the heavy chain variable region specified by SEQ ID NO: 130 and the light chain variable region specified by SEQ ID NO: 134.

[0020] In some such methods, the subject has a body weight of less than 65 kg and is administered the antibody at a dose of 3500 mg every 4 weeks.

[0021] In some such methods, before administering an antibody within the range of 3000-5000 mg, a loading dose of 2000 mg of the antibody is administered and until reaching a dose of 3500 mg, 2000 One or more subsequent doses that are above mg but less than 3500 mg may be titrated up gradually (u ptitration), and all doses are separated by intervals of 3 to 5 weeks. are.

[0022] In some such methods, the 3500 mg dose is administered at the next interval after the 2000 mg dose. In some such methods, the dose is gradually increased over multiple intervals prior to administration of the 3500 mg dose. In some such methods, the subject has a body weight greater than 65 kg and is administered an antibody at a dose of 4500 mg every 4 weeks.

[0023] In some such methods, prior to administering the antibody at 4500 mg, a loading dose of the antibody at 2000 mg is administered, and gradual titration may be performed using one or more subsequent doses that are above 2000 mg but less than 4 500 mg until the 4500 mg dose is reached, and all doses are separated by intervals of 3 to 5 weeks. are.

[0024] In some such methods, the 4500 mg dose is administered at the next interval after the 2000 mg dose. In some such methods, the dose is gradually increased over multiple intervals prior to administration of the 4500 mg dose. are.

[0025] Some such methods further include monitoring the subject for changes in movement, cognitive impairment, autonomic dysfunction, gastrointestinal dysfunction, visual hallucinations or psychological symptoms.

[0026] In some such methods, monitoring comprises (a) a subject having or suspected of having synucleinopathy Provided for mobile devices programmed to receive and transmit data obtained from internal and / or external sensors of the mobile device related to movement disorders, subsequently the subject performs a series of movements to reveal a movement disorder if there is one, wherein internal or external sensors of the device obtain data related to the movement, (b) collecting data transmitted from the mobile device, and (c) comparing the data obtained from the subject with reference data to assess the presence or degree of movement disorder in the subject. Including. Including.

[0027] In some such methods, the mobile device is programmed to receive and transmit data from at least two external sensors attached to the subject's upper and lower limbs, In some such methods, the mobile device obtains data from sensors on the subject's upper and lower limbs. In some such methods, the mobile device is carried by the subject and obtains data from internal sensors. In some such methods the series of movements includes tapping, sitting and standing of the device. In some such methods, monitoring indicates a reduction in movement disorder or cognitive impairment in response to administration of an antibody. In some such methods, the synucleinopathy is Parkinson's disease. In some such methods, the synucleinopathy is dementia with Lewy bodies. In some such methods the synucleinopathy is multiple system atrophy

[0028] In some such methods, the synucleinopathy is progressive supranuclear palsy. Some In some such methods, the synucleinopathy is dementia with Lewy bodies. In some such methods in which the synucleinopathy is multiple system atrophy (multiple system atrophy ). In some such methods, the synucleinopathy is progressive supranuclear palsy. Some In such a method, synucleinopathy is REM sleep behavior disorder. In some such methods, synucleinopathy is Alzheimer's disease with Lewy bodies in the tonsils. In some such methods, the subject has the disease.

[0029] In some such methods, the subject has not received symptomatic treatment for Parkinson's disease in combination with an antibody. In some such methods, the

[0030] In another aspect, the present invention provides a method of performing treatment or prevention of a subject having Lewy body disease, the method comprising administering to the subject an antibody against alpha-synuclein at a dose of 1300-1700 mg intravenously at intervals of 3-5 weeks, or

[0031] administering to the subject another regimen that delivers the antibody with substantially the same area under the curve. In some such methods, the dose is 1500 mg. In some such methods, the antibody is administered to a plurality of subjects having Lewy body disease,

[0032] and the subjects receive one or two fixed doses. In some such methods, the subjects receive a dose of 18-25 mg / kg. In some such methods, the subjects receive a dose of 20 mg / kg. In some such methods, the interval is 28 days. In some such methods, the The interval is 4 weeks. In some such methods, the subject receives the antibody every 4 weeks for at least 52 weeks. Receive.

[0033] Some such methods further include monitoring the subject for changes in movement, cognitive impairment, autonomic dysfunction, gastrointestinal dysfunction, visual hallucinations or psychological symptoms.

[0034] In some such methods, monitoring involves (a) providing the subject with a mobile device programmed to receive and transmit data from internal and / or external sensors regarding movement impairment of a subject having or suspected of having Lewy body disease such that the subject then performs a series of movements to reveal movement impairment, if any, and internal or external sensors of the device acquire data regarding the movement ; (b) collecting the data transmitted from the mobile device, and (c) comparing the data obtained from the subject with control data to assess the presence or degree of movement impairment in the subject. including.

[0035] In some such methods, the mobile device is programmed to receive and transmit data from at least two external sensors attached to the upper and lower limbs of the subject.

[0036] In some such methods, the mobile device acquires data from sensors on the upper and lower limbs of the subject. In some such methods, the mobile device is carried by the subject and acquires data from internal sensors. In some such methods, a series of Movements include tapping, sitting, and standing of the device. In some such methods, mo nitating refers to reducing movement disorders or reducing cognitive impairment in response to administering an antibody.

[0037] In some such methods, the antibody binds within residues 115 - 130 of alpha-synuclein.

[0038] In some such methods, the antibody binds within residues 118 - 126 of alpha-synuclein.

[0039] In some such methods, the antibody binds within residues 117 - 123 of alpha-synuclein.

[0040] In some such methods, the antibody is NI-202.21D11. In some such methods, the antibody comprises three heavy chain CDRs specified by SEQ ID NOs: 139 - 141 and three light chain CDRs specified by SEQ ID NOs: 143 - 145 respectively. In some such methods, the antibody comprises a heavy chain variable region specified by SEQ ID NO: 138 and a light chain variable region specified by SEQ ID NO: 14 2.

[0041] In some such methods, the antibody comprises three light chain Kabat CDRs of SEQ ID NO: 5 and three heavy chain Kabat CDRs of SEQ ID NO: 10. In some such methods, the anti body comprises a heavy chain variable region of any one of SEQ ID NOs: 8 - 11 and a light chain variable region of any one of SEQ ID NOs: 3 - 5, preferably the heavy chain variable region is the heavy chain variable region of SEQ ID NO: 10 and the light chain variable region is the light chain variable region of SEQ ID NO: 5.

[0042] ​​​​​​​In some such methods, the antibody is an antibody of the human IgG1 isotype.

[0043] In some such methods, the antibody comprises a heavy chain constant region of SEQ ID NO: 35 in which the C-terminal lysine may be absent, and a light chain constant region of SEQ ID NO: 30. In some such methods the antibody comprises a heavy chain constant region of SEQ ID NO: 35 in which the C-terminal lysine may be absent, and a light chain constant region of SEQ ID NO: 13.

[0044] In some such methods, the antibody comprises three heavy chain CDRs designated by SEQ ID NOs: 146 to 148 respectively and three light chain CDRs designated by SEQ ID NOs: 149 to 151 respectively. In some such methods, the antibody comprises a heavy chain designated by SEQ ID NO: 37 and a light chain designated by SEQ ID NO: 32, and the C-terminal lysine of SEQ ID NO: 37 may be absent.

[0045] In some such methods, the antibody binds within residues 1 to 20 of alpha-synuclein. In some such methods, the antibody binds within residues 4 to 15 of alpha-synuclein.

[0046] In some such methods, the antibody is NI-202.12F4. In some such methods, the antibody comprises three heavy chain CDRs designated by SEQ ID NOs: 131 to 133 respectively and three light chain CDRs designated by SEQ ID NOs: 135 to 137 respectively. In some such methods, the antibody comprises a heavy chain variable region designated by SEQ ID NO: 130 and a light chain variable region designated by SEQ ID NO: 134.

[0047] In some such methods, synucleinopathy is Parkinson's disease. In some such In some ways, synucleinopathy is dementia with Lewy bodies. In some of those ways synucleinopathy is multiple system atrophy. In some of those ways, synucleinopathy is progressive supranuclear palsy. In some of those ways, synucleinopathy is REM sleep behavior disorder. In some of those ways, synucleinopathy is Alzheimer's disease with Lewy bodies in the amygdala. In some of those ways, the subject has the disease.

[0048] In some of those ways, the subject has not received symptomatic treatment for Parkinson's disease in combination with an antibody. In some of those ways, the antibody is

[0049] DEFINITION The term "antibody" includes intact antibodies and their binding fragments. Typically, the fragments compete with the intact antibodies from which they are derived for specific binding to the target. Examples of fragments include isolated heavy chains, isolated light chains, Fab, Fab’, F(ab’)2, F(ab )c, Fv, single-chain antibodies, and single-domain antibodies. The term "antibody" also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy chain / light chain pairs and two different binding sites [see, for example, Songsi

[0050] The basic antibody structural unit is a tetramer of subunits. Each tetramer is a polypeptide It contains two identical pairs, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids that is mainly involved in antigen recognition. When first expressed, this variable region is typically linked to a cleavable signal peptide. A variable region without a signal peptide may be referred to as a mature variable region. Thus, for example, a light-chain mature variable region means a light-chain variable region without a light-chain signal peptide. The carboxy-terminal portion of each chain defines a constant region that is mainly involved in effector functions. The constant region can include any or all of the CH 1 region, hinge region, CH2 region, and CH3 region. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, defining the antibody isotypes as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are linked by a "J" region of about 12 or more amino acids, and the heavy chain also

[0051] contains a "D" region of about 10 or more amino acids. [Generally, see Fundamental Immunology ( Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7] (which is incorporated by reference in its entirety for all purposes). The mature variable regions of each light-chain / heavy-chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except for bispecific or dual - specificity antibodies, the two binding sites are identical. By reference, the entire reference is incorporated for all purposes).

[0052] sites are identical. Except for bispecific or dual - specificity antibodies, the two binding ​The locations are the same. All of the chains exhibit the same general structure of relatively conserved framework regions (FRs) that are linked by three hypervariable regions, also called complementarity determining regions (CDRs). Each pair of CDRs from the two chains are aligned by the framework regions to enable binding to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chains contain the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid assignments to each region are defined by Kabat, Sequences of Proteins of Immunological Interest ( National Institutes of Health, Bethesda, MD, 1987 and 1991), or by Chothia & Les k, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or different light chains are assigned the same number. The percentage of sequence identity is determined using antibody sequences maximally aligned according to the Kabat numbering convention. After alignment, when the target antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the target antibody region and the reference antibody region is calculated by multiplying by 100 the number of positions occupied by the same amino acid in both the target antibody region and the reference antibody region divided by the total number of aligned positions in the two regions (gaps are not counted).

[0053] and the reference antibody region, and dividing by the total number of aligned positions in the two regions (gaps are not counted). It is converted into an integer value.

[0054] For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are classified as follows: Group I (hydrophobic side chains): norleucine, Met, Ala, Val, Le u, Ile; Group II (neutral hydrophilic side chains): Cys, Ser, Thr; Group III (acidic side chains ): Asp, Glu; Group IV (basic side chains): Asn, Gln, His, Lys, Arg ; Group V (residues affecting chain orientation): Gly, Pro; and Group VI (aromatic side chains): Tr p, Tyr, Phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions consist of exchanging a member of one of these classes for a member of another class.

[0055] The antibodies of the present invention typically bind to their designated targets with an affinity constant of at least 10 6 10 7 10 8 10 9 or 1 0 10 M -1 . Such binding is specific binding in that it is detectably stronger and distinguishable from non-specific binding that occurs to at least one unrelated target. Specific binding results from the formation of a bond between specific functional groups or a specific spatial fit (e.g., lock and key type), while non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily imply that a monoclonal antibody binds to one and only one target.

[0056] The term "symptom" refers to subjective manifestations of a disease, such as a change in gait, as perceived by the subject. refers to evidence of a kind. "Symptom" refers to objective evidence of a disease as observed by a physician.

[0057] If an individual has at least one known risk factor (e.g., genetic, biochemical, family history, environmental exposure), and is at statistically significantly higher risk of developing a disease than an individual without the risk factor, the individual has an increased risk of the disease. Statistical significance means p ≦ 0.05 .

[0058] Unless it is clear from the context that it is not, the term "about" encompasses values that fall within either the standard deviation of the mean of the value being described or + / - 5% of the value being described, whichever is greater. .

[0059] The term "9E4 antibody" refers to any antibody in which each CDR is substantially that of 9E4, and thus includes murine, chimeric, veneered, and humanized 9E4 . References to other antibodies, such as 5C1 and 1H7, have the corresponding meaning.

[0060] The antibodies of the present invention can be administered in combination with another treatment for the same indication as the antibody, which means that the other treatment is administered at least once during the period in which the antibody is administered, and such period begins one month before the first administration of the antibody and ends one month after the last administration of the antibody . The other treatment can be administered at repeated intervals during this period, and the repeated intervals can be the same as or different from the intervals at which the antibody is administered. The other treatment can be symptomatic treatment . . The other treatment can be administered at repeated intervals during this period, and the repeated intervals can be the same as or different from the intervals at which the antibody is administered. The other treatment can be symptomatic treatment .

[0061] Treatment is symptomatic when it affects only one or more symptoms of a disease, not its cause, i.e., its etiology. ​​

[0062] As used herein, the term "mobile device" refers to any portable device that includes sensors and data recording equipment suitable for obtaining a set of activity measurement data. Typically, a mobile device includes sensors for measuring activity. This may also require a display for electronically simulating activity tests on the mobile device, in addition to a data processor and storage device. Further, data is recorded from the activity of interest and compiled into a data set, and the data set is evaluated by the method of the present invention either on the mobile device itself or on a second device. Depending on the particular configuration envisioned, the mobile device may need to include data transmission equipment to transmit the acquired data set from the mobile device to one or more further devices. Particularly well-suited as mobile devices according to the present invention are smartphones, smartwatches, wearable sensors, portable multimedia devices or tablet computers. Alternatively, a portable sensor having a data recording device and, optionally, processing equipment may be used. Further, depending on the type of activity test being performed, the mobile device is configured to indicate an instruction to the subject regarding the activity to be performed for the test.

[0063] Unless the context clearly dictates otherwise, references to a range include any integer within the range.

[0064] A treatment regimen refers to a combination of parameters that characterize the administration of an immunotherapeutic agent, including any or all of dosage, frequency of administration, route of administration, and total duration of administration. ​​​​​​​​​​​​​

[0065] Brief Explanation of Arrays Array No. 1 is the m9E4VL variable region.

[0066] Array No. 2 is the 63102889Hu9E4VLFr variable region.

[0067] Array No. 3 is the Hu9E4VLv1 variable region.

[0068] Array No. 4 is the Hu9E4VLv2 (without revertant mutation) variable region.

[0069] Array No. 5 is the Hu9E4VLv3 variable region.

[0070] Array No. 6 is the m9E4VH variable region.

[0071] Array No. 7 is the 1791009Hu9E4VHFr variable region.

[0072] Array No. 8 is the Hu9E4VHv1 variable region.

[0073] Array No. 9 is the Hu9E4VHv2 variable region.

[0074] Array No. 10 is the Hu9E4VHv3 variable region.

[0075] Array No. 11 is the Hu9E4VHv4 (without revertant mutation) variable region.

[0076] Array No. 12 is the amino acid sequence of native human wild-type alpha-synuclein.

[0077] Array No. 13 is the humanized 9E4 light chain constant region (with R) (common for v1, v2, v3 )

[0078] Array No. 14 is the nucleotide sequence of the humanized 9E4 light chain constant region (with R) (v1, v2, is common for v3.

[0079] SEQ ID NO: 15 is the amino acid sequence of an exemplary human IgG1 constant region.

[0080] SEQ ID NO: 16 is the nucleic acid sequence encoding an exemplary human IgG1 constant region.

[0081] SEQ ID NO: 17 is the Hu9E4VLv1 nucleotide sequence variable region.

[0082] SEQ ID NO: 18 is the Hu9E4VLv2 nucleotide sequence (without revertant mutations) variable region. is.

[0083] SEQ ID NO: 19 is the Hu9E4VLv3 nucleotide sequence variable region.

[0084] SEQ ID NO: 20 is the Hu9E4VHv1 nucleotide sequence variable region.

[0085] SEQ ID NO: 21 is the Hu9E4VHv2 nucleotide sequence variable region.

[0086] SEQ ID NO: 22 is the Hu9E4VHv3 nucleotide sequence variable region.

[0087] SEQ ID NO: 23 is the Hu9E4VHv4 nucleotide sequence (without revertant mutations) variable region. is.

[0088] SEQ ID NO: 24 is the Hu9E4VL signal peptide (common for v1, v2, v3). is.

[0089] SEQ ID NO: 25 is the Hu9E4VL signal peptide nucleotide sequence (common for v1, v2, v3). is.

[0090] SEQ ID NO: 26 is the Hu9E4VH signal peptide (common for v1, v2, v3). is.

[0091] SEQ ID NO: 27 is the Hu9E4VH signal peptide nucleotide sequence (common for v1, v2, v3 , v4).

[0092] SEQ ID NO: 28 is a Hu9E4VL alternative.

[0093] SEQ ID NO: 29 is a Hu9E4VH alternative.

[0094] SEQ ID NO: 30 is the amino acid sequence of an exemplary human kappa light chain constant region.

[0095] SEQ ID NO: 31 is the nucleotide of the humanized 9E4 light chain constant region (without R).

[0096] sequence (common for v1, v2, v3).

[0097] SEQ ID NO: 32 is humanized 9E4 light chain type 3 (variable region + constant region, with arginine) .

[0098] SEQ ID NO: 33 is humanized 9E4 light chain type 3 (variable region + constant region, without arginine) .

[0099] SEQ ID NO: 34 is humanized 9E4 heavy chain type 3 (variable region + constant region).

[0100] SEQ ID NO: 35 is the humanized 9E4 heavy chain constant region (G1m3 allotype; BIP type).

[0101] SEQ ID NO: 36 is humanized 9E4 heavy chain type 3 (variable region + constant region).

[0102] SEQ ID NO: 37 is humanized 9E4 heavy chain type 3 (variable region + alternative constant region G1m3 allotype p).

[0103] SEQ ID NO: 38 is the m5C1 antibody mature heavy chain variable region nucleotide sequence.

[0104] SEQ ID NO: 39 is the amino acid sequence of the m5C1 antibody heavy chain variable region.

[0105] SEQ ID NO: 40 is the nucleotide sequence of the m5C1 antibody heavy chain signal peptide.

[0106] SEQ ID NO: 41 is the amino acid sequence of the m5C1 antibody heavy chain signal peptide.

[0107] SEQ ID NO: 42 is the nucleotide sequence of the m5C1 antibody mature light chain variable region.

[0108] SEQ ID NO: 43 is the amino acid sequence of the m5C1 antibody light chain variable region.

[0109] SEQ ID NO: 44 is the nucleotide sequence of the m5C1 antibody light chain signal peptide.

[0110] SEQ ID NO: 45 is the amino acid sequence of the m5C1 antibody light chain signal peptide.

[0111] SEQ ID NO: 46 is the 5C1 immunogen.

[0112] SEQ ID NO: 47 is the m5C1 heavy chain CDR1.

[0113] SEQ ID NO: 48 is the m5C1 heavy chain CDR2.

[0114] SEQ ID NO: 49 is the m5C1 heavy chain CDR3.

[0115] SEQ ID NO: 50 is the m5C1 light chain CDR1.

[0116] SEQ ID NO: 51 is the m5C1 light chain CDR2.

[0117] SEQ ID NO: 52 is the m5C1 light chain CDR3.

[0118] SEQ ID NO: 53 is the mouse 5C1 heavy chain variable region having a sequence encoding a signal peptide It is a domain nucleotide sequence.

[0119] SEQ ID NO: 54 is a mouse 5C1 heavy chain variable region sequence having a signal peptide.

[0120] SEQ ID NO: 55 is a mouse 5C1 light chain variable region nucleotide sequence having an array encoding a signal peptide. It is a domain nucleotide sequence.

[0121] SEQ ID NO: 56 is a mouse 5C1 light chain variable region sequence having a signal peptide.

[0122] SEQ ID NO: 57 is a human VH acceptor FR (accession number AAY42876.1).

[0123] SEQ ID NO: 58 is humanized 5C1H1.

[0124] SEQ ID NO: 59 is humanized 5C1H2.

[0125] SEQ ID NO: 60 is humanized 5C1H3.

[0126] SEQ ID NO: 61 is humanized 5C1H4.

[0127] SEQ ID NO: 62 is humanized 5C1H5.

[0128] SEQ ID NO: 63 is a nucleic acid sequence encoding humanized 5C1 H1.

[0129] SEQ ID NO: 64 is a nucleic acid sequence encoding humanized 5C1 H2.

[0130] SEQ ID NO: 65 is a nucleic acid sequence encoding humanized 5C1H3.

[0131] SEQ ID NO: 66 is a nucleic acid sequence encoding humanized 5C1 H4.

[0132] SEQ ID NO: 67 is a nucleic acid sequence encoding humanized 5C1H5.

[0133] Sequence number 68 is a human VL acceptor FR (Accession number CAB51293.1).

[0134] Sequence number 69 is humanized 5C1L1.

[0135] Sequence number 70 is humanized 5C1L2.

[0136] Sequence number 71 is humanized 5C1L3.

[0137] Sequence number 72 is humanized 5C1L4.

[0138] Sequence number 73 is a nucleic acid sequence encoding humanized 5C1 L1.

[0139] Sequence number 74 is a nucleic acid sequence encoding humanized 5C1 L2.

[0140] Sequence number 75 is a nucleic acid sequence encoding humanized 5C1L3.

[0141] Sequence number 76 is a nucleic acid sequence encoding humanized 5C1 L4.

[0142] Sequence number 77 is a nucleic acid sequence encoding an exemplary human kappa light chain constant region.

[0143] Sequence number 78 is the non-amyloid component (NAC) domain of alpha-synuclein as reported by Jensen et al.

[0144] Sequence number 79 is the non-amyloid component (NAC) domain of alpha-synuclein as reported by Ueda et al.

[0145] Sequence number 80 is the m1H7 antibody heavy chain variable nucleotide sequence.

[0146] SEQ ID NO: 81 is the m1H7 heavy chain variable amino acid sequence.

[0147] SEQ ID NO: 82 is the m1H7 light chain variable nucleotide sequence.

[0148] SEQ ID NO: 83 is the m1H7 light chain variable amino acid sequence.

[0149] SEQ ID NO: 84 is the mature m1H7 heavy chain variable nucleotide sequence.

[0150] SEQ ID NO: 85 is the mature m1H7 heavy chain variable amino acid sequence.

[0151] SEQ ID NO: 86 is the mature m1H7 light chain variable nucleotide sequence.

[0152] SEQ ID NO: 87 is the mature m1H7 light chain variable amino acid sequence.

[0153] SEQ ID NO: 88 is the m1H7 heavy chain CDR1 (Kabat definition).

[0154] SEQ ID NO: 89 is the m1H7 heavy chain CDR2 (Kabat definition).

[0155] SEQ ID NO: 90 is the m1H7 heavy chain CDR3 (Kabat definition).

[0156] SEQ ID NO: 91 is the m1H7 light chain CDR1 (Kabat definition).

[0157] SEQ ID NO: 92 is the m1H7 light chain CDR2 (Kabat definition).

[0158] SEQ ID NO: 93 is the m1H7 light chain CDR3 (Kabat definition).

[0159] SEQ ID NO: 94 is the Hu1H7VHv1 nucleotide sequence.

[0160] SEQ ID NO: 95 is the Hu1H7VHv1 amino acid sequence.

[0161] SEQ ID NO: 96 is the Hu1H7VHv2 nucleotide sequence.

[0162] SEQ ID NO: 97 is the Hu1H7VHv2 amino acid sequence.

[0163] SEQ ID NO: 98 is the Hu1H7VHv3 nucleotide sequence.

[0164] SEQ ID NO: 99 is the Hu1H7VHv3 amino acid sequence.

[0165] SEQ ID NO: 100 is the Hu1H7VHv4 nucleotide sequence.

[0166] SEQ ID NO: 101 is the Hu1H7VHv4 amino acid sequence.

[0167] SEQ ID NO: 102 is the Hu1H7VHv5 nucleotide sequence.

[0168] SEQ ID NO: 103 is the Hu1H7VHv5 amino acid sequence.

[0169] SEQ ID NO: 104 is the Hu1H7VH signal peptide nucleotide sequence.

[0170] SEQ ID NO: 105 is the Hu1H7VH signal peptide amino acid sequence.

[0171] SEQ ID NO: 106 is the Hu1H7VH signal peptide nucleotide sequence.

[0172] SEQ ID NO: 107 is the Hu1H7VH signal peptide amino acid sequence.

[0173] SEQ ID NO: 108 is the Hu1H7VLv1 nucleotide sequence.

[0174] SEQ ID NO: 109 is the Hu1H7VLv1 amino acid sequence.

[0175] SEQ ID NO: 110 is the Hu1H7VLv2 nucleotide sequence.

[0176] SEQ ID NO: 111 is the Hu1H7VLv2 amino acid sequence.

[0177] SEQ ID NO: 112 is the Hu1H7VLv3 nucleotide sequence.

[0178] SEQ ID NO: 113 is the Hu1H7VLv3 amino acid sequence.

[0179] SEQ ID NO: 114 is the Hu1H7VLv4 nucleotide sequence.

[0180] SEQ ID NO: 115 is the Hu1H7VLv4 amino acid sequence.

[0181] SEQ ID NO: 116 is the Hu1H7VL signal peptide nucleotide sequence.

[0182] SEQ ID NO: 117 is the amino acid sequence of the BAC02037 (GI- 21670055) human acceptor used for the heavy chain framework.

[0183] SEQ ID NO: 118 is the amino acid sequence of the AAY33358 GI- 63102905) human acceptor used for the light chain framework.

[0184] SEQ ID NO: 119 is Hu1H7VH that has no revertant mutations or CDR mutations. and

[0185] SEQ ID NO: 120 is Hu1H7VL that has no revertant mutations or CDR mutations. and

[0186] SEQ ID NO: 121 is a Hu1H7VH substitute.

[0187] SEQ ID NO: 122 is a Hu1H7VL substitute.

[0188] Sequence number 123 is a Hu1H7 VH CDR3 substitute.

[0189] Sequence number 124 is a humanized 1H7 light chain type 3 (variable region + constant region, with arginine). It is.

[0190] Sequence number 125 is a humanized 1H7 light chain type 3 (variable region + constant region, without arginine). It is.

[0191] Sequence number 126 is a humanized 1H7 heavy chain type 3 (variable region + constant region).

[0192] Sequence number 127 is a humanized 1H7 heavy chain type 3 (variable region + constant region G1m3 allotype). It is.

[0193] Sequence number 128 is a humanized 1H7 heavy chain constant region (IgG2).

[0194] Sequence number 129 is a humanized 1H7 heavy chain constant region (G1m1 allotype).

[0195] Sequence number 130 is the NI-202.12F4-VHA1b variable heavy chain (VH) sequence.

[0196] Sequence number 131 is the NI-202.12F4-VHA1b CDR1 sequence.

[0197] Sequence number 132 is the NI-202.12F4-VHA1b CDR2 sequence.

[0198] Sequence number 133 is the NI-202.12F4-VHA1b CDR3 sequence.

[0199] Sequence number 134 is the NI-202.12F4-VLa1 variable light chain (VL) sequence.

[0200] Sequence number 135 is the NI-202.12F4-VLa1 CDR1 sequence.

[0201] Sequence number 136 is the NI-202.12F4-VLa1 CDR2 sequence.

[0202] Sequence number 137 is the NI-202.12F4-VLa1 CDR3 sequence.

[0203] Sequence number 138 is the NI-202.21D11-VH sequence.

[0204] Sequence number 139 is the NI-202.21D11-VH CDR1 sequence.

[0205] Sequence number 140 is the NI-202.21D11-VH CDR2 sequence.

[0206] Sequence number 141 is the NI-202.21D11-VH CDR3 sequence.

[0207] Sequence number 142 is the NI-202.21D11-VK sequence.

[0208] Sequence number 143 is the NI-202.21D11 VK CDR1 sequence.

[0209] Sequence number 144 is the NI-202.21D11 VK CDR2 sequence.

[0210] Sequence number 145 is the NI-202.21D11 VK CDR3 sequence.

[0211] Sequence number 146 is the 9E4 VH CDR1 sequence.

[0212] Sequence number 147 is the 9E4 VH CDR2 sequence.

[0213] Sequence number 148 is the 9E4 VH CDR3 sequence.

[0214] Sequence number 149 is the 9E4 VL CDR1 sequence.

[0215] Sequence number 150 is the 9E4 VL CDR2 sequence.

[0216] Sequence number 151 is the 9E4 VL CDR3 sequence.

Mode for Carrying Out the Invention

[0217] I. Overview The present invention provides a dosing regimen for immunotherapy of synucleinopathy. The subject receives an antibody against alpha-synuclein intravenously at a dose of 3000 - 5000 mg or 1300 - 1700 mg every 3 - 5 weeks. Prior to such a dose, one or more lower loading doses may be administered. against alpha-synuclein at a dose of 3000 - 5000 mg or 1300 - 1700 m g every 3 - 5 weeks. Prior to such a dose, one or more lower loading doses may be administered.

[0218] II. Target Molecule Native human wild-type alpha-synuclein is a 140-amino acid peptide having the following amino acid sequence: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTG VTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (sequence number 12) and has the GenBank accession number: P37840. The protein has three recognized domains: an N-terminal KTKE repeat domain covering amino acids 1 - 61 ; an NAC (non-amyloid component) domain spanning approximately amino acids 60 - 95; and a C-terminal acidic domain spanning approximately amino acids 98 - 140.

[0219] Unless it is clear from the context that this is not the case, references to alpha-synuclein or fragments thereof refer to the native human wild-type amino acid sequence as indicated above, and human allelic variants thereof, particularly human allelic variants associated with Lewy body disease (e.g., variant E46K, A30P and A53T; the first letter indicates the amino acid in SEQ ID NO: 12, the number indicates the codon position in SEQ ID NO: 12, and the second letter indicates the amino acid in the allelic variant). Such variants can be present, as appropriate, individually or in any combination in any of the aspects of the invention described below.

[0220] III. Synucleinopathies Synucleinopathies are characterized by the excessive accumulation of alpha-synuclein, which may or may not be accompanied by the formation of Lewy bodies (LB). For example, Lewy body disease (LBD) is characterized by the degeneration of the dopaminergic system, motor changes, cognitive impairment, and the formation of Lewy bodies. [McKeith et al., Neurology (1996) 47:1113-24]. Lewy bodies are spherical protein deposits found in nerve cells. Their presence in the brain disrupts the normal function of the brain and interferes with the action of chemical messengers including acetylcholine and dopamine. Synucleinopathies include Parkinson's disease (including idiopathic Parkinson's disease), dementia with Lewy bodies (DLB) [also known as diffuse Lewy body disease (DLBD)], Lewy body variant of Alzheimer's disease (LBV), Alzheimer's-Parkinson complex disease, progressive supranuclear palsy (PSP), and multiple system atrophy (MSA; ​ For example, olive-bridge cerebellar atrophy, striatonigral degeneration, and Shy-Drager syndrome) are included. DLB shares symptoms of both Alzheimer's disease and Parkinson's disease . DLB is mainly different from Parkinson's disease in the location of Lewy bodies. In DLB , Lewy bodies are mainly formed in the cortex. In Parkinson's disease, they are mainly formed in the substantia nigra. Other synucleinopathies include pure autonomic failure, Lewy body type dysphagia, sporadic LBD, and hereditary LBD (e.g., alpha-synuclein gene mutation, mutations in PARK3 and PARK4).

[0221] IV. Immunotherapeutic agents Passive immunotherapy involves treatment with antibodies that specifically bind to alpha-synuclein .

[0222] 1. Antibodies A. Binding specificity and functional characteristics The methods of the present invention can include antibodies that specifically bind to epitopes within residues 1-15, 4-15, 1-20, 91-99, 115-130, 117-123, or 118-126 of human alpha-synuclein . The antibodies can be monoclonal antibodies or polyclonal antibodies . Polyclonal sera can be specific for the epitope or range in that they lack antibody binding outside the epitope or amino acid range within alpha-synuclein . The antibodies can be, among other possibilities, non-human, chimeric, humanized, fully human , or human antibodies .

[0223] Some antibodies are bispecific, with one arm binding to alpha-synuclein (alp an antibody against ha-synculein, and the other arm binds to a receptor expressed at the blood-brain barrier, such as the insulin receptor, the insulin-like growth factor (IGF) receptor, the leptin receptor, or the lipoprotein receptor, or preferably the transferrin receptor (Friden et al., PNAS 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such bispecific antibodies can be transported across the blood-brain barrier by receptor-mediated transcytosis. The brain uptake of bispecific antibodies can be further enhanced by engineering the bispecific antibody to reduce its affinity for the blood-brain barrier receptor. The reduced affinity for the receptor results in a broader distribution in the brain (see, e.g., Atwal. et al. Sci. Trans. Med. 3, 84ra43, 2011; Yu et al. Sci. Trans. Med. 3, 84ra44, 2011). Exemplary bispecific antibodies can also include: (1) a dual variable domain antibody (DVD-Ig) in which each light and heavy chain contains two variable domains in tandem through a short peptide linker [Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig Molecule, Antibody Engineering, Springer Berlin Heidelberg (2010)]; (2) a tetravalent bispecific antibody having two binding sites for each target antigen . Med. 3, 84ra43, 2011; Yu et al. Sci. Trans. Med. 3, 84ra44, 2011).

[0224] TM ​​​​​​​​​Tandab, a fusion of two single-chain diabodies that results in a bispecific antibody ;(3) Flexibody, a combination of an scFv with a diabody that results in a multivalent molecule ;(4) Triabody, a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments obtained by the "dimerization and docking domain" in protein kinase A; (5) So-called scorpion molecules containing two scFvs fused to both ends of the human Fc region. Examples of platforms useful for preparing bispecific antibodies include BiTE ( Micromet), DART (MacroGenics), Fcab and Mab2 ( F-star), Fc-engineered IgGl (Xencor) or Duo Body (based on Fab arm exchange, Genmab). Another exemplary antibody is mAb 9E4, which binds to an epitope within residues 118 - 126 of human alpha-synuclein. The cell line JH17 .9E4.3.37.1.14.2, which produces antibody 9E4, has an ATCC accession number PTA-822 1 and was deposited on February 26, 2007 with the American Type Culture Collection (ATCC, P.O. Box 1549

[0225] , Manassas, Va. 20108) under the terms of the Budapest Treaty. The mouse light and heavy chain variable sequences are SEQ ID NO: 1 and SEQ ID NO: 6, respectively. Mouse mAb 9E4 has SEQ ID NO: 1 and SEQ ID NO: 6 for the mouse light and heavy chain variable sequences, respectively. Mouse mAb 9E4 has SEQ ID NO: 1 and SEQ ID NO: 6 for the mouse light and heavy chain variable sequences, respectively. Mouse mAb 9E4 has SEQ ID NO: 1 and SEQ ID NO: 6 for the mouse light and heavy chain variable sequences, respectively. Mouse mAb 9E4 has SEQ ID NO: 1 VH-CDR1 of SEQ ID NO: 46, VH-CDR2 of SEQ ID NO: 147, VH-CD R3, VL-CDR1 of SEQ ID NO: 149, VL-CDR2 of SEQ ID NO: 150, and the sequence includes VL-CDR3 of SEQ ID NO: 151.

[0226] Another exemplary antibody is mAb 5C1. The mouse light and heavy chain variable sequences are SEQ ID NO: 43 and SEQ ID NO: 39, respectively.

[0227] Another exemplary antibody is mAb 1H7. The JH17.1H7. 4.24.34 cell line that produces antibody 1H7 has the ATCC accession number PTA-8220 and was deposited with the American Type Culture Collection (ATCC, P.O. Box 1549, Manassas , Va. 20108) on February 26, 2007 under the provisions of the Budapest Treaty. The mouse light and heavy chain variable sequences are SEQ ID NO: 87 and SEQ ID NO: 85, respectively.

[0228] Such antibodies are described in WO06 / 020581 and U.S. applications Ser. Nos. 61 / 591,835 , 61 / 711,207, 13 / 750,983, 61 / 711,2 04, 61 / 719,281, 61 / 840,432, 61 / 872 ,366, 14 / 049,169, 61 / 553,131, 61 / 7 11,204, 61 / 843,011, and 13 / 662,261 (all of which are incorporated herein by reference in their entirety for all purposes). are described in .

[0229] Another exemplary antibody is NI-202.12F4, and NI-202.12F4 is an Binds to an epitope within residues 4 - 15 of alpha - synuclein, and the major contributing factor to the epitope was reported to be residue 10 in a human antibody (see US8,940,276; WO2012177972, US9,580,493). NI - 202.12F4 contains the variable heavy chain (VHA1b) sequence of SEQ ID NO: 130 and the variable light chain (V La1) sequence of SEQ ID NO: 134. NI - 202.12F4 contains the VHA1b - CDR 1 of SEQ ID NO: 131, the VHA1b - CDR2 of SEQ ID NO: 132, the VH A1b - CDR3 sequence of SEQ ID NO: 133 (Ala - His), the VLa1 - CDR1 sequence of SEQ ID NO: 135, the V La1 - CDR2 sequence of SEQ ID NO: 136, and the VLa1 - CDR3 sequence of SEQ ID NO: 137. Another exemplary antibody is NI - 202.21D11, which binds to an epitope within residues 117 - 123 of alpha - synuclein and is reported to most contribute to the epitope at residues 121 and 122 (see US9,580,493 ). NI - 202.21D11 contains the variable heavy chain (VH) sequence of SEQ ID NO: 138 and the variable light chain (VK) sequence of SEQ ID NO: 142. NI - 202.21D11 contains the VH - CDR1 of SEQ ID NO: 1 39, the VH - CDR2 of SEQ ID NO: 140, the VH - CD R3 sequence of SEQ ID NO: 141, the VK - CDR1 sequence of SEQ ID NO: 143, the VK - CDR2 sequence of SEQ ID NO: 144 , and the VK - CDR3 sequence of SEQ ID NO: 145.

[0230] B. Humanized Antibodies A humanized antibody is a genetically engineered antibody in which the CDRs from a non - human "donor" antibody are grafted onto a human "acceptor" antibody sequence (e.g., Queen et al., US5,530, 101 and US 5,585,089; Winter et al., US 5,225,539, Carter , US 6,407,213, Adair, US 5,859,205, US 6,881,557, F oote, US 6,881,557 (see). The acceptor antibody sequence can be, for example, a mature human anti- body variable region sequence, a complex of such sequences, a consensus sequence of human antibody sequences (e.g., Kabat, 1991, the light and heavy chain variable region consensus sequences cited above), or a germline variable region sequence. Thus, the humanized antibodies of the present invention have, in whole or in substantial part, 9 three light chain CDRs and three heavy chain CDRs as defined by Kabat from a mouse antibody (donor antibody) such as 9E4, 5C1 or 1H7, and, if present, a mature variable region framework sequence and a constant region from a whole or substantially human antibody sequence. Similarly, a humanized heavy chain has three heavy chain CDRs as defined by Kabat from the heavy chain of the donor antibody, and, if present, a mature heavy chain variable sequence and a heavy chain constant region sequence from a whole or substantially human antibody heavy chain sequence. Similarly, a humanized light chain has three light chain CDRs as defined by Kabat from the light chain of the donor antibody, and, if present, a mature light chain variable sequence and a light chain constant region sequence from a whole or substantially human antibody light chain sequence. The mature variable region framework sequence or the constant region sequence of the antibody chain is substantially a human mature variable region framework sequence or a human when at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % of the corresponding residues defined by Kabat are identical. The heavy chain includes a heavy chain having a constant region sequence. Similarly, a humanized light chain includes a light chain having three light chain CDRs as defined by Kabat from the light chain of the donor antibody, and, if present, a mature light chain variable sequence and a light chain constant region sequence from a whole or substantially human antibody light chain sequence. The mature variable region framework sequence or the constant region sequence of the antibody chain is substantially a human mature variable region framework sequence or a human when at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % of the corresponding residues defined by Kabat are identical. The mature variable region framework sequence of the antibody chain or the constant region sequence of the antibody chain is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % identical to the corresponding residues defined by Kabat, and is substantially a human mature variable region framework sequence or a human constant region sequence, respectively. constant region sequence when the percentage of identical residues is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 It is from the constant region array. The CDR is substantially from the corresponding donor CDR when it shows at least 85% identity to the corresponding donor CDR, except for the case of CDRH2 which requires at least 65% identity. y)

[0231] Certain amino acids from human mature variable region framework residues can be selected for substitution based on their possible effects on CDR conformation and / or binding to the antigen. Investigation of such possible effects is by modeling, examination of the characteristics of amino acids at specific positions, or empirical observation of the effects of substitution or mutagenesis of specific amino acids.

[0232] For example, if an amino acid is different between a mouse mature variable region framework residue and a selected human mature variable region framework residue, the human framework amino acid can be replaced by the equivalent framework amino acid from the mouse antibody if it is reasonably predicted to (1) bind directly to the antigen by non-covalent bond, (2) be adjacent to the CDR region, (3) otherwise interact with the CDR region (e.g., be within about 6 Å of the CDR region), (4) mediate the interaction between the heavy chain and the light chain.

[0233] An exemplary humanized mouse 9E4 antibody includes three exemplified humanized light chain mature variable regions ( Hu9E4VLv1~v3; SEQ ID NOs: 3~5) and four exemplified humanized heavy chain mature variable regions (Hu9E4VHv1~v4; SEQ ID NOs: 8~11).

[0234] The exemplary light and heavy chain mature variable regions can pair in any combination. Exemplary combinations are Hu9E4VLv3 (SEQ ID NO: 5) and Hu9E4VHv3 (SEQ ID NO: 10). The exemplary heavy chain constant region is SEQ ID NO: 35 with or without a C-terminal lysine. This constant region can be linked to any of the heavy chain variable regions. The exemplary light chain constant region is SEQ ID NO: 30. This constant region can be linked to any of the light chain variable regions. The exemplary full-length heavy chain has the amino acid sequence of SEQ ID NO: 37 with or without a C-terminal lysine. The exemplary full-length light chain has the amino acid sequence of SEQ ID NO: 32. An exemplary combination is an antibody comprising SEQ ID NO: 37 (with or without a C-terminal lysine) and SEQ ID NO: 32. Nucleic acids encoding the various heavy and light chains described immediately above are provided at SEQ ID NOS: 17 - 23. The exemplified humanized antibody has heavy and light chains characterized by SEQ ID NO: 37

[0235] (with or without a C-terminal lysine) and SEQ ID NO: 32. The exemplary humanized form of the murine 5C1 antibody comprises four exemplified humanized light chain mature variable regions (5C1L1 - L4; SEQ ID NOS: 69 - 72), and five exemplified humanized heavy chain mature variable regions (5C1H1 - H4; SEQ ID NOS: 58 - 62). The exemplary light and heavy chain mature variable regions can pair in any combination. The exemplary heavy chain constant region is SEQ ID NO: 35 with or without a C-terminal lysine. This constant region can be linked to any of the heavy chain variable regions. The exemplary light chain constant region is SEQ ID NO: 30. This constant region can be linked to any of the light chain variable regions. Nucleic acids encoding the various heavy and light chains described immediately above are provided at SEQ ID NOS: 73 - 76 and 63 - 67.

[0236] Exemplary humanized murine 1H7 antibody comprises four exemplary humanized light chain mature variable regions ( Hu1H7VLv1-v4; SEQ ID NOs: 109, 111, 113, and 115 respectively) , and five exemplary humanized heavy chain mature variable regions (Hu1H7VHv1-v5; each respectively SEQ ID NOs: 95, 97, 99, 101, and 103). Exemplary light and heavy chain mature variable regions can pair in any combination. An exemplary combination is Hu1H7VHv 3 (SEQ ID NO: 99) and Hu1H7VLv3 (SEQ ID NO: 113). Exemplary heavy chain constant regions are SEQ ID NO: 35 with or without a C-terminal lysine. This constant region can be linked to either of the heavy chain variable regions. Exemplary light chain constant regions are SEQ ID NO: 30. This constant region can be linked to either of the light chain variable regions. Exemplary full-length heavy chains have the amino acid sequence of SEQ ID NO: 127 (with G1m3). Exemplary full-length light chains have the amino acid sequence of SEQ ID NO: 125 (without arginine). An exemplary combination is an antibody comprising SEQ ID NO: 127 and SEQ ID NO: 125. Nucleic acids encoding the various heavy and light chains described immediately above are provided in SEQ ID NOs: 108, 110, 112, 114, 94, 96, 98, 100, 10 2.

[0237] Such humanized antibodies are described in WO06 / 020581 and US13 / 750,983, 14 / 049,169, 13 / 662,261, and 61 / 843,011.

[0238] In these or other antibodies described herein, one or more terminal residues are ​​In particular, the terminal lysine of the heavy chain constant region may be cleaved during post-translational processing.

[0239] C. Chimeric and Veneered Antibodies The present invention further relates to chimeric and veneered non-human antibodies, in particular 9E4, 5C1 or 1 Offers H7.

[0240] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) are different. An antibody that combines light and heavy chain constant regions from any antibody. Typically The light and heavy chain constant regions are of human origin, although the constant regions may be modified as needed (e.g., To facilitate testing of non-human antibodies in appropriate animal models, for example, different animal models such as rats. Such antibodies may originate from a non-human species that provides the variable region. The antibody has substantially or entirely retained the binding specificity of human (or human equivalent) antibodies, and is approximately two-thirds human (or human equivalent) of the antibody. The sequences are of a non-human species.

[0241] A veneered antibody comprises some, and usually all, of the CDRs of a non-human antibody and a non-human variable region. Retain some of the framework residues but add other possible residues that may contribute to B or T cell epitopes. Variable region framework residues, e.g., exposed residues (Padlan, Mol. Immunol. 28:489, 1999). 91) is replaced with residues from the corresponding positions in a human antibody sequence. The result is a method for identifying a CDR that is wholly or substantially from a non-human antibody and that is An antibody in which the variable region framework has been made more human-like by substitutions. 4 is included in the present invention.

[0242] D. Selection of Constant Regions The heavy and light chain variable regions of chimeric, veneered or humanized antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and / or complement-dependent cytotoxicity are desired. For example, human isotopes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotopes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa. An exemplary human light chain kappa constant region has the amino acid sequence of SEQ ID NO: 13. Some such light chain kappa constant regions can be encoded by a nucleic acid sequence. The N-terminal arginine of SEQ ID NO: 13 may be omitted, in which case the light chain kappa constant region has the amino acid sequence of SEQ ID NO: 30. Some such light chain kappa constant regions can be encoded by a nucleic acid sequence. An exemplary human IgG1 heavy chain constant region has the amino acid sequence of the heavy chain constant region component of SEQ ID NO: 15 (with or without C-terminal lysine) or SEQ ID NO: 37 (with or without C-terminal lysine). Some such heavy chain constant regions can be encoded by a nucleic acid sequence. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the heavy and light chain mature variable domains are linked through a spacer. It can be linked to at least a portion of the human constant region. The choice of constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and / or complement-dependent cytotoxicity are desired. For example, human isotopes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotopes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa. An exemplary human light chain kappa constant region has the amino acid sequence of SEQ ID NO: 13. Some such light chain kappa constant regions can be encoded by a nucleic acid sequence. The N-terminal arginine of SEQ ID NO: 13 may be omitted, in which case the light chain kappa constant region has the amino acid sequence of SEQ ID NO: 30. Some such light chain kappa constant regions can be encoded by a nucleic acid sequence. An exemplary human IgG1 heavy chain constant region has the amino acid sequence of the heavy chain constant region component of SEQ ID NO: 15 (with or without C-terminal lysine) or SEQ ID NO: 37 (with or without C-terminal lysine). Some such heavy chain constant regions can be encoded by a nucleic acid sequence. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the heavy and light chain mature variable domains are linked through a spacer. It has the amino acid sequence of SEQ ID NO: 13. Some such light chain kappa constant regions can be encoded by a nucleic acid sequence. The N-terminal arginine of SEQ ID NO: 13 may be omitted, in which case the light chain kappa constant region has the amino acid sequence of SEQ ID NO: 30. Some such light chain kappa constant regions can be encoded by a nucleic acid sequence. In that case, the light chain kappa constant region has the amino acid sequence of SEQ ID NO: 30. Some such light chain kappa constant regions can be encoded by a nucleic acid sequence. An exemplary human IgG1 heavy chain constant region has the amino acid sequence of the heavy chain constant region component of SEQ ID NO: 15 (with or without C-terminal lysine) or SEQ ID NO: 37 (with or without C-terminal lysine). It has the amino acid sequence of the heavy chain constant region component of SEQ ID NO: 15 (with or without C-terminal lysine) or SEQ ID NO: 37 (with or without C-terminal lysine). Some such heavy chain constant regions can be encoded by a nucleic acid sequence. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the heavy and light chain mature variable domains are linked through a spacer. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the heavy and light chain mature variable domains are linked through a spacer. as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the heavy and light chain mature variable domains are linked through a spacer. or as a single-chain antibody in which the heavy and light chain mature variable domains are linked through a spacer. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the heavy and light chain mature variable domains are linked through a spacer.

[0243] Human constant regions exhibit allotype variations and isallo type variations among different individuals, i.e., the constant regions have one or more polymorphic sites in different individuals. That is, the constant regions have one or more polymorphic sites in different individuals. may differ in nature. An isotype differs from an allotype in that the sera that recognize the isotype bind to the non-polymorphic regions of one or more other isotypes. Thus, for example, another heavy chain constant region is the heavy chain constant region of the IgG1 G1m3 allotype and has an amino acid sequence encoding the constant region of SEQ ID NO: 37. Still another heavy chain constant region has an amino acid sequence encoding the constant region of SEQ ID NO: 37 except that it lacks the C-terminal lysine. One or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in part or all of the molecule. Substitutions are made in the constant region to reduce or increase effector functions such as

[0244] complement-mediated cytotoxicity or ADCC (see, for example, Winter et al., US5,624,821; Tso et al., US5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006) or to prolong the half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 to increase the half-life of the antibody (EU numbering is used for the constant region in this paragraph). Substitutions at any or all of positions 234, 235, 236 and / or 237 reduce the affinity for Fcγ receptors, particularly the FcγRI receptor (see, for example, US6,624,821). Some antibodies have effector functions that are reduced or eliminated, such as complement-mediated cytotoxicity or ADCC, while maintaining antigen binding activity. These antibodies are referred to as "effector function-deficient antibodies" or "null antibodies" (see, for example, US6,624,821; Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006). Substitutions can be made at other positions in the constant region, such as positions 289, 290, 298, 301, 303, 305, 307, 311, 312, 317, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, Arrangements at positions 234, 235, and 237 of human IgG1 were made to reduce function. The amino acid sequence at positions 234, 236 and / or 237 in human IgG2 is and position 235 may be substituted by glutamine (e.g., US5 ,624,821).

[0245] E. Expression of Recombinant Antibodies Antibodies can be produced by recombinant expression. Nucleic acid encoding the antibody can be expressed in a desired cell type (e.g., The recombinant vector may be codon-optimized for expression in a recombinant host (e.g., CHO or Sp2 / 0). The acid construct typically contains a naturally associated or heterologous promoter region. The antibody chain coding sequence includes an expression control sequence operably linked to the coding sequence of the antibody chain, the expression control sequence including The control sequence is a eukaryotic sequence in a vector capable of transforming or transfecting a eukaryotic host cell. When the vector is introduced into a suitable host, the host can express the nucleic acid The method is suitable for high level expression of nucleotide sequences and for recovery and purification of cross-reactive antibodies. The vector(s) encoding the antibody chains are also maintained under suitable conditions. To allow amplification of the copy number of the nucleic acid encoding the It may contain a selectable gene.

[0246] E. coli is a particularly useful prokaryotic host cell for the expression of antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Yeast hosts include, for example, yeast strains such as yeast strains containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 It has an expression control sequence, a replication origin, a termination sequence, etc. Typical promoters include 3- Phosphoglycerate kinase and other glycolytic enzymes may be mentioned. Inducible yeast promoters include, in particular, alcohol dehydrogenase, cytochrome C, and maltose and promoters from enzymes involved in galactose utilization.

[0247] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof . See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and such host cell lines include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2 / 0 and NS0. It may be advantageous to use non-human cells. Expression vectors for these cells may include expression control sequences such as origins of replication, promoters, enhancers [Queen et al., Immunol. Rev. 89:49 (1986)], and ribosome binding sites, RNA splice sites, polyadenylation sites, and processing information sites such as transcription terminator sequences. Suitable expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0248] Once vectors encoding the antibody heavy and light chains are introduced into cell cultures, cell pools can be screened for growth productivity and the quality of the product in serum-free media . Next, the top-producing cell pool can then be subjected to FACS-based single cell cloning to generate monoclonal clones. Specific productivity higher than 50 pg or 100 pg per cell per day, corresponding to a productivity greater than 7.5 g per liter of culture, may be advantageous. The antibodies produced by single cell clones can also be tested for turbidity, filtration characteristics, PAGE, IEF, UV scan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore. The selected clones are then stored in multiple vials and can be cryopreserved for later use. Once the antibody is expressed, it can be purified according to standard procedures in the art, which may include Protein A capture, column chromatography (e.g., hydrophobic interaction or ion exchange), low pH for virus inactivation, etc. [See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)]. Methodologies for commercial production of antibodies may include codon optimization, promoter, transcription element, and terminator selection, serum-free single cell cloning, cell banking, use of selectable markers for copy number amplification, CHO terminator, serum free single cell cloning, and improvement of protein titer (e.g., US5,786,464, US6,114,148, US6,063

[0249]

[0250] ​ , 598, US 7,569,339, WO 2004 / 050884, WO 2008 / 01 2142, WO 2008 / 012142, WO 2005 / 019442, WO 2008 / 107388, and WO 2009 / 027471, and US 5,888,809 are referred to).

[0251] V. Complex The complex of the present invention (also known as a pharmaceutical composition) comprises an antibody (e.g., a chimeric, veneered or humanized form of murine 9E4 (ATCC deposit number PTA - 8221)) or an antigen - binding fragment thereof, a buffer, one or more sugars and / or polyols, and a surfactant and has a pH in the range of about 5 to about 7.5. The complex can be prepared for storage in liquid form or in lyophilized form . When stored in lyophilized form, the complex can be reconstituted to the concentrations and properties described herein using a liquid (e.g., sterile water). When it is described that adding water to produce a complex with the specified component concentrations and pH makes the lyophilized composition reconstitutable, it means that the lyophilized complex can be so reconstituted simply by adding water (i.e., without supplying additional amounts of components or adding acids or bases to change the pH). The concentrations and properties of the pre - lyophilized liquid complex can also be as described below when the lyophilized complex is reconstituted to the same volume as the pre - lyophilized complex of the complex . If the volumes are different, the concentration of the complex should be adjusted proportionally . For example, if the reconstituted volume is half of the pre - lyophilized volume , the concentration of the components in the pre - lyophilized complex should be half of the concentration in the reconstituted complex . ​​

[0252] Some formulations contain a bulking agent, which may be the same as the sugar / polyol component or Typically the formulation is sterile, which may be, for example, 0.2 μm Or by sterile filtration using a 0.22 μm filter. Some formulations have a bioburden of ≦about 0.1 EU / The formulation of the present invention also contains the following when frozen and thawed: and / or Other formulations are generally stable due to aggregation, and remain stable at 40°C after reconstitution of the lyophilized cake. In some formulations, less than about 10% of the antibody remains in the formulation. In some formulations, about 5% or less of the antibodies are aggregated in the formulation. It exists as an object.

[0253] In some formulations, the antibody is present at a concentration in the range of about 5 mg / mL to about 100 mg / mL. In some formulations, the antibody is present at a concentration in the range of about 5 mg / mL to about 50 mg / mL. In some formulations, the antibody is present in a range of about 25 mg / mL to about 50 mg / mL. For example, the antibody is present at a concentration of about 35-45 mg / ml or about 40 mg / ml. The antibody may be present in a concentration of about 50 mg / vial to about 500 mg / vial, or The antibody may be present in a more sterile liquid dosage form. The antibody may be present in a dosage form of about 40 mg / vial to about 5 For example, the antibody may be present in a lyophilized dosage form of about 250-300 mg / vial. In sterile liquid or lyophilized dosage form of 50 mg / vial or approximately 200 mg / vial May be present.

[0254] The antibodies used in the disclosed formulations are cytotoxic agents, radiotherapy agents, immunomodulatory agents , a second antibody (e.g., to form an antibody heteroconjugate), or any other bioactive agent that promotes or enhances the activity of the conjugated antibody, such as a therapeutic moiety, can be conjugated. However, the antibodies are typically used in their naked form (i.e., not conjugated to any of these moieties ). .

[0255] The conjugated antibody can be any of the above antibodies, including any of the chimeric, veneered or humanized forms of the antibody 9E4 .

[0256] Buffers are used in the disclosed formulations to achieve a suitable pH for the antibody , such as histidine, succinic acid, and citrate buffers. Some formulations have a pH in the range of about 5.5 to about 7, e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6 .0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. Some formulations have a pH between about 5.5 and about 6.5. Some formulations have a pH of about 6.0 and other formulations have a pH of about 6.5. In some formulations , the citrate buffer or succinate buffer is present at a concentration in the range of about 10 mM to about 30 mM , e.g., about 15 - 25 mM or about 20 mM. Some citrate buffers contain sodium citrate dehydrate and citric acid monohydrate at concentrations in the ranges of about 15 mM to about 20 mM and about 2 mM to about 6 mM, respectively .

[0257] Sugars and / or polyols suitable for the formulation include trehalose, sucrose Examples include trehalose, sucrose, mannitol, or combinations thereof. The sugar / polyol acts as a filler, cryoprotectant, and / or tonicity modifier. For example, some formulations contain trehalose present at a concentration within the range of about 220 mM to about 260 mM, sucrose present at a concentration within the range of about 220 mM to about 260 mM, or a mixture of sucrose present at a concentration within the range of about 20 mM to about 40 mM and mannitol present at a concentration within the range of about 200 mM to about 220 mM. Some formulations contain trehalose present at a concentration of about 230 mM or 240 mM. Other formulations contain sucrose present at a concentration of about 230 mM or 240 mM. Other formulations contain a mixture of sucrose present at a concentration of about 50 mM and mannitol present at a concentration of about 200 mM. Another formulation contains a mixture of sucrose present at a concentration of about 28 mM and mannitol present at a concentration of about 212 mM. Some such formulations are characterized by a weight osmolarity within the range of about 250 - 400, 300 - 400, or 300 - 350 mOsm / kg, for example 335 mOsm / kg. The formulation may contain a surfactant to reduce antibody aggregation and absorption to the surface. Suitable surfactants include polysorbate 20 (PS20) present at a concentration within the range of about 0.005 wt% to about 0.05 wt%. PS20 protects against a significant increase in aggregation or turbidity that would otherwise occur in formulations of the 9E4 antibody. Polysorbate 20 may be present at a concentration within the range of about 0.01% to about 0.05%. For example, the concentration may be 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.

[0258] ​ It may be 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%. In some formulations, or in some formulations, polysorbate 20 is approximately in the range of about 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, or 0.5 g / L. Some formulations contain polysorbate 20 at a concentration of 0.2 g / L (i.e., 0.163 mmol / L).

[0259] Exemplary formulations (liquid, pre - lyophilized or reconstituted after lyophilization) are characterized by a pH in the range of about 5.5 to about 7 and contain (a) a chimeric, humanized, or human - type antibody 9E4 at a concentration in the range of about 10 mg / ml to about 50 mg / ml, or fragments thereof that specifically compete with 9E4 for binding to the antigen; (b) a citrate buffer or succinate buffer present at a concentration in the range of about 10 mM to about 30 mM; (c) trehalose present at a concentration in the range of about 220 mM to about 260 mM, sucrose present at a concentration in the range of about 220 mM to about 260 mM, and a mixture of sucrose present at a concentration in the range of about 20 mM to about 40 mM and mannitol present at a concentration in the range of about 200 mM to about 220 mM, one or more sugars and polyols selected therefrom (the "sugar / polyol"); and (d ) polysorbate 20 present at a concentration in the range of about 0.005 wt% to about 0.05 wt%. For example, the formulation contains (a) a light chain having the amino acid sequence shown as SEQ ID NO: 32 and a heavy chain containing the amino acid sequence shown as SEQ ID NO: 37 with or without a C - terminal lysine, and the antibody is present at a concentration of about 40 mg / mL; (b) citrate at a concentration of about 20 mM ; (c) trehalose at a concentration of about 240 mM, sucrose at a concentration of about 240 mM, a mixture of sucrose at a concentration of about 30 mM and mannitol at a concentration of about 210 mM; and (d) polysorbate 20 at a concentration of about 0.02 wt%. ; (c) trehalose present at a concentration in the range of about 220 mM to about 260 mM, sucrose present at a concentration in the range of about 220 mM to about 260 mM, and a mixture of sucrose present at a concentration in the range of about 20 mM to about 40 mM and mannitol present at a concentration in the range of about 200 mM to about 220 mM, one or more sugars and polyols selected therefrom (the "sugar / polyol"); and (d ) polysorbate 20 present at a concentration in the range of about 0.005 wt% to about 0.05 wt%. For example, the formulation contains (a) a light chain having the amino acid sequence shown as SEQ ID NO: 32 and a heavy chain containing the amino acid sequence shown as SEQ ID NO: 37 with or without a C - terminal lysine, and the antibody is present at a concentration of about 40 mg / mL; (b) citrate at a concentration of about 20 mM ; (c) trehalose at a concentration of about 240 mM, sucrose at a concentration of about 240 mM, a mixture of sucrose at a concentration of about 30 mM and mannitol at a concentration of about 210 mM; and (d) polysorbate 20 at a concentration of about 0.02 wt%. ; (c) trehalose present at a concentration in the range of about 220 mM to about 260 mM, sucrose present at a concentration in the range of about 220 mM to about 260 mM, and a mixture of sucrose present at a concentration in the range of about 20 mM to about 40 mM and mannitol present at a concentration in the range of about 200 mM to about 220 mM, one or more sugars and polyols selected therefrom (the "sugar / polyol"); and (d ) polysorbate 20 present at a concentration in the range of about 0.005 wt% to about 0.05 wt%. For example, the formulation contains (a) a light chain having the amino acid sequence shown as SEQ ID NO: 32 and a heavy chain containing the amino acid sequence shown as SEQ ID NO: 37 with or without a C - terminal lysine, and the antibody is present at a concentration of about 40 mg / mL; (b) citrate at a concentration of about 20 mM ; (c) trehalose at a concentration of about 240 mM, sucrose at a concentration of about 240 mM, a mixture of sucrose at a concentration of about 30 mM and mannitol at a concentration of about 210 mM; and (d) polysorbate 20 at a concentration of about 0.02 wt%. ; (c) trehalose at a concentration of about 240 mM, sucrose at a concentration of about 240 mM, a mixture of sucrose at a concentration of about 30 mM and mannitol at a concentration of about 210 mM; and (d) polysorbate 20 at a concentration of about 0.02 wt%. chain and a heavy chain containing the amino acid sequence shown as SEQ ID NO: 37 with or without a C - terminal lysine, and the antibody is present at a concentration of about 40 mg / mL; (b) citrate at a concentration of about 20 mM ; (c) trehalose at a concentration of about 240 mM, sucrose at a concentration of about 240 mM, a mixture of sucrose at a concentration of about 30 mM and mannitol at a concentration of about 210 mM; and (d) polysorbate 20 at a concentration of about 0.02 wt%. An acid buffer; (c) trehalose at a concentration of about 230 mM; (d) poly sorbate 20; and may include a pH of about 6.0.

[0260] Some lyophilized formulations include (a) a humanized antibody 9E4 or an antigen-binding fragment thereof; ( b) citrate; (c) trehalose; and polysorbate 20. The lyophilized formulation may contain about 200 mg of the antibody. Some lyophilized formulations can be reconstituted using sterile water. Some lyophilized formulations contain 100 - 300 or 150 - 250 mg of the 9E4 antibody, 15 - 35 or 20 - 25 mg of sodium citrate anhydrous, 1.65 - 2.75 or 2 - 2.3 mg of citric acid monohydrate, 360 - 500 or 400 - 4 70 mg of trehalose anhydrous, and 0.5 - 1.5 or 0.75 - 1.25 m g of polysorbate 20. An exemplary lyophilized formulation contains 200 mg of the 9E4 antibody ( e.g., a humanized 9E4 antibody), 25 mg of sodium citrate anhydrous, 2.15 mg of citric acid monohydrate, 435 mg of trehalose anhydrous, and 1 mg of polysorbate 20 Another exemplary lyophilized formulation contains 200 mg of the 9E4 antibody (e.g., a humanized 9E 4 antibody), 25 mg of sodium citrate anhydrous, 3.15 mg of citric acid monohydrate, 4 35 mg of trehalose anhydrous, and 1 mg of polysorbate 20. Such a formulation can be reconstituted to a volume of about 5 ml. Other lyophilized formulations have the same ratios as any of those disclosed in this paragraph but different amounts (e.g., 400 mg of the antibody, 50 mg of citrate sodium, 4.3 mg of citric acid monohydrate, 870 mg of trehalose anhydrous, and 2 mg of polysorbate 20) of the same components. and contain the same components.

[0261] The lyophilized formulation may have a concentration of about 30-50 or 35-45 mg / mL, e.g., about 40 mg / mL (b) an antibody concentration of about 10 to 30 or 15 to 25 mM, preferably about 20 mM; (c) a citrate buffer present at about 160 to 330 or 200 to 260 mM, e.g. (d) trehalose present at a concentration of about 0.1 to 0.3 or 0.15 to Polysorbate 20, present at a concentration of 0.25 g / L, e.g., about 0.2 g / L; and e) can be reconstituted to a pH of about 5.5 to 6.5, for example about 6.0.

[0262] The liquid or reconstituted lyophilized formulation may be substantially isotonic, having a pH of about 250-350. An osmolality of about 335 mOsm / kg water is implied. Some formulations have an osmolality of 270-300 mOsm / kg. The liquid or reconstituted lyophilized formulation also has an osmolality of >350 mOs The blood may be hypertonic (<250 mOsm / kg water) or hypotonic (<250 mOsm / kg water).

[0263] Any of the formulations described may contain pharmaceutical excipients other than those described as ingredients herein. Such formulations may be made without the use of excipients or carriers, etc. ..., which may affect the properties of the formulation. The composition consists of the listed ingredients or contains the listed ingredients, if small amounts of other ingredients are present. The formulation may be described as consisting essentially of a drug for administration to a human. Manufacturing procedures approved or approvable by the FDA for Good Manufacturing Practices (GMP) It can be made as follows.

[0264] VI. Diagnostic Criteria for Synucleinopathies The method of the present invention is generally performed on subjects who have been diagnosed with Lewy body disease by a qualified healthcare professional, or who have an elevated risk as demonstrated by genetic or biochemical markers of the disease, family history, or prodromal symptoms, compared to the general population. Such individuals include anyone who has previously been prescribed treatment or prevention for Lewy body disease. The diagnosis of synucleinopathy can be based on the criteria recognized in the art for the possibility or high probability of Lewy body disease, such as DSM-V or DSM IV-TR, the Lewy Body dementia association, and the Parkinson’s disease society. However, the diagnosis can also be based on the presence of any signs or symptoms of Lewy body disease that lead the treating physician to conclude that the subject is likely to have Lewy body disease. Exemplary criteria for diagnosing the possibility or high probability of PD are shown below. or a record of conditions (e.g., a well-located focal brain injury or use of a neuroleptic within the past six months) known to cause parkinsonism and plausibly associated with the subject's symptoms. Group A: Rest tremor, bradykinesia, rigidity, and the occurrence of asymmetry Features of Group B: Suggestion of a selective diagnosis Marked postural instability in the first three years after symptom onset The phenomenon of freezing in the first three years Hallucinations unrelated to medication in the first three years Dementia preceding motor symptoms or in the first year Supranuclear gaze palsy (other than restriction of upward gaze) or slowing of vertical saccades Severe symptomatic autonomic dysfunction unrelated to medication

[0265]

[0266] Criteria for the diagnosis of possible Parkinson's disease: At least two of the four features in Group A are present; at least one of these is tremor or bradykinesia, none of the features in Group B are present, or the symptoms have been present for less than 3 years and none of the features in Group B have been present to date; and, a substantial and sustained response to levodopa or dopamine agonist has been recorded, or the subject .

[0267] Criteria for the diagnosis of highly probable Parkinson's disease: At least three or four features in Group A are present, none of the features in Group B are present, and a substantial and sustained response to levodopa or dopamine agonist has been recorded .

[0268] Exemplary criteria for the diagnosis of Lewy body dementia are: The presence of dementia At least two of three core features: Fluctuations in attention and concentration, Recurrent well-formed visual hallucinations, and Spontaneous Parkinsonian motor signs. Suggestive clinical features include the following: Rapid eye movement (REM) sleep behavior disorder Severe neuroleptic sensitivity Low dopamine transporter uptake in the basal ganglia demonstrated by SPECT or PET imaging

[0269] In the absence of two core features, a diagnosis of highly probable DLB is also, in addition to dementia ​can be made when at least one suggestive feature is present along with a core feature 。

[0270] The likelihood of DLB can be diagnosed along with the presence of a core or suggestive feature in addition to dementia 。

[0271] Early warning signs of Lewy body disease include, for example, EEG slowing, neuropsychiatric manifestations (depression, dementia, hallucinations, anxiety, apathy, anhedonia), autonomic neural changes (orthostatic hypotension, bladder disorders, constipation, fecal incontinence, sialorrhea, dysphagia, sexual dysfunction, changes in cerebral blood flow), perceptual changes (abnormal sensations of smell, pain, color discrimination), sleep disorders [REM sleep behavior disorder (RBD), restless legs syndrome / periodic limb movement, hypersomnia, insomnia], as well as various other signs and symptoms (fatigue, diplopia, blurred vision, seborrhea, weight loss / gain ). Genetic markers of risk for PD include mutations in the alpha-synuclein or Parkin, UCHL1, and CYP2D6 genes, particularly mutations at positions 30 and 53 of the alpha -synuclein gene. None of these genetic markers or early warning signs by themselves are a diagnosis of Lewy body disease but they can contribute to the diagnosis of Lewy body disease individually or in combination 。 。 。

[0272] VII. Treatment Regimen In a treatment application, a regimen (dose, frequency, and route of administration) known or presumed to be effective in improving at least one sign or symptom of the disease or inhibiting at least further worsening in a subject diagnosed with synucleinopathy 。 An antibody is administered. In prophylactic applications, it is known or presumed to be effective for inhibiting or delaying the onset of at least one sign or symptom of a disease. In a regimen where there is an increased risk of synucleinopathy but not yet sufficient symptoms to be diagnosed as having the disease, an antibody is administered to a subject.

[0273] In an animal model with a control or a clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial), when a more favorable outcome is demonstrated in the treatment group compared to the control group at a level of p < 0.2, 0.1, 0.05, 0.01, or even 0.001, the regimen is considered to be therapeutically or prophylactically effective. However, due to the variability in genetics, subject characteristics and environment, and disease subtypes among individual subjects, a regimen that is effective in one subject may not be effective in another subject, or may be effective to a different extent.

[0274] Exemplary dosage ranges of the antibody are 3000 - 5000 mg of an antibody against alpha - synuclein, administered intravenously at intervals of 3 - 5 weeks, such as every 4 weeks. In some subjects, the dosage is 3500 - 4500 mg every 3 - 5 weeks, such as every 4 weeks. Subjects can receive the same or different dosages (e.g., depending on the subject's body weight). In some methods, a subject receives one of two fixed dosages. For example, a subject with a body weight of less than 65 kg may receive 3500 mg, and a subject with a body weight of 65 kg or more may receive 4500 mg. In some For example, 50-70 mg / kg, 45 mg / kg, 60 mg / kg or 65 mg / Dosage is usually given at intervals of 3 to 5 weeks, for example, every 28 days or every 4 weeks, or is administered on multiple occasions per calendar month. Subjects must receive at least 6, 9, 12, or 30 such treatments at such intervals. or 18 doses, or for as long as symptoms of the condition persist or for the remainder of the subject. In some regimens, an initial loading dose of 2000 mg is administered. After administration, the dose should be increased by at least 2000 mg until the intended target dose is reached, but no more than the intended target dose. For example, a subject may receive an initial loading dose of 2000 mg. After this, an upward titration to a 3500 mg dose or a 4500 mg dose can be made. Titration may be at a single subsequent dose or to reach a target dose or dose within a target range. For example, subjects may be administered multiple doses in increments up to 2000 Subjects may receive an initial dose of 20 mg followed by subsequent doses of 3500 mg. A starting dose of 0.00 mg followed by one or more doses of 2000 mg or more but less than 3500 mg Subjects may receive 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 Alternatively, the subject may receive an initial dose of 4,500 mg followed by subsequent doses of 4,500 mg. , 2000 mg starting dose followed by 2000 mg or more but less than 4500 mg thereafter In some regimens, subjects may receive a dose of 3000 mg and a subsequent dose of 4500 mg. Patients will receive a dose of 000 to 5000 mg of the antibody intravenously every 4 weeks for at least 52 weeks. For subjects receiving a multiple-dose regimen with doses within a specified range, such as 00-5000 mg In each administration, subjects may receive the same or different doses within the specified ranges. In some regimens, the subject receives the same dosage within the specified range for each administration.

[0275] In another exemplary regimen, an antibody at a dosage of 1300 - 1700 mg is administered intravenously to the subject at intervals of 3 to 5 weeks. An exemplary dosage is 1500 mg. The subject may receive a single fixed dosage or two or more different dosages within this range, for example, based on the subject's weight. Some subjects administered within this range receive an antibody at 18 - 25 mg / kg, for example, 20 mg / kg. As in other methods, the intervals can be 3 to 5 weeks, for example, every 4 weeks or calendar monthly. The subject may receive at least 6, at least 9, at least 12, or at least

[0276] 18 administrations, or may be administered at such intervals for as long as symptoms persist or for the remainder of the subject's life. For any of the above treatment regimens, the area under the curve can be calculated to indicate the amount of antibody delivered over time. Alternative treatment regimens can be devised to deliver a substantially the same area under the curve (e.g., within 25, 20, or 15% ), for example, using different administration routes, frequencies, or dosages. Preferably, such alternative treatment regimens do not substantially exceed the Cmax of the specified regimen (e.g., 25, 20, or 15% or less). Other administration routes include topical, oral, subcutaneous, intra - arterial, intracranial, intrathecal, intraperitoneal, intranasal, or intramuscular.

[0277] Any treatment regimen may involve monitoring the subject being treated for changes in motor and / or cognitive impairment. Preferably, such monitoring occurs at the start of treatment. ​Includes at least one assessment before and after. Preferably, monitoring indicates a reduction in motor and / or cognitive impairment in response to treatment compared to before starting treatment or, at least indicates a reduction in the rate of decline compared to the rate of decline in a control patient who has not received any previous rate of decline or any immunotherapy in the subject. The subject may also be monitored for changes in other signs or symptoms, particularly autonomic dysfunction, gastrointestinal dysfunction, visual hallucinations or one or more psychological symptoms.

[0278] The symptoms of the subject, such as motor symptoms such as tremors, rigidity and slowness of movement, may also be monitored. A wearable system or on-body sensor may be used to assess and quantify the motor symptoms of the subject. An "on-body sensor" may be used in a laboratory setting or in free-living conditions [S. Del Din, et al., J. of NeuroEngineering and Rehabilitation, 2016 13:46].

[0279] The subject may be monitored using mobile device-based monitoring. The mobile device may be a smartphone, smartwatch, wearable sensor, portable multi media device or tablet computer. An in-built mobile device sensor may be used to record the subject's daily activities. The subject may carry a mobile device to record daily activities. Mobile device-based assessment and sensors, for example, in subjects receiving treatment for Parkinson's disease, walking and It may also be used for remote passive monitoring of mobility. For example, see Lipsmeier, F., et al.. Mov Disord. 2017; 32 (suppl 2);W. Y. Cheng e t al., 2017 IEEE / ACM International Conference on Connected Health: Applications, Systems and Engineering Technologies (CHASE), Philadelphia, PA, 2017, pp. 249-2 50. Sensor data may be analyzed by machine learning-based activity profiling. Walking and mobility may correlate with the MDS-UPDRS used in the clinic to evaluate the severity of Parkinson's disease.

[0280] Some mobile device-based monitoring provides a mobile device programmed to receive and transmit data obtained from internal and / or external sensors of the mobile device regarding movement disorders of a subject having or suspected of having synucleinopathy, and then the subject performs a series of movements to reveal the movement disorder if any, and the internal or external sensors of the device obtain data regarding the movement, (b) collecting the data transmitted from the mobile device, and (c) comparing the data obtained from the subject with control data to assess the presence or extent of movement disorder in the subject. In some mobile device-based monitoring, the mobile device is for the upper and lower limbs of the subject and may include assessing the presence or degree of movement disorder in the subject. In some mobile device-based monitoring, the mobile device is for the upper and lower limbs of the subject Receives and transmits data from at least two external sensors attached thereto and is programmed to do so. In some mobile device-based monitoring, the mobile device obtains data from sensors on the upper and lower limbs of interest. In some mobile device-based monitoring, the mobile device is carried by the subject and obtains data from internal sensors. In some mobile device-based monitoring, a series of movements includes tapping, sitting, and standing of the device.

[0281] The regimen of the present invention can be administered in combination with another agent effective in the treatment or prevention of the disease being treated. The other agent can be another immunotherapeutic agent or other agent for treating Parkinson's disease described herein, and can include levodopa, benzaseride, carbidopa, dopamine agonists, non-ergot dopamine agonists, catechol-O-methyl (「COMT」) inhibitors such as entacopone or tolcopone, monoamine oxidase (「MAO」) inhibitors such as rasagaline, amantadine, etc., and anticholinergics can be used in combination with the regimen of the present invention. Some such other agents reduce one or more symptoms of the disease without affecting the causative factor. .

[0282] All publications (including GenBank accession numbers, UniProtKB / Swiss-Prot accession numbers, etc.), patents, and patent applications cited are specifically incorporated by reference in their entirety for all purposes as if each individual publication, patent, and patent application are incorporated herein by reference in their entirety for all purposes to the same extent as if each were individually indicated. Sequences or websites associated with Genbank and UniProtKB / Swiss-Prot accession numbers, or organizations if there are any variations in the disease criteria, this application is valid as of its effective filing date are incorporated herein by reference in their entirety for all purposes to the same extent as if each were individually indicated. Sequences or websites associated with Genbank and UniProtKB / Swiss-Prot accession numbers, or organizations if there are any variations in the disease criteria, this application is valid as of its effective filing date is meant.

[0283] VIII. Examples

Examples

[0284] Phase II Clinical Trial for Alpha-Synuclein Antibody Study Design: The Phase II study is conducted on subjects with Parkinson's disease for an alpha-synuclein antibody having a heavy chain variable region specified by SEQ ID NO: 10 and a light chain variable region specified by SEQ ID NO: 5. The study has two treatment groups and one control group. Subjects are randomized to the groups at a ratio of 1:1:1 (N = 300). The initial phase of the study is a 52-week double-blind placebo-controlled treatment. During the initial phase of the study, subjects do not receive other treatments for Parkinson's disease (including symptomatic treatments ). Subjects in one treatment group receive a fixed dose of 1500 mg of the antibody intravenously every 4 weeks . Subjects in the other treatment group receive 3500 mg or 4500 mg of the antibody intravenously every 4 weeks depending on body weight, with subjects weighing less than 65 kg receiving a lower dose and subjects weighing 65 kg or more receiving a higher dose. Subjects in the second group receive a loading dose of 2000 mg and then additional escalating titration doses of 2000 mg or more until they reach the target dose of 3500 mg or 4500 mg as appropriate . The administration continues for one year. Then, the study has an extension period . Subjects in one treatment group receive a fixed dose of 1500 mg of the antibody intravenously every 4 weeks . Subjects in the other treatment group receive 3500 mg or 4500 mg of the antibody intravenously every 4 weeks depending on body weight, with subjects weighing less than 65 kg receiving a lower dose and subjects weighing 65 kg or more receiving a higher dose. Subjects in the second group receive a loading dose of 2000 mg and then additional escalating titration doses of 2000 mg or more until they reach the target dose of 3500 mg or 4500 mg as appropriate . The administration continues for one year. Then, the study has an extension period . The administration continues for one year. Then, the study has an extension period And during the extension period, initially, subjects in the placebo group receive one of two treatment regimens from the initial phase, and subjects from the initial-phase treatment group continue to receive the same treatment as before. During the extension phase of the study, subjects may receive systematic treatment using the antibodies of the study in addition to levodopa, but do not receive other treatments for Parkinson's disease.

[0285] Objective: The primary objective is to evaluate the efficacy of an alpha-synuclein antibody having a heavy-chain variable region designated by SEQ ID NO: 10 and a light-chain variable region designated by SEQ ID NO: 5 using the Movement Disorder Society (MDS)-sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS). The secondary objectives are to - evaluate the effect of the antibody on Parkinson's disease signs and symptoms, including changes in DaT-SPECT signal, and motor and cognitive impairments - evaluate the safety and tolerability of the antibody for up to 104 weeks, and - evaluate the pharmacokinetics of the antibody

Example

[0286] Passive monitoring of mobility in early-stage Parkinson's disease patients in a Phase I alpha-synuclein antibody clinical trial using smartphone sensors

[0287] 1. Summary - Walking and mobility in early-stage Parkinson's disease (PD) patients were measured using smartphone-based passive monitoring. An alpha-synuclein antibody designated by SEQ ID NO: 10 ​​​An alpha-synuclein antibody having a heavy chain variable region and a light chain variable region designated by SEQ ID NO: 5 In a multiple escalating dose clinical trial of the antibody, 44 PD patients and 35 age- and sex-matched healthy individuals were assessed for up to 24 weeks and up to 6 weeks, respectively, using a smartphone-based assessment [Lipsmeier, F., et al.. Mov Disord. 2017; 32 (suppl 2);W . Y. Cheng et al., 2017 IEEE / ACM International Conference on Connected Health: A pplications, Systems and Engineering Technologies (CHASE), Philadelphia, PA, 201 7, pp. 249-250].

[0288] For "passive monitoring", subjects carried a smartphone as part of their daily routine and continuously recorded movement data using the smartphone's sensors. A total of over 30,000 hours of passive monitoring data were collected. To classify the sensor signals into activity profiles a deep neural network (DNN) trained on previously published data was used to build a human activity recognition (HAR) model. The activity profiles of the participants determined by the HAR model showed significant differences between PD patients and healthy controls in terms of the percentage of walking time and the frequency with which the controls changed postures (sitting and standing from sitting and standing positions).

[0289] 2. Methods

[0290] A. Data collection ​The analysis focused only on investigating the differences between the HC cohort and the PD cohort and did not examine antibody -related effects. In total, 24,104 hours of passive monitoring data were recorded for the P D cohort, and 8,614 hours for the HC cohort. Rai , A et al., [MobiCom’12, August 22-26, 2012] in line with the approach, accelerometer data when the standard deviation of the Euclidean norm less than 0.03m / s 2 was longer than 30 minutes was filtered -removed, as it is likely that the smartphone was not carried by the subject during these spans. This step removed 14% of the passive monitoring data .

[0291] B. HAR used a 9-layer neural network model structure. A similar structure has been previously used for HAR and has been shown to outperform traditional machine learning methods (F. J. Ordonez and D. R oggen, Sensors 2016, 16, 115). The HAR model was trained on two public datasets (G. M . Weiss and J. W. Lockhart, Proceedings of the AAAI-12 Workshop on Activity Cont ext Representation: Techniques and Languages, Toronto, CA. 2012; A. Stisen, et a l., 13th ACM Conference on Embedded Networked Sensor Systems, Seoul, Korea, 2015 ) to classify six activities: walking, stairs, jogging, sitting, standing, and lying ​。The continuous accelerometer data was downsampled to 20 Hz and segmented into 4 - second windows where 75% of adjacent ones overlapped.

[0292] 3. Results

[0293] A. Verification of human activity recognition performance: To ensure that the HAR model could accurately translate sensor data into activity profiles, the performance of the model was first analyzed in a hold - out validation set. The HAR model was able to correctly distinguish between stationary activities (sitting, standing, lying down) and walking activities (walking, stairs, jogging) with an accuracy higher than 98%. Additional verification on labeled walking and rest data from the test data also showed that the HAR model was successful in profiling walking segments with 96.9% accuracy and rest segments with 99.5% accuracy.

[0294] B. Comparison of activity profiles The mobility of each subject was quantified by calculating the percentage of the total passive monitoring coverage time of the subject that the subject spent engaged in walking activities (walking, stairs, jogging). The overall percentages of different walking activities with respect to the total coverage for the PD and HC cohorts were calculated. In contrast to 15.1% of the HC cohort, a median walking span of 9.7% of all coverage spans was detected in the PD cohort. The HC cohort had a significantly higher level of walking activity per subject than the PD cohort, and the P - value of the Mann - Whitney test was 2.43E - 8.

[0295] C. Comparison of the number of transitions from sitting to standing and from standing to sitting One manifestation of the functional impact of PD is in the sit-to-stand and stand-to-sit (STS) events (it-to-stand and stand-to-sit). This has been observed (A. Zijlstra, et al., J. NeuroEngineering and Rehabilitation 2012 , 9:75). From the activity profiles, coverage-normalized STS events were calculated for each subject . The median number of STS per hour for PD patients was 1.44, which was significantly lower than that of the HC subjects at 1.74. The p-value of the Mann-Whitney test between the two groups was 1.60 E-8.

[0296] 4. Conclusions

[0297] The results from this study indicate that it is feasible to measure walking and mobility in early-stage PD patients using smartphone-based passive monitoring . The sensor data collected during passive monitoring provides ecologically valid insights into the patient's daily behavior and function that were previously inaccessible. Significant differences were observed between PD patients and healthy controls (HC). ​

Claims

1. Treating or preventing a subject having or at risk for a synucleinopathy The method of carrying out the method of claim 1, further comprising administering to the subject a dose of 3000 to 5000 mg of alpha-synuclein. or administering to the subject an antibody against administering another regimen that delivers the antibody to the area.

2. 2. The method of claim 1, wherein the dose is between 3500 and 4500 mg.

3. administered to a plurality of subjects having or at risk for synucleinopathy.

2. The method of claim 1, wherein the subject receives one or two fixed doses.

4. Subjects weighing less than 65 kg will receive a dose of 3,500 mg of antibody, and subjects weighing 65 kg or more will receive a dose of 3,500 mg of antibody.

4. The method of claim 3, wherein the subject having a body weight receives a dose of 4500 mg.

5. 2. The method of claim 1, wherein the dose is 45-75 mg / kg.

6. 2. The method of claim 1, wherein the dose is 50-70 mg / kg.

7. 2. The method of claim 1, wherein the interval is 28 days.

8. 10. The method of claim 1, wherein the subject receives at least 6 doses of the antibody, spaced 3-5 weeks apart.

9. 10. The method of claim 1, wherein the subject receives at least 12 doses of the antibody at intervals of 3 to 5 weeks.

10. 10. The method of claim 1, wherein the subject receives at least 18 doses of the antibody at intervals of 3 to 5 weeks.

11. 11. The method of any one of claims 8 to 10, wherein the interval is 4 weeks.

12. 10. The method of claim 1, wherein the subject receives the antibody every 4 weeks for at least 52 weeks.

13. Changes in movement, cognitive impairment, autonomic dysfunction, gastrointestinal dysfunction, visual hallucinations or psychological symptoms 10. The method of claim 1, further comprising monitoring the subject with

14. To monitor, (a) of a subject having or suspected of having a synucleinopathy Obtained from internal and / or external sensors of the mobile device related to movement disorders providing subjects with mobile devices programmed to receive and transmit data; The subjects were then asked to perform a series of exercises to reveal any motor impairments present. , sensors inside or outside the device collect data about your movement; (b) collect data transmitted from your mobile device; and (c) comparing the data obtained from the subject with the control data to identify the presence or absence of a movement disorder in the subject; To assess the presence or extent of The method of claim 13, comprising:

15. The mobile device is attached to at least two external 15. The method of claim 14, further comprising: The method described.

16. The mobile device acquires data from sensors on the subject's upper and lower extremities.

16. The method according to claim 14 or 15.

17. A mobile device is carried by a subject and acquires data from sensors inside the device. The method according to claim 14 or 15,

18. 16. The method of claim 14, wherein the sequence of movements includes tapping the device, sitting down and standing up. The method described above.

19. Monitoring may include reducing motor impairment or reducing cognitive impairment in response to administering the antibody. The method of claims 13 to 18, which indicates a reduction.

20. The antibody of claim 1, wherein the antibody binds within residues 115-130 of alpha synuclein. method.

21. The antibody of claim 1, wherein the antibody binds within residues 118-126 of alpha synuclein. method.

22. The antibody of claim 1, wherein the antibody binds within residues 117-123 of alpha synuclein. method.

23. The method of any one of claims 1 to 19, wherein the antibody is NI-202.21D11.

24. The antibody comprises three heavy chain CDRs, each of which is designated as SEQ ID NOs: 139-141, and each of which is designated as SEQ ID NOs: 139-142.

20. Any of claims 1 to 19, comprising the three light chain CDRs set forth in SEQ ID NOs: 143 to 145. Any method according to the present invention.

25. The antibody has a heavy chain variable region designated as SEQ ID NO: 138 and a heavy chain variable region designated as SEQ ID NO:

142.

20. The method of any one of claims 1 to 19, comprising a light chain variable region.

26. The antibody comprises three light chain Kabat CDRs of SEQ ID NO:5 and three heavy chain Kabat CDRs of SEQ ID NO:

10.

2. The method of claim 1 , comprising a Kabat CDR.

27. The antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 8 to 11 and a heavy chain variable region of any one of SEQ ID NOs: 3 to 5. and preferably the heavy chain variable region is the heavy chain variable region of SEQ ID NO:

10. The method of claim 1, wherein the light chain variable region is the light chain variable region of SEQ ID NO:

5.

28. 28. The method of claim 1, wherein the antibody is of the human IgG1 isotype. method.

29. The antibody comprises a heavy chain constant region of SEQ ID NO:35, optionally without the C-terminal lysine of SEQ ID NO:

35.

29. The method of claim 28, comprising a light chain constant region and a light chain constant region of SEQ ID NO:

30.

30. The antibody comprises a heavy chain constant region of SEQ ID NO:35, optionally without the C-terminal lysine of SEQ ID NO:

35. The method of claim 28, comprising a light chain constant region and a light chain constant region of SEQ ID NO:

13.

31. 22. The method of any one of claims 1 to 21, wherein the antibody is 9E4.

32. The antibody comprises three heavy chain CDRs designated as SEQ ID NOs: 146-148, respectively, and 22. Any of claims 1 to 21, comprising the three light chain CDRs set forth in SEQ ID NOs: 149 to 151. Any method according to the present invention.

33. the antibody comprises a heavy chain designated as SEQ ID NO:37 and a light chain designated as SEQ ID NO:32; 22. The method according to claim 1, wherein the C-terminal lysine of SEQ ID NO: 37 is optionally absent. How to.

34. The method of claim 1, wherein the antibody binds within residues 1-20 of alpha-synuclein. 。

35. The method of claim 34, wherein the antibody binds within residues 4-15 of alpha-synuclein. Law.

36. The method of any one of claims 1 to 19, wherein the antibody is NI-202.12F4.

37. The antibody comprises three heavy chain CDRs, each of which is designated as SEQ ID NOs: 131-133, and each of which is designated as SEQ ID NOs: 132-134.

20. Any of claims 1 to 19, comprising the three light chain CDRs set forth in SEQ ID NOs: 135 to 137. Any method according to the present invention.

38. The antibody has a heavy chain variable region designated as SEQ ID NO: 130 and a heavy chain variable region designated as SEQ ID NO:

134.

20. The method of any one of claims 1 to 19, comprising a light chain variable region.

39. The subject has a body weight of less than 65 kg and is administered a dose of 3500 mg of the antibody every 4 weeks; 39. The method of any one of claims 1 to 38.

40. A loading dose of 2000 mg of antibody was administered prior to administration of the antibody in the range of 3000-5000 mg. The body is then dosed with 2000 mg or more, but not more than 3500 mg, until a dose of 3500 mg is reached. Optionally, an upward titration may be performed with one or more subsequent doses that are less than 1 g, all of which are 40. The method of any one of claims 1 to 39, separated by an interval of 3 to 5 weeks.

41. The 3500 mg dose is administered at the next interval after the 2000 mg dose.

41. The method according to claim 40.

42. The dose is gradually increased over multiple intervals prior to administration of a dose of 3500 mg.

41. The method according to claim 40.

43. The subject weighs more than 65 kg and is administered a dose of 4500 mg of the antibody every 4 weeks.

39. The method of any one of claims 1 to 38,

44. A loading dose of 2000 mg of antibody was administered prior to administration of 4500 mg of antibody. One or more doses of 2000 mg or more but less than 4500 mg are taken until a dose of 0 mg is reached. An escalation titration with one or more subsequent doses may be performed, with all doses administered for 3 to 5 days.

39. The method of any one of claims 1 to 38, wherein the first and second electrodes are separated by a partition.

45. The 4500 mg dose is administered at the next interval after the 2000 mg dose.

45. The method according to claim 44.

46. The dose is gradually increased over multiple intervals prior to administration of a 4500 mg dose.

45. The method according to claim 44.

47. Changes in movement, cognitive impairment, autonomic dysfunction, gastrointestinal dysfunction, visual hallucinations or psychological symptoms 47. The method of any of claims 44 to 46, further comprising monitoring the subject with

48. To monitor, (a) of a subject having or suspected of having a synucleinopathy Obtained from internal and / or external sensors of the mobile device related to movement disorders providing subjects with mobile devices programmed to receive and transmit data; The subjects were then asked to perform a series of exercises to reveal any motor impairments present. , sensors inside or outside the device collect data about your movement; (b) collect data transmitted from your mobile device; and (c) comparing the data obtained from the subject with the control data to identify the presence or absence of a movement disorder in the subject; To assess the presence or extent of 48. The method of claim 47, comprising:

49. The mobile device is attached to at least two external 49. The method of claim 48, further comprising the steps of: The method described.

50. The mobile device acquires data from sensors on the subject's upper and lower extremities.

50. The method according to claim 48 or 49.

51. A mobile device is carried by the subject and acquires data from an internal sensor.

50. The method of claim 48 or 49.

52. 50. The method of claim 48 or 49, wherein the sequence of movements includes tapping the device, sitting down and standing up. The method according to

53. Monitoring may include reducing motor impairment or reducing cognitive impairment in response to administering the antibody.

53. The method of any of claims 47 to 52, indicating a reduction.

54. 54. The method of any one of claims 1 to 53, wherein the synucleinopathy is Parkinson's disease.

55. 54. The method according to any one of claims 1 to 53, wherein the synucleinopathy is dementia with Lewy bodies. Method of posting.

56. 54. The method of any one of claims 1 to 53, wherein the synucleinopathy is multiple system atrophy.

57. 54. The method of any one of claims 1 to 53, wherein the synucleinopathy is progressive supranuclear palsy. 。

58. The method according to any one of claims 1 to 53, wherein the synucleinopathy is REM sleep behavior disorder. Law.

59. 1 to 5, wherein the synucleinopathy is Alzheimer's disease accompanied by Lewy bodies in the amygdala.

53. A method according to any one of claims 53 to 53.

60. 60. The method of any of claims 1 to 59, wherein the subject has the disease.

61. 10. The method of claim 1, wherein the subject is not receiving symptomatic treatment for Parkinson's disease in combination with the antibody.

61. A method according to any one of claims 60 to 60.

62. 61. The method of any of claims 1-60, wherein the antibody is administered in combination with levodopa.

63. 1. A method of treating or preventing a subject having Lewy body disease, comprising administering to the subject 1300 to 1500 mg of Antibody against alpha-synuclein was administered intravenously at a dose of 1700 mg at 3-5 week intervals. or administering to the subject another regimen that delivers the antibody with substantially the same area under the curve. The method includes:

64. 64. The method of claim 63, wherein the dose is 1500 mg.

65. A method for administering a therapeutic agent to a plurality of subjects having Lewy body disease, the subjects receiving one or two fixed doses of the therapeutic agent.

64. The method of claim 63,

66. 64. The method of claim 63, wherein the subject receives a dose of 18-25 mg / kg.

67. 67. The method of claim 66, wherein the subject receives a dose of 20 mg / kg.

68. 64. The method of claim 63, wherein the interval is 28 days.

69. 64. The method of claim 63, wherein the subject receives at least 6 doses of the antibody at intervals of 3 to 5 weeks.

70. 64. The method of claim 63, wherein the subject receives at least 12 doses of the antibody at intervals of 3-5 weeks. 。

71. 64. The method of claim 63, wherein the subject receives at least 18 doses of the antibody at intervals of 3-5 weeks. 。

72. 71. The method of any one of claims 68 to 70, wherein the interval is 4 weeks.

73. 64. The method of claim 63, wherein the subject receives the antibody every 4 weeks for at least 52 weeks.

74. Changes in movement, cognitive impairment, autonomic dysfunction, gastrointestinal dysfunction, visual hallucinations or psychological symptoms 64. The method of claim 63, further comprising monitoring the subject with

75. To monitor, (a) movement of a subject having or suspected of having Lewy body disease Data obtained from internal and / or external sensors of the mobile device regarding the fault provide users with mobile devices programmed to receive and transmit data, The subjects then performed a series of exercises to reveal any motor deficits, if any, and sensors inside or outside the device to capture data about your movements; (b) collect data transmitted from your mobile device; and (c) comparing the data obtained from the subject with the control data to identify the presence or absence of a movement disorder in the subject; To assess the presence or extent of 75. The method of claim 74, comprising:

76. The mobile device is attached to at least two external 76. The method of claim 75, further comprising: The method described.

77. The mobile device acquires data from sensors on the subject's upper and lower extremities.

77. The method according to claim 75 or 76.

78. A mobile device is carried by the subject and acquires data from an internal sensor.

77. The method of claim 75 or 76.

79. 77. The method of claim 75, wherein the sequence of movements includes tapping the device, sitting down and standing up. The method described above.

80. Monitoring may include reducing motor impairment or reducing cognitive impairment in response to administering the antibody.

80. The method of any of claims 74 to 79, indicating a reduction.

81. 64. The method of claim 63, wherein the antibody binds within residues 115-130 of alpha synuclein. How to.

82. 64. The method of claim 63, wherein the antibody binds within residues 118-126 of alpha synuclein. How to.

83. 64. The method of claim 63, wherein the antibody binds within residues 117-123 of alpha synuclein. How to.

84. The method according to any one of claims 63 to 80, wherein the antibody is NI-202.21D11. 。

85. The antibody comprises three heavy chain CDRs, each of which is designated as SEQ ID NOs: 139-141, and each of which is designated as SEQ ID NOs: 139-142.

81. The method of claim 63 to 80, comprising the three light chain CDRs set forth in SEQ ID NOs: 143 to 145. Any method as described above.

86. The antibody has a heavy chain variable region designated as SEQ ID NO: 138 and a heavy chain variable region designated as SEQ ID NO:

142.

81. The method of any of claims 63 to 80, comprising a light chain variable region.

87. The antibody comprises three light chain Kabat CDRs of SEQ ID NO:5 and three heavy chain Kabat CDRs of SEQ ID NO:

10.

64. The method of claim 63, comprising Kabat CDRs.

88. The antibody comprises a heavy chain variable region of any one of SEQ ID NOs: 8 to 11 and a heavy chain variable region of any one of SEQ ID NOs: 3 to 5. and preferably the heavy chain variable region is the heavy chain variable region of SEQ ID NO:

10. The method of claim 63, wherein the light chain variable region is the light chain variable region of SEQ ID NO:

5.

89. 89. The method of claim 63, wherein the antibody is of the human IgG1 isotype. How to.

90. The antibody comprises a heavy chain constant region of SEQ ID NO: 35, optionally without the C-terminal lysine, and The method of claim 89, comprising the light chain constant region of sequence number 30.

91. The antibody comprises a heavy chain constant region of SEQ ID NO: 35, optionally without the C-terminal lysine, and The method of claim 89, comprising the light chain constant region of sequence number 13.

92. The antibody comprises three heavy chain CDRs designated as SEQ ID NOs: 146-148, respectively, and 83. The method of claim 63 to 82, comprising the three light chain CDRs set forth in SEQ ID NOs: 149 to 151. Any method as described above.

93. the antibody comprises a heavy chain designated as SEQ ID NO:37 and a light chain designated as SEQ ID NO:32; 83. The method of any one of claims 63 to 82, wherein the C-terminal lysine of SEQ ID NO: 37 is optionally absent. Method of posting.

94. The method of claim 63, wherein the antibody binds within residues 1-20 of alpha-synuclein. Law.

95. The method of claim 94, wherein the antibody binds within residues 4-15 of alpha-synuclein. Law.

96. The method of any one of claims 63 to 80, wherein the antibody is NI-202.12F4.

97. The antibody comprises three heavy chain CDRs, each of which is designated as SEQ ID NOs: 131-133, and each of which is designated as SEQ ID NOs: 132-134.

81. The method of claim 63 to 80, comprising the three light chain CDRs set forth in SEQ ID NOs: 135 to 137. Any method as described above.

98. The antibody has a heavy chain variable region designated as SEQ ID NO: 130 and a heavy chain variable region designated as SEQ ID NO:

134.

81. The method of any of claims 63 to 80, comprising a light chain variable region.

99. 99. The method of any of claims 63 to 98, wherein the synucleinopathy is Parkinson's disease. 。

100. 99. The method according to any one of claims 63 to 98, wherein the synucleinopathy is dementia with Lewy bodies. The method described.

101. 99. The method of any one of claims 63 to 98, wherein the synucleinopathy is multiple system atrophy.

102. The method according to any one of claims 63 to 98, wherein the synucleinopathy is progressive supranuclear palsy. Law.

103. 99. The method of any of claims 63 to 98, wherein the synucleinopathy is REM sleep behavior disorder. method.

104. 63. The method according to claim 62, wherein the synucleinopathy is Alzheimer's disease associated with Lewy bodies in the amygdala.

98. The method according to any one of claims 98 to 98.

105. 105. The method of any of claims 63 to 104, wherein the subject has the disease.

106. 63. The subject is not receiving symptomatic treatment for Parkinson's disease in combination with the antibody.

105. The method of claim 10, wherein

107. The method of any of claims 63 to 105, wherein the antibody is administered in combination with levodopa. Law.