Anti-A-beta protein antibodies, methods and uses thereof

Abstract

Herein is reported an antibody that binds to human A-beta protein wherein the antibody comprises a heavy chain variable domain (VH) and a light chain variable domain, the heavy chain variable domain and the light chain variable domain comprising a CDR selected from the group consisting of: (1) a CDR of SEQ ID NO: 85, 86, 87, 81, 82 and 83; or (2) a CDR of SEQ ID NO: 85, 89, 87, 81, 82, and 83; or (3) a CDR of SEQ ID NO: 85, 86, 87, 81, 82 and 91; or (4) CDRs of SEQ ID NO: 85, 89, 87, 81, 82 and 91.

Description

[0001] This invention relates to antibodies against human A-β protein (anti-A-β protein antibodies), methods for their preparation, pharmaceutical compositions containing these antibodies, and their uses. Compared with known anti-A-β protein antibodies, the antibodies according to the present invention exhibit improved technical and biological properties. Background Technology

[0002] Approximately 70% of all dementia cases are caused by Alzheimer's disease (AD), which is associated with selective damage to brain regions and neural circuits crucial for cognition. AD is characterized by neurofibrillary tangles (particularly in pyramidal neurons in the hippocampus) and numerous amyloid plaques, which primarily contain a dense core of amyloid deposits and defusion loops.

[0003] Extracellular neuritis plaques contain a large number of predominantly fibrous peptides called "amyloid protein," "amyloid β," "A-β," "Aβ4," "β-A4," or "Aβ"; see Selkoe, Ann. Rev. Cell Biol. 10 (1994) 373-403; Koo, Proc. Natl. Acad. Sci. USA 96 (1999) 9989-9990; US 4,666,829; Glenner BBRC 12 (1984) 1131). This amyloid protein is derived from "Alzheimer's precursor protein / β-amyloid precursor protein" (APP). APP is a complete membrane glycoprotein (see Sisodia, Proc. Natl. Acad. Sci. USA 89(1992) 6075), and is cleaved by a plasma membrane protease (α-secretase) within the AP sequence via endonuclease hydrolysis (see Sisodia (1992), ibid.). Furthermore, further secretase activity, particularly β-secretase and γ-secretase activity, leads to the extracellular release of amyloid-β (Aβ) containing 39 amino acids (Aβ39), 40 amino acids (Aβ40), 41 amino acids (Aβ41), 42 amino acids (Aβ42), or 43 amino acids (Aβ43) (see Sinha, Proc. Natl. Acad. Sci. 96 (1999) 11094-1053; Price, Science 282 (1998) 1078-1083; WO 00 / 72880 or Hardy, TINS ​​20 (1997) 154).

[0004] It is worth noting that the A-β protein exists in several naturally occurring forms, among which the human forms are designated Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43, as described above. The most important form, Aβ42, has the following amino acid sequence (starting from the N-terminus): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV VIA (SEQ ID NO: 45). In Aβ41, Aβ40, and Aβ39, the C-terminal amino acids A, IA, and VIA are deleted, respectively. In Aβ43, additional threonine residues are contained at the C-terminus of the sequence depicted above.

[0005] Nucleation of Aβ40 fibrils has been shown to take significantly longer than that of Aβ42 fibrils (see, for example, Lansbury, Jr., PT and Harper, JD, Ann. Rev. Biochem. 66(1997) 385-407). As reviewed in Wagner (J. Clin. Invest. 104(1999) 1239-1332), Aβ42 is more frequently found to be associated with neuritis plaques and is considered to be more fibrogenic in vitro. Aβ42 has also been suggested to act as a “seed” for the nucleation-dependent polymerization of ordered, non-crystalline Aβ peptides (see, for example, Jarrett, Cell 93(1993) 1055-1058). The formation of extracellular plaques with modified APP processing and / or protein deposits is known not only in Alzheimer's pathology but also in subjects with other neurological and / or neurodegenerative disorders. These disorders include, among others, Down syndrome, Dutch hemorrhage with amyloidosis, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Creutzfeldt-Jakob disease, HIV-related dementia, and motor neuron disease.

[0006] To date, only a limited range of medical interventions have been described for amyloid-related diseases. For example, cholinesterase inhibitors such as galantamine, rivastigmine, or donepezil have been discussed as beneficial for Alzheimer's patients with only mild to moderate disease. However, adverse events have been reported due to the cholinergic effects of these drugs. While these cholinergic-enhancing therapies do produce some symptomatic benefits, the treatment response is unsatisfactory for most patients receiving treatment. It is estimated that only about 5% of treated patients experience significant cognitive improvement, and there is little evidence that treatment significantly alters the course of this progressive disease.

[0007] Therefore, there remains a huge clinical need for more effective treatments, especially those that can stop or slow disease progression.

[0008] In addition, NMDA receptor antagonists, such as memantine, have been used. However, adverse events have been reported due to their pharmacological activity. Furthermore, this treatment with these NMDA receptor antagonists may be considered merely a symptomatic approach rather than a method for improving the condition.

[0009] Furthermore, immunomodulatory approaches for treating amyloid-related disorders have been proposed. WO 99 / 27944 discloses a conjugate comprising several portions of an A-β peptide and a carrier molecule, wherein the carrier molecule is intended to enhance the immune response. WO 00 / 72880 mentions another active immunization approach, which also uses an A-β fragment to induce an immune response.

[0010] Furthermore, passive immunization methods using general anti-A-β antibodies have been proposed in WO 99 / 27944 or WO 01 / 62801, and specific humanized antibodies against several parts of A-β have been described in WO 02 / 46237, WO 02 / 088306, and WO 02 / 088307. WO 00 / 77178 describes an antibody that binds to the transitional state adopted by β-amyloid protein during hydrolysis. WO 03 / 070760 discloses an antibody molecule that recognizes two discontinuous amino acid sequences on the A-β peptide.

[0011] WO 2014 / 033074 relates to blood-brain barrier shuttles that bind receptors on the blood-brain barrier and their methods of use. Blood-brain barrier drug delivery of IgG fusion proteins with monoclonal antibodies against transferrin receptors has been reported by Pardridge, W. et al. (Exp. Opin. Drug Deliv. 12 (2015) 207-222) and Yu, YJ et al. (Sci. Translat. Med. 6 (2014) 261ra154-261ra154). They reported the crossing of the blood-brain barrier by therapeutic bispecific antibodies in non-human primates. Sumbria, RK et al. (Mol. Pharm. 10 (2013) 3507-3513) reported the breakdown of amyloid plaques in the brains of transgenic Alzheimer's mice after daily subcutaneous administration of a tetravalent bispecific antibody targeting transferrin receptors and Aβ amyloid peptides. Niewoehner, J. et al. (Neuron 81(2014) 49-609) reported increased brain penetration and potency of therapeutic antibodies using monovalent molecular shuttles.

[0012] WO 2016 / 207240 reports an anti-transferrin receptor antibody with a designed dissociation rate targeting the human transferrin receptor and its use as a blood-brain barrier shuttle module.

[0013] WO 2017 / 055542 reports trivalent bispecific antibodies against human CD20 and human transferrin receptor, their production methods, pharmaceutical compositions containing these antibodies, and their uses.

[0014] WO 2007 / 068429 reports antibodies against amyloid β with glycosylation in the variable region. The purified antibody molecule formulation is characterized by at least one antigen-binding site comprising glycosylated asparagine (Asn) in the variable region (VH) of the heavy chain. WO 2007 / 068429 also reports mixtures of antibodies containing one or two glycosylated antigen-binding sites having glycosylated asparagine (Asn) in the variable region of the heavy chain, i.e., isotype mixtures of antibodies containing glycosylated Asn in the variable region (VH) of the heavy chain.

[0015] WO 2017 / 055540 reports trivalent bispecific antibodies against human A-β and human transferrin receptors, their production methods, pharmaceutical compositions containing these antibodies, and their uses.

[0016] EP 2 368 907 reports on anti-Aβ antibodies and their uses.

[0017] Edwards, E. et al. reported a strategy for controlling the glycosylation of therapeutic antibodies during bioprocessing (Biotechnol. Bioeng. 119 (2022) 1343-1358). Summary of the Invention

[0018] This article reports antibodies against human A-β protein (anti-A-β protein antibodies), their production methods, pharmaceutical compositions containing these antibodies, and their uses.

[0019] The antibodies according to the invention are variants of the anti-A-β antibody gantenerumab. Compared to their parent antibodies, they possess improved technical and biological properties. Among other improvements are improved manufacturing characteristics, such as improved production titers, improved production yields, and improved process robustness.

[0020] This invention is based, at least in part, on the discovery that the properties of gantingrulimab have been improved by introducing specific mutations in the heavy chain CDR2 and by shortening the heavy chain CDR3. Without being bound by this theory, it is assumed that the introduced modifications improve properties by reducing the heterogeneity of glyco-occupation in the Fab and removing deamidation hotspots, thereby reducing the aggregation tendency of the parental antibody, gantingrulimab.

[0021] The present invention is based at least in part on the discovery that when combined with the modified heavy chain according to the present invention, the light chain of the parent antibody gintinguishumab does not require modification in order to maintain binding affinity and specificity.

[0022] The anti-A-β protein antibody according to the present invention has a significant sugar occupancy of up to 100% of the glycosylation sites in the heavy chain CDR2.

[0023] Among other things, the anti-A-β protein antibody according to the present invention has improved properties in terms of target binding, exploitability, IHC / plaque binding and PK behavior.

[0024] This invention covers at least the following embodiments:

[0025] 1. An antibody that binds to human A-β protein, wherein the antibody has one or more of the following properties:

[0026] -Specifically binds to human A-β protein of SEQ ID NO: 45;

[0027] -Has a glycosylation site in the heavy chain CDR2, which has a sugar occupancy of at least 95% as determined by CE-SDS;

[0028] - Species that do not contain any non-glycosylated glycosylation sites;

[0029] - Having an EC50 value of 0.5 nM or less for human A-β protein of SEQ ID NO: 45;

[0030] - At staining concentrations of up to and including 1 μg / mL, it binds to A-β plaques in vitro and does not bind to non-A-β protein molecules in brain samples;

[0031] - Compared with gantinguishumab, it binds more strongly to A-β plaques in vivo;

[0032] -As determined by one-way ANOVA, the relative in vivo occupancy of plaques in the cortex and hippocampus exceeds 0.2;

[0033] - Exhibits thermal stability exceeding 68°C (DLS T) agg / DLS T m ).

[0034] 2. An antibody that binds to human A-β protein, wherein the antibody comprises a heavy chain variable domain (VH) and a light chain variable domain, the heavy chain variable domain and the light chain variable domain comprising CDRs selected from: (1)

[0036] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0037] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86;

[0038] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0039] (d) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0040] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0041] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0042] or (2)

[0044] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0045] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89;

[0046] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0047] (d) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0048] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0049] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0050] or (3)

[0052] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0053] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86;

[0054] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0055] (d) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0056] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0057] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91;

[0058] or (4)

[0060] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0061] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89;

[0062] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0063] (d) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0064] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0065] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91.

[0066] 3. The antibody according to Example 2, wherein the antibody has one or more of the following characteristics:

[0067] -Specifically binds to human A-β protein of SEQ ID NO: 45;

[0068] -Has a glycosylation site in the heavy chain CDR2, which has a sugar occupancy of at least 95% as determined by CE-SDS;

[0069] - Species that do not contain any non-glycosylated glycosylation sites;

[0070] - Having an EC50 value of 0.5 nM or less for human A-β protein of SEQ ID NO: 45;

[0071] - At staining concentrations of up to and including 1 μg / mL, it binds to A-β plaques in vitro and does not bind to non-A-β protein molecules in brain samples;

[0072] - Compared with gantinguishumab, it binds more strongly to A-β plaques in vivo;

[0073] -As determined by one-way ANOVA, the relative in vivo occupancy of plaques in the cortex and hippocampus exceeds 0.2;

[0074] - Exhibits thermal stability exceeding 68°C (DLS T) agg / DLS T m ).

[0075] 4. The antibody according to any one of Examples 1 to 3, wherein the antibody is a monoclonal antibody.

[0076] 5. The antibody according to any one of Examples 1 to 4, wherein the antibody is a humanized antibody or a chimeric antibody.

[0077] 6. The antibody according to any one of Examples 1 to 5, wherein the antibody is an antibody fragment that binds to human A-β protein.

[0078] 7. The antibody according to any one of Examples 1 to 6, wherein the antibody comprises:

[0079] (a) A VH sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 84;

[0080] (b) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 80; or

[0081] (c) VH sequences as defined in (a) and VL sequences as defined in (b).

[0082] 8. The antibody according to any one of Examples 1 to 7, wherein the antibody comprises the VH sequence of SEQ ID NO: 84 and the VL sequence of SEQ ID NO: 80.

[0083] 8a. The antibody according to any one of Examples 1 to 7, wherein the antibody comprises: the VH sequence of SEQ ID NO: 84, wherein a pyroglutamic acid (pE) residue replaces a glutamine (Q) residue as the first N-terminal amino acid residue; and the VL sequence of SEQ ID NO: 80.

[0084] 9. An antibody that specifically binds to human A-β protein, comprising the VH sequence of SEQ ID NO:84 and the VL sequence of SEQ ID NO:80.

[0085] 10. The antibody according to any one of Examples 1 to 9, wherein the antibody is:

[0086] a) Full-length antibody against the human IgG1 subclass.

[0087] b) Full-length antibody against human IgG4 subclass.

[0088] c) Full-length antibodies against the human IgG1 subclass, containing mutants L234A, L235A, and P329G.

[0089] d) A full-length antibody against the human IgG1 subclass, containing mutations L234A, L235A, and P329G in both heavy chains, and mutations T366W and S354C in one heavy chain, and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0090] e) A full-length antibody against the human IgG1 subclass, containing mutations L234A, L235A, and P329G in both heavy chains, and mutations T366W and Y349C in one heavy chain, and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain.

[0091] f) A full-length antibody against the human IgG4 subclass, containing mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0092] g) A full-length antibody against the human IgG4 subclass, containing mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain.

[0093] h) A full-length antibody against the human IgG1 subclass, containing mutations L234A, L235A, P329G, I253A, H310A, and H435A in both heavy chains, and mutations T366W and S354C in one heavy chain, and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0094] i) A full-length antibody against the human IgG1 subclass, containing mutations L234A, L235A, P329G, I253A, H310A, and H435A in both heavy chains, and mutations T366W and Y349C in one heavy chain, and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain.

[0095] j) A full-length antibody against the human IgG1 subclass, containing mutations L234A, L235A, P329G, M252Y, S254T, and T256E in both heavy chains, and mutations T366W and S354C in one heavy chain, and mutations T366S, L368A, Y407V, and Y349C in the corresponding other heavy chain.

[0096] k) A full-length antibody against the human IgG1 subclass, containing mutations L234A, L235A, P329G, M252Y, S254T, and T256E in both heavy chains, and mutations T366W and Y349C in one heavy chain and mutations T366S, L368A, Y407V, and S354C in the corresponding other heavy chain, or

[0097] l) A full-length antibody against a human IgG1 subclass, possessing mutations L234A, L235A, P329G, H310A, H433A, and Y436A in both heavy chains, and possessing mutations i) T366W and ii) S354C or Y349C in one heavy chain, and mutations i) T366S, L368A, and Y407V and ii) Y349C or S354C in the corresponding other heavy chain, or

[0098] One of m)a) to l) and not containing a C-terminal lysine residue.

[0099] 10a. The antibody according to Example 10, wherein the antibody mediates the in vitro uptake of Aβ-coated beads by iPSC-derived microglia.

[0100] 10b. The antibody according to Example 10a, wherein the antibody has an Fc region of c), d), e), h), i), j), k), or l).

[0101] 11. The antibody according to any one of Examples 1 to 10b, wherein the antibody binds to the human A-β protein of SEQ ID NO: 45 with an affinity of 0.4 nM or less as determined / measured by surface plasmon resonance.

[0102] 12. The antibody according to any one of Examples 1 to 11, wherein the antibody is a full-length antibody of the human IgG1 subclass.

[0103] 13. The antibody according to any one of Examples 1 to 12, wherein the antibody comprises: i) a heavy chain comprising a heavy chain variable domain of SEQ ID NO: 84 and a heavy chain constant domain of SEQ ID NO: 01; and ii) a light chain comprising a light chain variable domain of SEQ ID NO: 80 and a light chain constant domain of SEQ ID NO: 29.

[0104] 13a. The antibody according to any one of claims 1 to 12, wherein the antibody comprises: a heavy chain comprising a heavy chain variable domain of SEQ ID NO: 84 and a heavy chain constant domain of SEQ ID NO: 01, wherein a pyroglutamic acid (pE) residue replaces a glutamine (Q) residue as a first N-terminal amino acid residue in the heavy chain variable domain; and a light chain comprising a light chain variable domain of SEQ ID NO: 80 and a light chain constant domain of SEQ ID NO: 29.

[0105] 14. The antibody according to any one of Examples 1 to 13a, wherein the antibody is a multispecific antibody.

[0106] 15. The antibody according to any one of Examples 1 to 14, wherein the antibody is a multispecific antibody, the multispecific antibody comprising at least one binding site that binds to human A-β protein and at least one binding site that does not bind to human A-β protein / second non-human A-β protein target.

[0107] 16. The antibody according to any one of Examples 14 to 15, wherein the at least one binding site that does not bind to human A-β protein binds to epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), α-synuclein, CD20, amyloid precursor protein (APP), glucocerebrosidase, human transferrin receptor 1, or human TREM2 protein.

[0108] 17. The antibody according to any one of Examples 14 to 16, wherein the bispecific antibody

[0109] i) Binds to both human A-β protein and human transferrin receptor, or

[0110] ii) Binds to both human A-β protein and human TREM2 protein, or

[0111] iii) Binds to both human A-β protein and human CD20 protein, or

[0112] iv) Binds to both human A-β protein and human α-synuclein, or

[0113] v) binds to both human A-β protein and human phospho-tau protein, or

[0114] vi) It binds to both human A-β protein and glucocerebroside lipase.

[0115] 18. The antibody according to any one of Examples 1 to 17, wherein the antibody is a bispecific antibody, the bispecific antibody comprising:

[0116] i) A first binding site comprising the heavy chain variable domain of SEQ ID NO: 84 and the light chain variable domain of SEQ ID NO: 80.

[0117] as well as

[0118] ii) Selected from the following second binding sites:

[0119] a) The heavy chain variable domain of SEQ ID NO: 68 and the light chain variable domain of SEQ ID NO: 72, or

[0120] b) The heavy chain variable domain of SEQ ID NO: 54 and the light chain variable domain of SEQ ID NO: 55, or

[0121] c) The heavy chain variable domain of SEQ ID NO: 56 and the light chain variable domain of SEQ ID NO: 57, or

[0122] d) The heavy chain variable domain of SEQ ID NO: 58 and the light chain variable domain of SEQ ID NO: 59, or

[0123] e) The heavy chain variable domain of SEQ ID NO: 60 and the light chain variable domain of SEQ ID NO: 61, or

[0124] f) The heavy chain variable domain of SEQ ID NO: 62 and the light chain variable domain of SEQ ID NO: 63, or

[0125] g) The heavy chain variable domain of SEQ ID NO: 64 and the light chain variable domain of SEQ ID NO: 65, or

[0126] h) The heavy chain variable domain of SEQ ID NO: 66 and the light chain variable domain of SEQ ID NO: 67, or

[0127] i) The heavy chain variable domain of SEQ ID NO: 92 and the light chain variable domain of SEQ ID NO: 93, or

[0128] j) The heavy chain variable domain of SEQ ID NO: 94 and the light chain variable domain of SEQ ID NO: 95, or

[0129] k) The heavy chain variable domain of SEQ ID NO: 96 and the light chain variable domain of SEQ ID NO: 97, or

[0130] l) The heavy chain variable domain of SEQ ID NO: 98 and the light chain variable domain of SEQ ID NO: 99, or

[0131] m)The heavy chain variable domain of SEQ ID NO: 100 and the light chain variable domain of SEQ ID NO: 101, or

[0132] n) The heavy chain variable domain of SEQ ID NO: 102 and the light chain variable domain of SEQ ID NO: 103, or

[0133] o) The heavy chain variable domain of SEQ ID NO: 104 and the light chain variable domain of SEQ ID NO: 105, or

[0134] p) The heavy chain variable domain of SEQ ID NO: 106 and the light chain variable domain of SEQ ID NO: 107, or

[0135] q) The heavy chain variable domain of SEQ ID NO: 108 and the light chain variable domain of SEQ ID NO: 109, or

[0136] r) The heavy chain variable domain of SEQ ID NO: 110 and the light chain variable domain of SEQ ID NO: 111, or

[0137] s) The heavy chain variable domain of SEQ ID NO: 112 and the light chain variable domain of SEQ ID NO: 113, or

[0138] t)The heavy chain variable domain of SEQ ID NO: 114 and the light chain variable domain of SEQ ID NO: 115, or

[0139] u) The heavy chain variable domain of SEQ ID NO: 116 and the light chain variable domain of SEQ ID NO: 117, or

[0140] v) The heavy chain variable structural domain of SEQ ID NO:118 and the light chain variable structural domain of SEQ ID NO:119.

[0141] 18a. The antibody according to any one of Examples 1 to 18, wherein the antibody specifically binds to human A-β protein, or wherein the antibody specifically binds to both human A-β protein and human TREM2.

[0142] 18b. The antibody according to any one of Examples 1 to 18a, wherein the antibody is a bivalent antibody.

[0143] 18c. The antibody according to any one of Examples 14 to 18b, wherein the antibody is a bivalent, trivalent, or tetravalent antibody.

[0144] 18d. The antibody according to any one of Examples 14 to 18c, wherein the antibody is a bivalent bispecific antibody.

[0145] 18e. The antibody according to Example 18d, wherein the antibody is a bivalent bispecific antibody in the CrossMab format.

[0146] 18f. The antibody according to any one of Examples 14 to 18e, wherein the antibody binds to human A-β protein and human TREM2 protein.

[0147] 18g. The antibody according to any one of Examples 14 to 18e, wherein the antibody binds to human A-β protein and human transferrin receptor 1.

[0148] 18h. The antibody according to any one of Examples 14 to 18c, wherein the antibody is a trivalent bispecific antibody.

[0149] 18i. The antibody according to Example 18h, wherein the antibody is a trivalent bispecific antibody in BS or TCB format.

[0150] 18j. The antibody according to any one of Examples 14 to 18c and 18g to 18h, wherein the antibody binds to human A-β protein and human transferrin receptor 1.

[0151] 18k. The antibody according to any one of Examples 14 to 18c, wherein the antibody is a tetravalent bispecific antibody.

[0152] 18l. The antibody according to Example 18k, wherein the antibody is a tetravalent bispecific antibody in a 2+2 configuration.

[0153] 18m. The antibody according to any one of Examples 14 to 18c and 18k to 18l, wherein the antibody binds to human A-β protein and human TREM2 protein.

[0154] 18n. The antibody according to any one of Examples 14 to 18c and 18k to 18l, wherein the antibody binds to human A-β protein and human transferrin receptor 1.

[0155] 18o. The antibody according to any one of Examples 14 to 18c, wherein the antibody is a trivalent trispecific antibody, wherein the antibody binds to targets of human A-β protein, human transferrin receptor 1, and a third non-human A-β and non-human transferrin receptor 1.

[0156] 18p. The antibody according to any one of Examples 14 to 18c and 18o, wherein the antibody is a trivalent trispecific antibody, wherein the antibody binds to human A-β protein, human transferrin receptor 1 and human TREM2.

[0157] 18q. The antibody according to any one of Examples 18o to 18p, wherein the antibody is a trivalent trispecific antibody in BS format.

[0158] 18r. The antibody according to any one of Examples 18o to 18p, wherein the antibody is a trivalent trispecific antibody in TCB format.

[0159] 19. An immunoconjugate comprising an antibody according to any one of Examples 1 to 18r, and a cytotoxic agent.

[0160] 20. An isolated nucleic acid molecule encoding an antibody according to any one of Examples 1 to 18r.

[0161] 21. A composition of isolated nucleic acid molecules, wherein each of the isolated nucleic acid molecules in the composition encodes a chain of an antibody according to any one of Examples 1 to 18.

[0162] 22. A host cell comprising a nucleic acid molecule according to Example 20 or a composition of a nucleic acid molecule according to Example 21.

[0163] 23. A method for generating an antibody that binds to human A-β protein, the method comprising culturing host cells as described in Example 22 in a culture medium under conditions suitable for antibody expression.

[0164] 24. The method according to Example 23, further comprising the step of recovering the antibody from the host cell and / or the culture medium.

[0165] 25. The method according to any one of Examples 23 to 24, further comprising the step of purifying the antibody using one or more chromatographic steps after recovery from the cells and / or the culture medium.

[0166] 26. An antibody produced by the method according to any one of Examples 23 to 25.

[0167] 27. A pharmaceutical composition comprising: an antibody according to any one of Examples 1 to 18 or an immunoconjugate according to Example 19, and a pharmaceutically acceptable carrier.

[0168] 28. The pharmaceutical composition according to Example 27 further comprises additional therapeutic agents.

[0169] 28a. The pharmaceutical composition according to Example 28, wherein the additional therapeutic agent is an anti-TREM2 antibody.

[0170] 28b. The pharmaceutical composition according to any one of Examples 27 to 28a, wherein the pharmaceutical composition is used to treat Alzheimer's disease.

[0171] 28c. The pharmaceutical composition according to any one of Examples 27 to 28a, wherein the pharmaceutical composition is used to treat Parkinson's disease.

[0172] 29. The antibody according to any one of Examples 1 to 18, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c, used as a pharmaceutical.

[0173] 29a. The antibody according to Example 29, wherein the drug is used to treat Alzheimer's disease.

[0174] 29b. The antibody according to Example 29, wherein the drug is used to treat Parkinson's disease.

[0175] 30. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for the treatment of an individual suffering from a disease associated with the production of amyloid protein in the brain.

[0176] 31. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for the treatment of an individual suffering from a disease associated with the formation of amyloid plaques in the brain.

[0177] 32. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for the treatment of amyloid diseases or disorders in the brain.

[0178] 33. The antibody according to any one of Examples 30 to 32, wherein the disease is selected from the group consisting of: dementia, Alzheimer's disease, motor neuron disease, Down syndrome, Creutzfeldt-Jakob disease, Dutch hemorrhage with amyloidosis, Parkinson's disease, HIV-associated dementia, ALS, or age-related neuronal disorders.

[0179] 34. The antibody according to any one of Examples 30 to 32, wherein the disease is Alzheimer's disease.

[0180] 34a. The antibody according to any one of Examples 30 to 32, wherein the disease is Parkinson's disease.

[0181] 35. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for inhibiting the formation of amyloid plaques in the brain.

[0182] 36. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28, for slowing the formation of amyloid plaques in the brain.

[0183] 37. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for preventing the formation of amyloid protein in the brain.

[0184] 38. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for the prevention of amyloid plaque formation in the brain.

[0185] 39. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for reducing amyloid plaques in the brain.

[0186] 40. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for the removal of amyloid plaques in the brain.

[0187] 41. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for breaking down amyloid plaques in the brain.

[0188] 42. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for inhibiting the formation of amyloid plaques in the brain.

[0189] 43. An antibody according to any one of Examples 1 to 18, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c, for breaking down amyloid plaques in the brain.

[0190] 44. Use of the antibody according to any one of Examples 1 to 18r, or the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c, for the preparation of a medicament.

[0191] 45. The use as described in Example 44, wherein the drug is used to treat an individual’s disease associated with the production of amyloid protein in the brain.

[0192] 46. ​​The use according to Example 44, wherein the drug is used to treat an individual’s disease associated with the formation of amyloid plaques in the brain.

[0193] 47. The use as described in Example 44, wherein the drug is used to treat amyloid disease or disorders in the brain.

[0194] 48. According to the use described in Example 47, the amyloid disease is selected from the group consisting of: dementia, Alzheimer's disease, motor neuron disease, Down syndrome, Creutzfeldt-Jakob disease, Dutch hereditary cerebral hemorrhage with amyloidosis, Parkinson's disease, HIV-related dementia, ALS, or age-related neuronal disorders.

[0195] 49. The use according to any one of Examples 47 or 48, wherein the drug is used to treat Alzheimer's disease.

[0196] 49a. The use according to any one of Examples 47 or 48, wherein the drug is used to treat Parkinson's disease.

[0197] 50. The use as described in Example 44, wherein the drug is used to inhibit the formation of amyloid plaques in the brain.

[0198] 51. The use as described in Example 44, wherein the drug is used to slow the formation of amyloid plaques in the brain.

[0199] 52. The use as described in Example 44, wherein the drug is used to prevent the formation of amyloid protein in the brain.

[0200] 53. The use according to Example 44, wherein the drug is used to prevent the formation of amyloid plaques in the brain.

[0201] 54. The use as described in Example 44, wherein the drug is used to reduce amyloid plaques in the brain.

[0202] 55. The use as described in Example 44, wherein the drug is used to remove amyloid plaques in the brain.

[0203] 56. The use as described in Example 44, wherein the drug is used to break down amyloid plaques in the brain.

[0204] 57. The use as described in Example 44, wherein the drug is used to inhibit the formation of amyloid plaques in the brain.

[0205] 58. The use as described in Example 44, wherein the drug is used to break down amyloid plaques in the brain.

[0206] 59. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c, for the treatment of an individual suffering from a disease associated with the production of amyloid protein in the brain.

[0207] 60. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c, for the treatment of an individual suffering from a disease associated with the formation of amyloid plaques in the brain.

[0208] 61. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for the treatment of amyloid diseases or disorders in the brain.

[0209] 62. The use of the antibody according to Example 61, wherein the amyloid disease is selected from the group consisting of: dementia, Alzheimer's disease, motor neuron disease, Down syndrome, Creutzfeldt-Jakob disease, Dutch hemorrhage with amyloidosis, Parkinson's disease, HIV-related dementia, ALS, or age-related neuronal disorders.

[0210] 63. Use of the antibody according to any one of Examples 61 to 62, wherein the drug is used to treat Alzheimer's disease.

[0211] 63a. Use of the antibody according to any one of Examples 61 to 62, wherein the drug is used to treat Parkinson's disease.

[0212] 64. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for inhibiting the formation of amyloid plaques in the brain.

[0213] 65. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for slowing the formation of amyloid plaques in the brain.

[0214] 66. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for the prevention of amyloid formation in the brain.

[0215] 67. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for the prevention of amyloid plaque formation in the brain.

[0216] 68. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for reducing amyloid plaques in the brain.

[0217] 69. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for the removal of amyloid plaques in the brain.

[0218] 70. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for the breakdown of amyloid plaques in the brain.

[0219] 71. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for inhibiting the formation of amyloid plaques in the brain.

[0220] 72. Use of the antibody according to any one of Examples 1 to 18r, the immunoconjugate according to Example 19, or the pharmaceutical composition according to any one of Examples 27 to 28c for the breakdown of amyloid plaques in the brain.

[0221] 73. A method for treating an individual with amyloid plaques in the brain, the method comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 18r, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c.

[0222] 74. A method for treating an individual with amyloid production in the brain, the method comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 18r, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c.

[0223] 75. A method for treating an individual predicted to have or develop amyloid plaques in the brain, the method comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 18r, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c.

[0224] 76. The method according to any one of Examples 73 to 75, further comprising administering an additional therapeutic agent to the individual.

[0225] 76a. The method according to Example 76, wherein the additional therapeutic agent is an anti-TREM2 antibody.

[0226] 77. A method of treating an individual with a disease associated with amyloid formation, the method comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 18r, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c to treat the disease associated with amyloid formation.

[0227] 78. A method of treating an individual with a disease associated with the formation of amyloid plaques in the brain, the method comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 18r, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c to treat the disease associated with the formation of amyloid plaques.

[0228] 79. A method of treating an individual with an amyloid disease or disorder in the brain, the method comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 18r, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c to treat the disease or disorder in the brain.

[0229] 80. The treatment method according to Example 79, wherein the amyloid disease or disorder in the brain is selected from the group consisting of: dementia, Alzheimer's disease, motor neuron disease, Down syndrome, Creutzfeldt-Jakob disease, Dutch hemorrhage with amyloidosis, Parkinson's disease, HIV-related dementia, ALS, or age-related neuronal disorders.

[0230] 81. The method according to any one of Examples 79 to 80, wherein the disease is Alzheimer's disease.

[0231] 81a. The method according to any one of Examples 79 to 80, wherein the disease is Parkinson's disease.

[0232] 82. A method of treating an individual with a disease associated with amyloid formation, the method comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 18r, an immunoconjugate according to Example 19, or a pharmaceutical composition according to any one of Examples 27 to 28c to treat the disease associated with amyloid formation.

[0233] ***

[0234] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of the features disclosed or claimed herein. Therefore, the specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of the features disclosed herein. For illustrative and descriptive purposes, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments. Detailed Implementation

[0235] This invention is based, at least in part, on the discovery that the properties of the antibody gantinuzumab have been improved by introducing specific mutations in the heavy chain CDR2 and by shortening the heavy chain CDR3. Without being bound by this theory, it is assumed that the introduced modifications improve properties by reducing the glyco-occupation heterogeneity in the Fab and removing deamidation hotspots, thereby reducing the aggregation tendency of the parent antibody gantinuzumab.

[0236] The present invention is based at least in part on the discovery that when combined with the modified heavy chain according to the present invention, the light chain of the parent antibody gintinguishumab does not require modification in order to restore binding affinity and specificity.

[0237] definition

[0238] Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0239] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as “according to Kabat numbering”. Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647–660) is used for the constant domain CL of the κ and λ isoform light chains, and Kabat’s EU index numbering system (see pp. 661–723) is used for the constant heavy chain domains (CH1, hinge, CH2, and CH3, which are further classified herein by way of “according to Kabat’s EU index numbering”).

[0240] The mortar-and-pot structure dimer module and its application in antibody engineering are described in Carter P., Ridgway JBB, Presta LG: Immunotechnology, February 1996, Vol. 2, No. 1, pp. 73-73(1).

[0241] General information about the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in: Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0242] Methods and techniques applicable to carrying out the present invention are described in, for example, Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I through III (1997); Glover, ND and Hames, BD (eds.), DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture – a practical approach, IRL Press Limited (1986); Watson, JD et al., Recombinant DNA, 2nd ed., CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J. (ed.), Cell Biology, 2nd ed., Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd ed., Alan R. Liss, Inc. NY (1987).

[0243] Recombinant DNA technology can be used to generate derivatives of nucleic acids. Such derivatives can be modified, for example, at one or more nucleotide positions by substitution, alteration, exchange, deletion, or insertion. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be readily performed by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD and Higgins, SG, Nucleic acid hybridization – a practical approach (1985) IRL Press, Oxford, England).

[0244] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms “a,” “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0245] The term "about" indicates a range of + / - 20% of the value that follows it. In one embodiment, the term "about" indicates a range of + / - 10% of the value that follows it. In another embodiment, the term "about" indicates a range of + / - 5% of the value that follows it.

[0246] The term "amyloid plaque" refers to aggregates of misfolded proteins that form in the spaces between nerve cells. These abnormally configured proteins are believed to play a central role in Alzheimer's disease. Amyloid plaques first appear in brain regions associated with memory and other cognitive functions.

[0247] As used in this article, the term “determine” also includes term measurement and analysis.

[0248] The term "contains" also includes the term "composes of".

[0249] The term "anti-(human) A-β protein antibody" refers to an antibody that binds to human A-β protein with sufficient affinity, making the antibody usable as a diagnostic and / or therapeutic agent targeting human A-β protein.

[0250] It is noteworthy that human A-β protein exists in several naturally occurring forms, among which the human forms are designated Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43. The most important form, Aβ42, has the amino acid sequence of SEQ ID NO: 45. In Aβ41, Aβ40, and Aβ39, the C-terminal amino acids A, IA, and VIA are deleted, respectively. In Aβ43, additional threonine residues are included at the C-terminus of SEQ ID NO: 45. In a preferred embodiment, the antibody according to the invention specifically binds to human A-β protein having the amino acid sequence of SEQ ID NO: 45.

[0251] The "central nervous system" or "CNS" refers to the complex of neural tissues that control bodily functions and includes the brain and spinal cord.

[0252] The term “antibody” is used in a broad sense throughout this document and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) and fragments thereof, as long as they exhibit the desired human A-β protein binding activity.

[0253] As detailed above, the modified antibodies according to the present invention can be bispecific antibodies or multispecific antibodies. In some embodiments, the antibodies according to the present invention are bispecific antibodies in one of the following formats:

[0254] - Full-length antibodies with domain exchange (CrossMab format):

[0255] A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein the first Fab fragment contains...

[0256] a) Only the CH1 and CL domains are interchangeable (i.e., the light chain of the first Fab segment contains the VL and CH1 domains, and the heavy chain of the first Fab segment contains the VH and CL domains).

[0257] b) Only the VH and VL domains are interchangeable (i.e., the light chain of the first Fab segment contains both VH and CL domains, and the heavy chain of the first Fab segment contains both VL and CH1 domains); or

[0258] c) The CH1 and CL domains are interchangeable, and the VH and VL domains are interchangeable (i.e., the light chain of the first Fab segment contains both the VH and CH1 domains, and the heavy chain of the first Fab segment contains both the VL and CL domains); and

[0259] The second Fab segment contains a light chain and a heavy chain, the light chain containing VL and CL domains, and the heavy chain containing VH and CH1 domains.

[0260] The full-length antibody with domain exchange comprises: a first heavy chain including a CH3 domain and a second heavy chain including a CH3 domain, wherein the two CH3 domains are engineered in a complementary manner by corresponding amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain;

[0261] - Full-length antibodies with additional heavy chain C-terminal binding sites (BS format):

[0262] A multispecific IgG antibody, including

[0263] a) A full-length antibody comprising two pairs, each having a full-length antibody light chain and a full-length antibody heavy chain, wherein each pair of the full-length heavy chain and the full-length light chain forms a binding site that specifically binds to a first antigen, or one binding site binds to the first antigen and the other binding site binds to a second antigen, and

[0264] b) An additional Fab fragment fused to the C-terminus of one of the heavy chains of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen, or, in the case where the full-length antibody binds to a first and a second antigen, the additional Fab fragment binds to a third antigen.

[0265] The additional Fab fragment that specifically binds to the second antigen i) contains domain crossovers such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are interchanged with each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are interchanged with each other, or ii) is a single-chain Fab fragment;

[0266] The full-length antibody comprises a first heavy chain including a CH3 domain and a second heavy chain including a CH3 domain, wherein the two CH3 domains are engineered in a complementary manner by corresponding amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain;

[0267] - Full-length antibodies with two additional heavy chain C-terminal binding sites: (2+2 format)

[0268] A multispecific IgG antibody, including

[0269] a) A full-length antibody comprising two pairs, each consisting of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of the full-length heavy chain and the full-length light chain specifically binds to a first antigen, and

[0270] b) Two additional Fab fragments, wherein each C-terminus of the heavy chain of the full-length antibody is fused to an additional Fab fragment, wherein the binding site of the additional Fab fragment specifically binds to the second antigen;

[0271] - Full-length antibodies with an additional N-terminal heavy chain site (TCB format):

[0272] A multispecific IgG antibody, including

[0273] a) A full-length antibody comprising two pairs, each having a full-length antibody light chain and a full-length antibody heavy chain, wherein each pair of the full-length heavy chain and the full-length light chain forms a binding site that specifically binds to a first antigen, or one binding site binds to the first antigen and the other binding site binds to a second antigen, and

[0274] b) An additional Fab fragment, wherein the additional Fab fragment is inserted between one of the Fab fragments of the full-length antibody and the Fc region, wherein the binding site of the additional Fab fragment specifically binds to a second antigen, or, in the case where the full-length antibody binds to a first antigen and a second antigen, the additional Fab fragment binds to a third antigen.

[0275] The additional Fab fragments that specifically bind to the second antigen include domain crossings such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are interchanged, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are interchanged.

[0276] - Single-arm single-chain form (= single-arm single-chain antibody):

[0277] An antibody comprising a first binding site and a second binding site, the first binding site specifically binding to a first epitope or antigen, and the second binding site specifically binding to a second epitope or antigen, wherein the individual chains are as follows:

[0278] - Light chain (variable light chain domain + light chain κ constant domain)

[0279] - Combination of light / heavy chains (variable light chain domain + constant light chain domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with club-shaped mutation)

[0280] - Heavy chain (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with mortise mutation);

[0281] - Two-arm single-chain form (= two-arm single-chain antibody):

[0282] An antibody comprising a first binding site and a second binding site, the first binding site specifically binding to a first epitope or antigen, and the second binding site specifically binding to a second epitope or antigen, wherein the individual chains are as follows:

[0283] - Combination light chain / heavy chain 1 (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with mortise mutation)

[0284] - Combination light chain / heavy chain 2 (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 with club-shaped mutation);

[0285] -Common light chain bispecific form (=common light chain bispecific antibody):

[0286] An antibody comprising a first binding site and a second binding site, the first binding site specifically binding to a first epitope or antigen, and the second binding site specifically binding to a second epitope or antigen, wherein the individual chains are as follows:

[0287] - Light chain (variable light chain structure domain + constant light chain structure domain)

[0288] -Heavy chain 1 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with mortar mutation)

[0289] - Heavy chain 2 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 with a club-like mutation).

[0290] As used herein, the term "substitute for each other" in relation to the corresponding heavy-chain and light-chain domains refers to the aforementioned domain crossings. Therefore, when the CH1 and CL domains "substitute for each other," it refers to the domain crossing mentioned under item (i) and the resulting heavy-chain and light-chain domain sequence. Similarly, when VH and VL "substitute for each other," it refers to the domain crossing mentioned in item (ii); and when the CH1 and CL domains "substitute for each other" and the VH and VL domains "substitute for each other," it refers to the domain crossing mentioned in item (iii).

[0291] An "antibody fragment" is a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the same antigen that the complete antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bispecific antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); DutaFab (bispecific Fab); and multispecific antibodies formed from antibody fragments.

[0292] “Affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can be measured using conventional methods known in the art, including those described herein.

[0293] The term "antibody-dependent cytotoxicity (ADCC)" refers to a function mediated by Fc receptor binding and specifically to the lysis of target cells by an antibody as reported herein in the presence of effector cells. ADCC can be measured by treating CD19-expressing erythroid cells (e.g., K562 cells expressing recombinant human CD19) with an antibody according to the invention in the presence of effector cells (such as freshly isolated PBMCs (peripheral blood mononuclear cells) from the erythrocyte sedimentation rate (ESR) amber layer) or purified effector cells, such as monocytes or NK (natural killer) cells. Target cells are labeled with 51Cr and subsequently incubated with the antibody. The labeled cells are incubated with effector cells, and the release of 51Cr in the supernatant is analyzed. A control involves incubating target endothelial cells with effector cells in the absence of an antibody. The ability of antibodies to induce the initial steps of ADCC is investigated by measuring the binding of antibodies to cells expressing Fcγ receptors, such as recombinant cells expressing FcγRI and / or FcγRIIA or NK cells (which essentially express FcγRIIIA).

[0294] "Multispecific antibody" refers to an antibody that has binding specificity for at least two different epitopes on the same antigen or for two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody or DutaFab) or combinations thereof (e.g., a full-length antibody plus one or more additional / fused Fv, scFv, or Fab fragments). Engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites have also been reported (see, for example, US 2002 / 0004587 A1).

[0295] The term “binding (to antigen)” refers to the binding of an antibody to its homoantigen. Binding can be determined in an in vitro assay. In some embodiments, binding is determined in a binding assay, wherein the antibody binds to a surface, and the binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). The affinity of binding is defined by the terms ka (association rate constant of the antibody from the antibody / antigen complex), kd (dissociation constant), and KD (kd / ka). Thus, binding implies a specific and detectable interaction between the antibody and its homoantigen, such as a binding affinity (KD) of 1E-4 M or lower. “Specific binding” means a binding affinity (KD) of 1E-8 M or lower, 1E-13 to 1E-8 M in some embodiments, and 1E-13 to 1E-9 M in some embodiments.

[0296] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0297] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called μ and μ, respectively.

[0298] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as lysozymes; antibiotics; toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various antitumor or anticancer drugs disclosed below.

[0299] The term “complement-dependent cytotoxicity (CDC)” refers to cell lysis induced by the antibody according to the invention in the presence of complement. CDC can be measured by treating human endothelial cells expressing CD19 with the antibody according to the invention in the presence of complement. Cells can be labeled with calcein. CDC is detected if the antibody induces 20% or more of target cell lysis at a concentration of 30 μg / ml. Binding to complement factor C1q can be measured in an ELISA. In such an assay, in principle, an ELISA plate is coated with the antibody according to the invention at multiple concentration ranges, wherein purified human C1q or human serum is added. C1q binding is detected by the antibody against C1q, followed by detection by a peroxidase-labeled conjugate. The detection of binding (maximum binding Bmax) to the peroxidase substrate ABTS® (2,2'-azido-di-[3-ethylbenzothiazoline-6-sulfonate (6)]) is measured as an optical density at 405 nm (OD405).

[0300] The term "effective function" refers to those biological activities attributable to the Fc region of an antibody, which vary depending on the antibody species. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0301] Fc receptor binding-dependent effector functions are mediated by the interaction of the antibody's Fc region with the Fc receptor (FcR), a specific cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes and to lyse antibody-coated erythrocytes and various other cellular targets (e.g., tumor cells) via antibody-dependent cell-mediated cytotoxicity (ADCC) (see, for example, Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: the Fc receptor for IgG antibodies is called FcγR. Fc receptor binding is described in, for example: Ravetch, JV and Kinet, JP, Annu. Revised, Immunol. 9 (1991) 457-492; Capel, PJ et al., Immunomethods 4 (1994) 25-34; de Haas, M. et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE et al., Ann. Hematol. 76 (1998) 231-248.

[0302] Cross-linking of the Fc region receptor of IgG antibodies (FcγRs) triggers multiple effector functions, including phagocytosis, antibody-dependent cytotoxicity, release of inflammatory mediators, and regulation of immune complex clearance and antibody production. Three classes of FcγRs have been identified in humans, including:

[0303] -FcγRI (CD64) binds to monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modifications to at least one residue in the Fc region of IgG at amino acid residues E233-G236, P238, D265, N297, A327, and P329 (according to Kabat's EU index number) reduce binding to FcγRI. Substitution of IgG2 residues at positions 233-236 with IgG1 and IgG4 reduces binding affinity to FcγRI by 10³-fold and eliminates the human monocyte response to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29 (1999) 2613–2624).

[0304] FcγRII (CD32) binds to complexed IgG with medium to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to activate the killing process. FcγRIIB appears to play a role in inhibition and is present on B cells, macrophages, mast cells, and eosinophils. On B cells, it appears to inhibit further immunoglobulin production and isoform conversion, such as to IgE. On macrophages, FcγRIIB is used to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B subtype may contribute to the inhibition of these cell activation through the binding of IgE to its individual receptor. It was found that, for example, antibodies (IgG Fc regions containing mutations in at least one amino acid residue E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (according to Kabat's EU index number)) exhibited reduced binding affinity to FcγRIIA;

[0305] -FcγRIII (CD16) binds to IgG with medium to low affinity and exists in two forms. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is expressed at high levels on neutrophils. Reduced binding to FcγRIIIA has been found, for example, for antibodies (according to Kabat's EU index number) that include the Fc region of IgG with mutations at at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376.

[0306] Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604 describe the location of the binding site on human IgG1 to the Fc receptor, the aforementioned mutation sites, and methods for measuring binding to FcγRI and FcγRIIA.

[0307] The "therapeutic effective amount" of a drug (e.g., a pharmaceutical preparation) refers to the amount that effectively achieves the desired therapeutic or preventive outcome at the necessary dosage and time period.

[0308] As used herein, the term "Fc receptor" refers to an activated receptor characterized by the presence of a receptor-associated cytoplasmic ITAM sequence (see, for example, Ravetch, JV and Bolland, S., Annu. Rev. Immunol. 19(2001) 275-290). Such receptors are FcγRI, FcγRIIA, and FcγRIIIA. The term "non-binding FcγR" means that at an antibody concentration of 10 μg / ml, the antibody binding to NK cells is 10% or less of the measured binding of the anti-OX40L antibody LC.001 as reported in WO 2006 / 029879.

[0309] Although IgG4 showed reduced FcR binding, antibodies against other IgG subclasses showed strong binding. However, Pro238, Asp265, Asp270, Asn297 (Fc carbohydrate loss), Pro329 and 234, 235, 236 and 237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues that, if altered, also provide reduced FcR binding (Shields, RL et al. J. Biol. Chem. 276 (2001) 6591-6604; Lund, J. et al., FASEB J. 9 (1995) 115-119; Morgan, A. et al., Immunology 86 (1995) 319-324; and EP 0 307 434). In some embodiments, the antibody according to the invention is an antibody of the IgG1 or IgG2 subclass and comprises mutant PVA236, GLPSS331, L234A / L235A, or P329G / L234A / L235A. In some embodiments, the antibody as reported herein is an antibody of the IgG4 subclass and comprises mutant L235E. In some embodiments, the antibody according to the invention further comprises mutant S228P.

[0310] The term "(human) Fc region polypeptide" refers to the C-terminal region of a human immunoglobulin heavy chain containing at least a portion of a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the human IgG heavy chain Fc region polypeptide extends from Cys226 or Pro230 to the C-terminus of the heavy chain. In a preferred embodiment, the Fc region polypeptide comprises the amino acid sequence of SEQ ID NO: 05 or a variant thereof. However, the C-terminal lysine (Lys447) of the Fc region polypeptide or the intact antibody heavy chain may or may not be present.

[0311] "Antibody Fc region" is a term familiar to those skilled in the art and is defined based on the cleavage of an antibody by papain. In some embodiments, the Fc region is a human Fc region. A "(human) Fc region" comprises two (human) heavy-chain Fc region polypeptides covalently linked to each other via hinge region cysteine ​​residues, forming interchain disulfide bonds.

[0312] The antibody according to the invention comprises an Fc region, and in some embodiments, comprises a human-derived Fc region. In some embodiments, the Fc region comprises all portions of the human constant region. The Fc region of the antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. Although the effect of the antibody on the complement system depends on certain conditions, binding to C1q is caused by the binding site defined in the Fc region. Such binding sites are known in the art and described in, for example, the following literature: Lukas, TJ, et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR, et al., Nature 288 (1980) 338-344; Thommesen, JE, et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE, et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (according to Kabat’s EU index numbering; unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242). Antibodies against subclasses IgG1, IgG2, and IgG3 typically exhibit complement activation, C1q binding, and C3 activation, while IgG4 does not activate the complement system, does not bind C1q, and does not activate C3.

[0313] The term "FcRn" refers to the human neonatal Fc receptor. The function of FcRn is to rescue IgG from lysosomal degradation, resulting in decreased clearance and increased half-life. FcRn is a heterodimeric protein composed of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a stoichiometric ratio of 1:2, with one IgG molecule binding to two FcRn molecules via its two heavy chains (Huber, AH et al., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in the endosome at acidic pH (pH < 6.5), while IgG is released at the neutral cell surface (pH approximately 7.4). This pH-sensitive interaction promotes FcRn-mediated protection of endocytosed IgG from intracellular degradation by binding to the receptor in the acidic environment of the endosome. FcRn then promotes IgG recycling to the cell surface, subsequently releasing it into the bloodstream when the FcRn-IgG complex is exposed to the extracellular neutral pH environment.

[0314] The term "FcRn binding portion of the Fc region" refers to the following portions of the antibody heavy chain polypeptide: approximately from EU position 243 to EU position 261, approximately from EU position 275 to EU position 293, approximately from EU position 302 to EU position 319, approximately from EU position 336 to EU position 348, approximately from EU position 367 to EU position 393 and EU position 408, and approximately from EU position 424 to EU position 440. In one embodiment, according to the EU numbering of Kabat, one or more of the following amino acid residues are modified F243, P244, and P245. P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E25 8. V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H28 5. N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L30 6. T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y31 9. I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V34 8. C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E38 2. S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 (EU numbers).

[0315] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0316] The term "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody. A full-length antibody comprises i) two full-length antibody light chains, each containing a variable domain and a constant domain; and ii) two full-length antibody heavy chains, each containing a variable domain, a first constant domain, a hinge region, a second constant domain, and a third constant domain. Full-length antibodies may contain other domains, such as, for example, additional scFv or scFab or domain-exchanged Fab conjugated to one or more chains of the full-length antibody (preferably conjugated to the C-terminus of one or more heavy chains).

[0317] The terms "host cell" and "host cell line" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include "transformations" and "transformed cells," which include primary transformed cells and their progeny, regardless of passage number. Progeny may not be identical to the parent cells but may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

[0318] The “human common framework” is a framework representing the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Typically, the subgroup is as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Bethesda MD (1991), NIH Publication 91-3242, Volumes 1-3. In some embodiments, for VL, this subgroup is subgroup κI as described in Kabat et al. (ibid.). In some embodiments, for VH, this subgroup is subgroup III as described in Kabat et al. (ibid.).

[0319] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody will substantially contain at least one of all, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0320] As used herein, the term “hypervariant region” or “HVR” refers to each of the following: a region of antibody variable domain that is hypervariable in sequence (“complementarity-determining region” or “CDR”) and / or forms a structurally defined loop (“hypervariant loop”) and / or contains antigen contact residues (“antigen contact site”). Generally, an antibody contains six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0321] HVR includes

[0322] (a) Hyperchromatic rings present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C and Lesk, AM, J. Mol. Biol. 196(1987) 901-917);

[0323] (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242).

[0324] (c) Antigen contact sites located at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); and

[0325] Combinations of (d)(a), (b) and / or (c), including amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0326] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domain are referenced in this paper to Kabat et al., with the same reference numbering above.

[0327] The term "derived from" means that the amino acid sequence is derived from the parent amino acid sequence by introducing a change at at least one position. Therefore, the derived amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position (numbered according to the Kabat EU index of the antibody Fc region). In some embodiments, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 15 amino acid residues. In some embodiments, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 10 amino acid residues. In some embodiments, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 6 amino acid residues. Similarly, the derived amino acid sequence has high amino acid sequence identity with its parent amino acid sequence. In some embodiments, the amino acid sequence derived from the parent amino acid sequence has 80% or more amino acid sequence identity. In some embodiments, the amino acid sequence derived from the parent amino acid sequence has 90% or more amino acid sequence identity. In some embodiments, the amino acid sequence derived from the parent amino acid sequence has 95% or more amino acid sequence identity. In a preferred embodiment, the amino acid sequence derived from the parent amino acid sequence has 98% or more amino acid sequence identity.

[0328] The term "(human) Fc region polypeptide" refers to an amino acid sequence identical to that of a "natural" or "wild-type" (human) Fc region polypeptide. The term "variant (human) Fc region polypeptide" refers to an amino acid sequence derived from a "natural" or "wild-type" (human) Fc region polypeptide due to at least one "amino acid alteration". A "variant (human) Fc region" consists of two Fc region polypeptides, both of which can be variant (human) Fc region polypeptides, or one can be a (human) Fc region polypeptide and the other a variant (human) Fc region polypeptide.

[0329] In some embodiments, the human Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region polypeptide or a variant thereof, or has the amino acid sequence of the human IgG2 Fc region polypeptide or a variant thereof, or has the amino acid sequence of the human IgG3 Fc region polypeptide or a variant thereof, or has the amino acid sequence of the human IgG4 Fc region polypeptide or a variant thereof. In some embodiments, the Fc region polypeptide is derived from the Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17 and has at least one amino acid mutation compared to the Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In some embodiments, the Fc region polypeptide contains / has about one to about ten amino acid mutations. In some embodiments, the Fc region polypeptide contains / has about one to about five amino acid mutations.

[0330] In some embodiments, the Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region polypeptide containing the PGLALA mutation and the mortar-cys mutation (SEQ ID NO: 12), wherein the C-terminal lysine residue is optionally deleted. In some embodiments, the Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region containing the PGLALA mutation and the mortar-cys mutation (SEQ ID NO: 16 or SEQ ID NO: 15), wherein the C-terminal lysine residue is optionally deleted.

[0331] In some embodiments, the Fc region polypeptide shares at least about 80% sequence homology with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In some embodiments, the Fc region polypeptide shares at least about 90% sequence homology with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In some embodiments, the Fc region polypeptide shares at least about 95% sequence homology with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In a preferred embodiment, the Fc region polypeptide shares at least about 97.5% homology with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17.

[0332] In some embodiments, the Fc region polypeptide has at least about 80% sequence identity with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In some embodiments, the Fc region polypeptide has at least about 90% sequence identity with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In some embodiments, the Fc region polypeptide has at least about 95% sequence identity with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In a preferred embodiment, the Fc region polypeptide has at least about 97.5% identity with the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17. In a preferred embodiment, the C-terminal lysine residue is deleted.

[0333] Variant Fc region polypeptides derived from the parental (human) Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17 are further defined by the amino acid changes contained compared to the parental or wild-type sequence. Thus, for example, the term P329G refers to an Fc region polypeptide derived from the (human) Fc region polypeptide, with a proline mutation to glycine at amino acid position 329 (according to Kabat numbering) relative to the human Fc region polypeptide of SEQ ID NO: 05 or SEQ ID NO: 17.

[0334] The human IgG1 Fc region polypeptide has the following amino acid sequence:

[0335] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 05), optionally having an additional lysine residue (K) added to the C-terminus.

[0336] The Fc region polypeptide derived from the human IgG1 Fc region with mutations L234A and L235A (LALA mutation) has the following amino acid sequence:

[0337] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 06), optionally having an additional lysine residue (K) added to the C-terminus.

[0338] The Fc region polypeptide derived from the human IgG1 Fc region with Y349C, T366S, L368A, and Y407V mutations (cys-mutation) has the following amino acid sequence:

[0339] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 07), optionally having an additional lysine residue (K) added to the C-terminus.

[0340] The Fc region polypeptide derived from the human IgG1 Fc region with the S354C, T366W (sodium-cys-mutant) mutation has the following amino acid sequence:

[0341] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 08), optionally having an additional lysine residue (K) added to the C-terminus.

[0342] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A mutations and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0343] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 09), optionally having an additional lysine residue (K) added to the C-terminus.

[0344] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A, S354C, and T366W mutations has the following amino acid sequence:

[0345] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10), optionally having an additional lysine residue (K) added to the C-terminus.

[0346] The Fc region polypeptide derived from the human IgG1 Fc region with the P329G mutation (PG mutation) has the following amino acid sequence:

[0347] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11), optionally having an additional lysine residue (K) added to the C-terminus.

[0348] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A mutations and P329G mutation (LALAPG mutation) has the following amino acid sequence:

[0349] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 12), optionally having an additional lysine residue (K) added to the C-terminus.

[0350] The Fc region polypeptide derived from the human IgG1 Fc region with P329G mutation and Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence:

[0351] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13), optionally having an additional lysine residue (K) added to the C-terminus.

[0352] The Fc region polypeptide derived from the human IgG1 Fc region with P329G mutation and S354C, T366W mutation has the following amino acid sequence:

[0353] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 14), optionally having an additional lysine residue (K) added to the C-terminus.

[0354] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0355] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15), optionally having an additional lysine residue (K) added to the C-terminus.

[0356] The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A, P329G mutations and S354C, T366W mutations has the following amino acid sequence:

[0357] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16), optionally having an additional lysine residue (K) added to the C-terminus.

[0358] The human IgG4 Fc region polypeptide has the following amino acid sequence:

[0359] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 17).

[0360] The Fc region polypeptide derived from the human IgG4 Fc region with S228P and L235E mutations has the following amino acid sequence:

[0361] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 18).

[0362] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E mutations and P329G mutations has the following amino acid sequence:

[0363] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 19).

[0364] The Fc region polypeptide derived from the human IgG4 Fc region with the S354C, T366W mutation has the following amino acid sequence:

[0365] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 20).

[0366] The Fc region polypeptide derived from the human IgG4 Fc region with mutations Y349C, T366S, L368A, and Y407V has the following amino acid sequence:

[0367] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 21).

[0368] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E and S354C, T366W mutations has the following amino acid sequence:

[0369] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 22).

[0370] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0371] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 23).

[0372] The Fc region polypeptide derived from the human IgG4 Fc region with the P329G mutation has the following amino acid sequence:

[0373] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 24).

[0374] The Fc region polypeptide derived from the human IgG4 Fc region with P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0375] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 25).

[0376] The Fc region polypeptide derived from the human IgG4 Fc region with P329G, S354C, and T366W mutations has the following amino acid sequence:

[0377] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 26).

[0378] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0379] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 27).

[0380] The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E, P329G and S354C, T366W mutations has the following amino acid sequence:

[0381] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 28).

[0382] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, the humanized antibody will substantially contain at least one of these variable domains, typically two variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, the humanized antibody may contain at least a portion of the antibody constant region derived from the human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0383] "Separated" antibodies are antibodies that have been isolated from components of their natural environment. In some embodiments, the antibodies are purified to a purity greater than 95% or 99%, as determined by analytical methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., size exclusion chromatography, ion exchange, or reversed-phase HPLC). For a review of methods used to assess, for example, antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0384] "Isolated" nucleic acids refer to nucleic acid molecules that have been isolated from components of their natural environment.

[0385] "Isolated nucleic acid encoding antibody against human A-β protein" means one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single plasmid or a single plasmid.

[0386] "Immune conjugates" are antibodies conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0387] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0388] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of a monoclonal antibody formulation, such variants are typically presented in small quantities), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0389] "Natural antibodies" are naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain. The light chains of antibodies can be classified into one of two types based on the amino acid sequence of their constant domains, called kappa (κ) and lamuda (λ).

[0390] The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information concerning the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of such therapeutic products.

[0391] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 is used to generate the value of the amino acid sequence identity %. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with user documentation to the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0392] When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which can be alternatively expressed as the percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B) is calculated as follows:

[0393] 100 multiplied by the fraction X / Y

[0394] Where X represents the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and Y represents the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % between A and B will not be equal to the amino acid sequence identity % between B and A. Unless otherwise specified, all amino acid sequence identity % values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0395] The term "pharmaceutical formulation" refers to a formulation in which the bioactive ingredients contained therein are in a form in which the active ingredients are permitted to be bioactive and which do not contain any components that would have unacceptable toxicity to a subject to whom the formulation will be administered.

[0396] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are non-toxic to the subjects, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0397] As used herein, the term "plasmid" refers to a nucleic acid molecule capable of replicating the other nucleic acid it contains. This term includes plasmids as self-replicating nucleic acid structures, as well as plasmids incorporated into the genome of a host cell into which they have been introduced. Some plasmids are capable of directing the expression of the nucleic acids they contain. Such plasmids are referred to herein as "expression plasmids."

[0398] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric antibodies, humanized antibodies, and human antibodies) prepared, expressed, created, or isolated by recombinant means. This includes antibodies isolated from host cells such as NSO, HEK, BHK, or CHO cells, or from transgenic animals (e.g., mice) carrying human immunoglobulin genes, or antibodies expressed using recombinant expression plasmids transfected into host cells. The amino acid sequences of the VH and VL regions of a recombinant antibody are as follows, although derived from and associated with human germline VH and VL sequences, but may not be present in the in vivo human antibody germline under natural conditions.

[0399] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of the disease in the treated individual, and may be performed for prevention or during the course of clinicopathological progression. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of disease or slow its progression.

[0400] As used in this application, the term "valence" indicates the presence of a specified number of binding sites in the (antibody) molecule. Therefore, the terms "bivalent," "trivalent," and "tetravalent" indicate the presence of two, three, and four binding sites in the (antibody) molecule, respectively.

[0401] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in the binding of the antibody to its antigen. The variable domains (VH and VL, respectively) of the antibody heavy and light chains typically have similar structures, with each domain containing four frame regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding to that antigen to screen libraries containing complementary VL or VH domains. See, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628). Homologous pairs of antibody heavy chain variable domains and antibody light chain variable domains form binding sites.

[0402] The term "variant" refers to a molecule having an amino acid sequence different from that of the corresponding parent molecule. Typically, such molecules have one or more alterations (mutations), insertions, or deletions. In some embodiments, the antibody according to the invention comprises at least a portion of the Fc region that is not naturally present. Such molecules have less than 100% sequence identity with the parent antibody. In some embodiments, the amino acid sequence of the variant antibody has from about 75% to less than 100% amino acid sequence identity with the amino acid sequence of the parent antibody, particularly from about 80% to less than 100%, particularly from about 85% to less than 100%, particularly from about 90% to less than 100%, and particularly from about 95% to less than 100%. In a preferred embodiment, the parent antibody and the variant antibody differ by one (single), two, three, five, seven, or ten amino acid residues.

[0403] amyloid hypothesis

[0404] Amyloid plaques form when protein sheets called β-amyloid aggregate. β-amyloid is produced when a larger protein called amyloid precursor protein (APP) is broken down. APP consists of 771 amino acids and is cleaved by two enzymes to produce β-amyloid. The larger protein is first cleaved by β-secretase and then by γ-secretase, resulting in β-amyloid sheets that may consist of 38, 40, or 42 amino acids. β-amyloid sheets of 42 amino acids are chemically more "sticky" than those of other lengths and are therefore more likely to form plaques. Studies have shown that three genetic abnormalities associated with early Alzheimer's disease each alter the function of γ-secretase in ways that lead to increased production of Aβ42.

[0405] How β-amyloid causes toxic damage to nerve cells is not fully understood, but some studies suggest it may break down into fragments and release free radicals that then attack neurons. Another theory is that β-amyloid forms tiny pores in the neuronal membrane, leading to unregulated calcium influx and consequently neuronal death. Regardless of the exact pathological process by which β-amyloid causes neuronal damage, the result is neuronal death.

[0406] These plaques form from a mixture of degenerated neurons and β-amyloid aggregates. These plaques cannot be broken down or removed by the body, so they gradually accumulate in the brain. This accumulation of amyloid protein leads to amyloidosis, which is considered a cause of many neurodegenerative diseases.

[0407] Amyloid plaque formation is one of the two defining characteristics of Alzheimer's disease, the other being neurofibrillary tangles. Beta-amyloid is also thought to be responsible for the formation of these tangles, which further damage neurons and lead to dementia symptoms. Technically, a person may have all the features of Alzheimer's disease, but if a brain biopsy or positron emission tomography (PET) scan does not show amyloid plaques or neurofibrillary tangles, Alzheimer's disease will not be diagnosed.

[0408] Description of embodiments of the present invention

[0409] Gantinrucumab is a fully human IgG1 antibody that binds with sub-nanomolar affinity to the conformational epitope of Aβ, consisting of both the N-terminal and central amino acids. It preferentially binds to the fibrillary form of the protein. The therapeutic mechanism of this antibody is that it concentrates and degrades amyloid plaques by recruiting microglia and activating phagocytosis. It prevents the formation of new plaques. Gantinrucumab preferentially interacts with aggregated brain Aβ (both parenchymal and vascular). The antibody induces phagocytosis of human Aβ deposits in AD brain slices co-cultured with human macrophages. It also neutralizes the long-term inhibitory effect of oligomeric Aβ42 on rat brain. In APP / PS1 transgenic mice, gantinrucumab binds to brain Aβ, reducing small plaques by recruiting microglia and preventing the formation of new plaques. Gantinrucumab does not alter systemic Aβ levels, indicating that the clearance of soluble Aβ is not interfered with. In the phase 3, multicenter, randomized, double-blind, placebo-controlled SCarlet RoAD trial, gantenerumab showed targeted involvement, resulting in plaque clearance and a reduction in phosphorylated tau levels in the cerebrospinal fluid (see, for example, Sumner, IL et al., Front. Neurosci. 12 (2018) No. 254; https: / / www.alzforum.org / therapeutics / gantenerumab).

[0410] Gantinlurumab has two sets of glycosylation sites: the first set covers the glycosylation sites in each of the Fab in the heavy chain CDR2, and the second set covers the glycosylation sites in each of the Fc regions at Asn 297 (according to Kabat numbering).

[0411] The glycosylation pattern, or sugar occupancy, of glycosylation sites differs between the two sets of glycosylation sites. In the Fc region, the sugar occupancy of each glycosylation site is homogeneous and comparable to each other and to other recombinant antibodies. Conversely, in the Fab region, the sugar occupancy of glycosylation sites is dissimilar; either both glycosylation sites are glycosylated, only one of these sites is glycosylated, or none of these sites are glycosylated. Therefore, the recombinant gantinumab is obtained as a mixture of three different glycosylation isoforms from the generating cells. Since sugar occupancy and glycosylation pattern affect pharmacokinetic properties and plaque binding, it is necessary to remove the non-glycosylated isoforms. Chromatographic methods, such as hydrophobic interaction chromatography (HIC), can be used to distinguish and separate the different glycoforms.

[0412] The inventors have now discovered that the properties of gantinuzumab have been improved by introducing a specific mutation in the heavy chain CDR2 and by shortening the heavy chain CDR3. Without being bound by this theory, it is assumed that the introduced modifications improve properties by reducing sugar content unevenness in the Fab and removing deamidation hotspots, thereby reducing the aggregation tendency of the parent antibody gantinuzumab.

[0413] Furthermore, the inventors have discovered that when combined with the modified heavy chain according to the present invention, the light chain of the parent antibody gintinguishumab does not require modification in order to maintain binding affinity and specificity.

[0414] Therefore, aspects of the present invention are antibodies against human A-β protein (anti-A-β protein antibodies), methods for producing the same, pharmaceutical compositions containing such antibodies, and their uses.

[0415] The antibodies according to the invention are variants of the anti-A-β antibody gantenerumab. Compared to their parent antibodies, they possess improved technical and biological properties. Among other improvements are improved production titers, improved production yields, improved glycosylation uniformity, reduced aggregation tendency, and improved process robustness.

[0416] The anti-A-β protein antibody according to the present invention has a significant sugar occupancy of up to 100% of the glycosylation sites in the heavy chain CDR2.

[0417] Among other things, the anti-A-β protein antibody according to the present invention has improved properties in terms of target binding, exploitability, IHC / plaque binding and PK behavior.

[0418] This invention is based, at least in part, on the finding that complete removal of glycosylation sites in the Fab of gantinuzumab leads to either reduced plaque binding or increased clearance. Therefore, modifications that result in reduced or even eliminated glycosylation at the Fab glycosylation sites of gantinuzumab are detrimental and need to be prevented.

[0419] Four different gantinumab variants with modified Fab sugar content have been generated:

[0420] Two variants with modified heavy chains (according to Kabat numbering):

[0421] - Complete Fab glycosylation was achieved by introducing a heavy chain S53T mutation (mAb 675);

[0422] - Complete removal of Fab glycosylation (mAb 663) by introducing the heavy chain N52Q mutation;

[0423] Two variants with modified heavy and light chains (according to Kabat numbering):

[0424] - Complete Fab glycosylation was achieved by introducing the heavy chain S53T mutation, and heavy chain modification was compensated by introducing the light chain M95H mutation (mAb 651).

[0425] - Complete removal of Fab glycosylation was achieved by introducing the N52Q mutation in the heavy chain, and the heavy chain modification was compensated by introducing the M95H mutation in the light chain (mAb 638).

[0426] The four variants of gantinumab are either fully Fab-glycosylated or completely unFab-glycosylated. Except for mAb 651, all variants have the same in vitro binding affinity for human A-β protein.

[0427]

[0428] Different glycosylation levels have been confirmed by hydrophilic interaction chromatography (HIC), as shown in Figures 1 to 3.

[0429] Figure 1 shows the partial glycosylation of the parental antibody gantingrulimab with the NAS sequence in the heavy chain CDR (two peaks at the alignment time of approximately 22–24 seconds).

[0430] Figure 2 shows the complete deglycosylation of the variant antibody mAb 663 with the sequence QAS in the heavy chain CDR (a peak at an alignment time of approximately 22 seconds).

[0431] Figure 3 shows the complete glycosylation of the variant antibody mAb 675 with the sequence QAS in the heavy chain CDR (a peak at an alignment time of approximately 23 seconds).

[0432] The antibody and gantinguissumab according to the present invention have been produced on a small scale, and the distribution of byproducts has been analyzed by protein A affinity chromatography after the first purification step and by preparative size exclusion chromatography after the second purification step. The results are shown in the table below.

[0433]

[0434] nd: Undetermined – Sufficient purity was achieved after protein A chromatography, therefore SEC was not performed.

[0435] The four variants were further characterized based on their binding properties and specificity. The qualitative results are shown in the table below:

[0436]

[0437] The features will be discussed in more detail below.

[0438] In vitro A-β plaque decoration

[0439] The parental antibody, gantinguishumab, showed strong specific plaque binding and some background nonspecific binding, especially at a concentration of 1 μg / ml (Figure 4).

[0440] MAb 675 (“142”) showed strong specific plaque binding and no detectable background nonspecific binding (Fig. 5).

[0441] MAb 663 (“143”) showed low specificity for plaque binding and some background nonspecific binding, especially at a concentration of 1 μg / ml (Figure 6).

[0442] MAb 651 (“144”) showed almost no specific plaque binding as well as no background binding and nonspecific binding, even at a concentration of 1 μg / ml (Figure 7).

[0443] MAb 638 showed low specificity for plaque binding and some background nonspecific binding, especially at a concentration of 1 μg / ml.

[0444] Therefore, mAb 675 unexpectedly showed equal specific plaque binding but significantly less background nonspecific binding compared to gantinguishumab. Thus, fully glycosylated sites in Fab were found to reduce in vitro background nonspecific binding at a concentration of 1 μg / mL.

[0445] The following table summarizes the staining results in a qualitative manner.

[0446]

[0447] In vivo A-β plaque decoration

[0448] Male APP / PS2 mice (9 months old, n = 3 per group) were administered 20 mg / kg of gantinumab and each of four variants. Antibody distribution in brain slices seven days post-injection was determined (detection antibody: goat anti-human IgG antibody conjugated to Alexa555 (Invitrogen (#A21433); A-β protein co-staining: Amylo Glo, Biosensis catalog number TR-300-AG; image settings: 30x magnification). Results are shown in Figures 8 and 9.

[0449] Therefore, mAb 675 (“142” in Figures 8 and 9) unexpectedly showed improved in vivo plaque binding compared to gantinumab. Thus, fully glycosylated sites in Fab have been found to increase A-β plaque binding in vivo.

[0450] Pharmacokinetics

[0451] The pharmacokinetics of four gantingrulimab variants were determined in rats and mice.

[0452] mAb 663, which lacks Fab glycosylation and has no modification in the variable domain of the light chain, was found to exhibit increased plasma clearance in rats. All other variants showed similar plasma clearance to gantinuzumab.

[0453] The results are shown in Figure 10.

[0454] Surprisingly, the clearance rates of mAb 638, mAb 675, and mAb 651 were lower than those of gantinusmab (historical data). It must be noted that the difference in plasma clearance rates cannot explain the difference in plaque decoration against mAb 638.

[0455] The single-dose pharmacokinetic (SDPK) characteristics of the gantinglumonab variant were further evaluated using a combination of FcRn and heparin affinity chromatography.

[0456] The combination of FcRn affinity chromatography and heparin affinity chromatography allows for the definition of retention time thresholds for both FcRn and heparin affinity columns, thereby defining a two-dimensional retention time region in which antibodies with slowed clearance (i.e., long systemic circulating half-life) can be identified. Therefore, this combination allows for the selection of antibodies with long systemic circulating half-lives, among other things.

[0457] For characterization, retention times on FcRn affinity columns and heparin affinity columns were normalized based on the retention times of control antibodies on the corresponding columns. This defined a relative retention time region primarily containing antibodies with reduced clearance. More specifically, this region was defined by relative retention times less than 1.78 on FcRn affinity columns (using oxidized (H2O2-treated) anti-Her3 antibody formulations as reference antibodies) and relative retention times less than 0.87 on heparin affinity columns (using anti-pTau antibody as reference antibody). Further details regarding this method can be found in WO 2018 / 197533.

[0458]

[0459] thermal stability

[0460] The thermal stability of mAb 675, mAb 663, and mAb 638 was determined by dynamic light scattering. Aggregation temperature (T) agg ) and melting temperature (T) m The results were determined by applying a heated ramp. Surprisingly, all three tested variants showed improved values ​​compared to the parental gantinuzumab.

[0461]

[0462] Stability assessment

[0463] mAb 675, mAb 663 and mAb 638 have been subjected to thermal stress by incubation at elevated temperatures in different buffer systems.

[0464] It has been found that mAb 638 exhibits significant monomer loss upon incubation in phosphate-buffered saline solution.

[0465] However, surprisingly, all three gantingrulimab variants showed improved stability in target binding compared to their parental counterpart, gantingrulimab.

[0466]

[0467] Reference: pH 6.0, 20 mM His / His*HCl, 140 mM NaCl; no stress (freezing -80℃)

[0468] Condition 1: pH 6.0, 20 mM His / His*HCl, 140 mM NaCl; stress at 40℃ for 14 days.

[0469] Condition 2: pH 7.4, PBS; 37℃ stress for 14 days.

[0470] Based on all data (100% uniform Fab sugar content, removal of deamidation sites, wild-type light chain), mAb675 was identified as the most favorable gantinus monoclonal antibody variant for performance improvement.

[0471] Exemplary anti-human A-β protein antibody according to the present invention

[0472] Therefore, the present invention covers at least the following embodiments:

[0473] In one aspect, the present invention provides an antibody that binds to human A-β protein.

[0474] In one aspect, the present invention provides isolated antibodies that bind to human A-β protein.

[0475] In one aspect, the present invention provides an antibody that specifically binds to human A-β protein.

[0476] In all aspects and certain embodiments of the invention, the anti-A-β antibody according to the invention has one or more of the following characteristics:

[0477] -Specifically binds to human A-β protein of SEQ ID NO: 45;

[0478] -Has a glycosylation site in the heavy chain CDR2, which has a sugar occupancy of at least 95% as determined by CE-SDS;

[0479] - Species that do not contain any non-glycosylated glycosylation sites;

[0480] - Having an EC50 value of 0.5 nM or less for human A-β protein of SEQ ID NO: 45;

[0481] - At staining concentrations of up to and including 1 μg / mL, it binds to A-β plaques in vitro and does not bind to non-A-β protein molecules in brain samples;

[0482] - Compared with gantinguishumab, it binds more strongly to A-β plaques in vivo;

[0483] -As determined by one-way ANOVA, the relative in vivo occupancy of plaques in the cortex and hippocampus exceeds 0.2;

[0484] - Exhibits thermal stability exceeding 68°C (DLS T) agg / DLS T m ).

[0485] In one aspect, the present invention provides an anti-A-β protein antibody comprising at least one, at least two, at least three, at least four, at least five, or all six critical receptors (CDRs), wherein the CDRs are selected from: (1)

[0487] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0488] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86;

[0489] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0490] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0491] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0492] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0493] or (2)

[0495] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0496] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89;

[0497] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0498] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0499] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0500] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0501] or (3)

[0503] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0504] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86;

[0505] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0506] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0507] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0508] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91;

[0509] or (4)

[0511] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0512] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89;

[0513] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0514] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0515] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0516] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91.

[0517] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VHCDR sequences selected from: (1)

[0519] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0520] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86; and

[0521] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0522] or (2)

[0524] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0525] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89; and

[0526] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0527] or (3)

[0529] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0530] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86; and

[0531] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0532] or (4)

[0534] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0535] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89; and

[0536] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87.

[0537] In a preferred aspect, the antibody comprises CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87.

[0538] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VLCDR sequences, wherein the VLCDR sequences are selected from: (1)

[0540] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0541] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0542] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0543] or (2)

[0545] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0546] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0547] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0548] or (3)

[0550] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0551] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0552] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91;

[0553] or (4)

[0555] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0556] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0557] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91.

[0558] In another aspect, the antibody of the present invention comprises:

[0559] (a) A VH domain containing at least one, at least two, or all three VH CDR sequences selected from: (1)

[0561] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0562] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86; and

[0563] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0564] or (2)

[0566] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0567] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89; and

[0568] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0569] or (3)

[0571] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0572] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86; and

[0573] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0574] or (4)

[0576] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0577] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89; and

[0578] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87,

[0579] and (b) a VL domain containing at least one, at least two, or all three VL CDR sequences selected from: (1)

[0581] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0582] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0583] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0584] or (2)

[0586] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0587] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0588] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0589] or (3)

[0591] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0592] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0593] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91;

[0594] or (4)

[0596] (a) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 81;

[0597] (b) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0598] (c) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91.

[0599] In another aspect, the present invention provides an anti-A-β protein antibody comprising: (1)

[0601] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0602] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86;

[0603] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0604] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0605] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0606] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0607] or (2)

[0609] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0610] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89;

[0611] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0612] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0613] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0614] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 83;

[0615] or (3)

[0617] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0618] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 86;

[0619] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0620] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0621] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0622] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91;

[0623] or (4)

[0625] (a) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 85;

[0626] (b) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 89;

[0627] (c) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 87;

[0628] (d) CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0629] (e) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 82; and

[0630] (f) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 91.

[0631] In another aspect, the anti-A-β protein antibody according to the invention comprises one or more of the CDR sequences of VH of SEQ ID NO: 84 or SEQ ID NO: 88. In another embodiment, the anti-A-β protein antibody comprises one or more of the CDR sequences of VL of SEQ ID NO: 80 or SEQ ID NO: 90. In another embodiment, the anti-A-β protein antibody comprises (1) the CDR sequence of VH of SEQ ID NO: 84 and the CDR sequence of VL of SEQ ID NO: 80; or (2) the CDR sequence of VH of SEQ ID NO: 88 and the CDR sequence of VL of SEQ ID NO: 80; or (3) the CDR sequence of VH of SEQ ID NO: 84 and the CDR sequence of VL of SEQ ID NO: 90; or (4) the CDR sequence of VH of SEQ ID NO: 88 and the CDR sequence of VL of SEQ ID NO: 90.

[0632] In a preferred aspect, the anti-A-β protein antibody comprises the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the VH domain of SEQ ID NO: 84 and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the VL domain of SEQ ID NO: 80.

[0633] In one aspect, the anti-A-β protein antibody according to the invention comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:84 and a frame having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the frame amino acid sequence of the VH domain of SEQ ID NO:84. In another aspect, the anti-A-β protein antibody according to the invention comprises three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:84 and a frame having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the frame amino acid sequence of the VH domain of SEQ ID NO:84. In one aspect, the anti-A-β protein antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 84 and a frame having at least 95% sequence identity with the frame amino acid sequence of the VH domain of SEQ ID NO: 84. In another aspect, the anti-A-β protein antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 84 and a frame having at least 98% sequence identity with the frame amino acid sequence of the VH domain of SEQ ID NO: 84.

[0634] In one aspect, the anti-A-β protein antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 80 and a frame having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the frame amino acid sequence of the VL domain of SEQ ID NO: 80. In another aspect, the anti-A-β protein antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 80 and a frame having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the frame amino acid sequence of the VL domain of SEQ ID NO: 80. In one aspect, the anti-A-β protein antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 80 and a frame having at least 95% sequence identity with the frame amino acid sequence of the VL domain of SEQ ID NO: 80. In another aspect, the anti-A-β protein antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 80 and a frame having at least (particularly at least) 98% sequence identity with the frame amino acid sequence of the VL domain of SEQ ID NO: 80.

[0635] In one aspect, the anti-A-β protein antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:86; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:83; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:84; and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:80; and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:80; The amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In a preferred embodiment, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:84, and the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:80.

[0636] In one aspect, the anti-A-β protein antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:86; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:83; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:84; and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:80; and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:80; The amino acid sequence of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; wherein the antibody specifically binds to human A-β protein. In a preferred embodiment, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:84, and the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:80.

[0637] On the other hand, the anti-A-β protein antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 84. In one aspect, the anti-A-β protein antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 84. In some aspects, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-A-β protein antibody containing this sequence retains its ability to bind to human A-β protein. In some aspects, in SEQ ID NO: 84, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). Optionally, the anti-A-β protein antibody comprises the VH sequence of SEQ ID NO: 84, including post-translational modifications of that sequence. In one specific aspect, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87. In another aspect, an anti-A-β protein antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 80. In one aspect, the anti-A-β protein antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 80. In some aspects, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-A-β protein antibody containing this sequence retains its ability to bind to human A-β protein. In some aspects, in SEQ ID NO: 80, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In some aspects, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR).Optionally, the anti-A-β protein antibody comprises the VL sequence of SEQ ID NO: 80, including post-translational modifications of that sequence. In one specific aspect, VL comprises one, two, or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83.

[0638] In another aspect, an anti-A-β protein antibody is provided, wherein the antibody comprises a VH sequence as described in any of the aspects provided above and a VL sequence as described in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 84 and SEQ ID NO: 80, respectively, including post-translational modifications of those sequences.

[0639] In another aspect of the invention, the anti-A-β protein antibody according to any of the foregoing aspects is a monoclonal antibody, including chimeric antibodies, humanized antibodies, or human antibodies. In one aspect, the anti-A-β protein antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, bisomatic antibodies, or F(ab')2 fragments. In another aspect, the antibody is a full-length antibody, such as a complete IgG1 antibody as defined herein or other antibody classes or isotypes.

[0640]

[0641] In some embodiments, amino acid sequence variants of the antibodies according to the invention are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibodies according to the invention. Amino acid sequence variants of the antibodies according to the invention can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody according to the invention or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody according to the invention. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0642] In some embodiments of all aspects and examples of the invention, the heavy chain variable domain of the antibody according to the invention comprises a pyroglutamic acid (pE) residue replacing a glutamine (Q) residue as a first residue.

[0643] a) Substitution, insertion, and deletion variants

[0644] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include HVR and FR. Conserved substitutions are shown under the “Preferred Substitutions” heading in the table below. Further substantial changes are provided under the “Exemplary Substitutions” heading in the table below and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activities (e.g., preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0645]

[0646] Amino acids can be grouped based on common side-chain characteristics:

[0647] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0648] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0649] (3) Acidic: Asp, Glu;

[0650] (4) Alkaline: His, Lys, Arg;

[0651] (5) Residues affecting chain orientation: Gly, Pro;

[0652] (6) Fang ethnic group: Trp, Tyr, Phe.

[0653] Non-conservative substitution would require swapping members of one of these categories for members of another category.

[0654] One type of substitution variant involves replacing one or more highly variable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more resulting variants selected for further research will alter (e.g., improve) certain biological properties (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody, relative to the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[0655] For example, HVR can be altered (e.g., substituted) to improve antibody affinity. Such alterations can occur in HVR “hotspots,” which are residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196) and / or residues that come into contact with the antigen (detecting the binding affinity of the resulting variant VH or VL). Methods for affinity maturation by constructing and reselecting from a secondary library have been described, for example, in Hoogenboom, HR et al., Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide site-directed mutagenesis). A secondary library is then created. This library is subsequently screened to identify any antibody variant with the desired affinity. Another approach to introducing diversity involves HVR-directed methods, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutations or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0656] In some embodiments, substitution, insertion, or deletion may occur within one or more HVRs, provided that such changes do not substantially reduce the antigen-binding ability of the antibody according to the invention. For example, conserved changes (e.g., conserved substitutions as provided herein) that do not substantially reduce binding affinity may be made in the HVRs. Such changes may occur outside the antigen-contact residues of the HVRs. In some embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or contains no more than one, two, or three amino acid substitutions.

[0657] A method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scan mutagenesis," as described by Cunningham, BC, and Wells, JA, Science 244 (1989) 1081-1085. In this method, residues or target groups of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they possess the desired properties.

[0658] Amino acid sequence insertions include the fusion of amino and / or carboxyl ends of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or peptide that increases the serum half-life of the antibody.

[0659] b) Fc region glycosylation variants

[0660] In some embodiments, the antibody according to the invention is modified to increase or decrease the degree of glycosylation in the antibody Fc region. Adding or deleting glycosylation sites to the antibody according to the invention can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0661] When an antibody contains an Fc region, the carbohydrates attached to it can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via N-bonding. See, for example: Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32. Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies reported herein can be modified to produce antibody variants with certain improved properties.

[0662] In some embodiments, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose intrinsically present at Asn297 of the glycan chain relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297 as determined by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have improved ADCC function. See, for example, US 2003 / 0157108 and US 2004 / 0093621. Antibody variants that are "defucosylated" or "fucose-deficient" include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003 / 0157108; and WO 2004 / 056312, especially in Example 11), and gene knockout cell lines, such as CHO cells with the α-1,6-fucosylation gene FUT8 knocked out (see, for example, Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003 / 085107).

[0663] Further antibody variants with dimeric oligosaccharides are provided, for example, wherein the diangular oligosaccharide linked to the Fc region of the antibody is dimeric with GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; US 6,602,684; and US 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0664] c) Fc region variant

[0665] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody according to the invention, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0666] In some embodiments, antibody variants with some, but not all, effector functions are considered herein, making them ideal candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, JV, and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in the following literature: US 5,500,362 (see, for example: Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be assessed in vivo, for example, in animal models (such as those disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656). A C1q binding assay may also be performed to confirm that the antibody does not bind to C1q and therefore lacks CDC activity.See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example: Gazzano-Santoro et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int. Immunol. 18 (2006: 1759-1769).

[0667] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6,737,056). Such Fc region variants include Fc regions with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc region variant, in which residues 265 and 297 are substituted with alanine (US 7,332,581).

[0668] Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, US 6,737,056; WO 2004 / 056312 and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0669] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbers of residues) in the Fc region.

[0670] In some embodiments, such as those described in US 6,194,551, WO 99 / 51642 and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184, alterations are made in the Fc region resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC).

[0671] Antibodies with prolonged half-lives and improved binding to the neonatal Fc receptor (FcRn), responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593; and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005 / 0014934. These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc region variants include Fc region variants with substitutions in one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as the substitution of Fc region residue 434 (US 7,371,826).

[0672] For other examples of Fc region variants, see Duncan, AR and Winter, G., Nature 322(1988) 738-740; US 5,648,260; US 5,624,821; and WO 94 / 29351.

[0673] d) Cysteine-engineered antibody variants

[0674] In some embodiments, it is desirable to generate cysteine-engineered antibodies, such as "thioMAbs," wherein one or more residues of the antibody are substituted with cysteine ​​residues. In some embodiments, the substituted residues are located at accessible sites on the antibody. As further described herein, by substituting those residues with cysteine, a reactive thiol group is positioned at an accessible site on the antibody and can be used to conjugate the antibody with other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to produce an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antibodies can be generated as described, for example, in US 7,521,541.

[0675] e) Antibody derivatives

[0676] In some embodiments, the antibody according to the invention may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers linked to the antibody may vary, and if more than one polymer is linked, they may be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for a limited therapy.

[0677] In some embodiments, a conjugate of an antibody and a non-protein portion that can be selectively heated by exposure to radiation is provided. In some embodiments, the non-protein portion is carbon nanotubes (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to a temperature at which cells near the antibody-non-protein portion are killed.

[0678] Recombination Method and Composition

[0679] Antibodies according to the invention can be generated using recombinant methods and compositions, for example, as described in US 4,816,567. The invention provides one or more isolated nucleic acid molecules encoding anti-human A-β antibodies according to the invention. Such nucleic acid molecules may encode an amino acid sequence of VL comprising an antibody according to the invention (e.g., the light and / or heavy chain of an antibody according to the invention) and / or an amino acid sequence of VH comprising an antibody according to the invention (e.g., the light and / or heavy chain of an antibody according to the invention). In some embodiments, one or more plasmids (e.g., expression plasmids) comprising such nucleic acid molecules are provided. In some embodiments, a host cell comprising such nucleic acid molecules is provided. In a preferred embodiment, the host cell comprises (e.g., having been transformed with): (1) a plasmid comprising a first nucleic acid molecule encoding an amino acid sequence of VL comprising an antibody and a second nucleic acid molecule encoding an amino acid sequence of VH comprising an antibody; or (2) a first plasmid comprising a nucleic acid molecule encoding an amino acid sequence of VL comprising an antibody and a second plasmid comprising a nucleic acid molecule encoding an amino acid sequence of VH comprising an antibody. In some embodiments, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, Sp2 / O cells). In some embodiments, a method for preparing an anti-human A-β protein antibody is provided, wherein the method includes culturing a host cell containing one or more nucleic acid molecules encoding antibodies as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0680] For the recombinant production of antibodies against human A-β protein, nucleic acid molecules encoding the antibody, such as those described above, are isolated and inserted into one or more plasmids for further cloning and / or expression in host cells. Such nucleic acid molecules can be readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0681] Suitable host cells for cloning or expressing nucleic acid molecules encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, for example, especially when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in *E. coli*.) Antibodies can be separated from the bacterial cell paste in a soluble fraction after expression and can be further purified.

[0682] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. These eukaryotic microorganisms include fungal and yeast strains whose glycosylation pathways have been "humanized," thereby enabling the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0683] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting cells of the meadow armyworm (Spodoptera frugiperda).

[0684] Plant cell cultures can also be used as hosts. See, for example, US 5959177, US 6040498, US 6420548, US 7125978, and US 6417429 (which describe PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0685] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney cell lines (such as 293 or 293 cells as described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells, such as those described, for example, in Mather, JP et al., Annals NY Acad. As described in Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0686] Methods and compositions for diagnosis and detection

[0687] In some embodiments, any of the anti-human A-β protein antibodies according to the invention can be used to detect the presence of A-β protein in a biological sample. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some embodiments, the biological sample comprises cells or tissue.

[0688] In some embodiments, an anti-human A-β protein antibody according to the invention is provided for diagnostic or detection methods. In some embodiments, a method for detecting the presence of A-β protein in a biological sample is provided. In some embodiments, the method includes contacting a biological sample with the anti-human A-β protein antibody according to the invention under conditions that allow the anti-human A-β protein antibody to bind to A-β protein, and detecting whether a complex is formed between the anti-human A-β protein antibody and A-β protein. Such methods can be in vitro or in vivo methods.

[0689] In some embodiments, labeled anti-human A-β protein antibodies according to the present invention are provided. Labeling includes, but is not limited to, labels or portions for direct detection (such as fluorescence, chromophores, electron-dense, chemiluminescence, and radioactive labeling), and portions for indirect detection (e.g., via enzymatic reactions or molecular interactions), such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I; fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, and umbelliferone; and luciferases, such as firefly luciferase and bacterial luciferase (US). 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucosylamylase; lysozyme; sugar oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as urate oxidase and xanthine oxidase; conjugated with enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase, or microperoxidase); biotin / antibiotin protein; spinning labeling; phage labeling; stable free radicals, etc.

[0690] pharmaceutical preparations

[0691] Pharmaceutical formulations of anti-human A-β antibodies according to the present invention are prepared as lyophilized formulations or aqueous solutions by mixing such antibodies of desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)). Pharmaceutically acceptable carriers are non-toxic to the recipient at the doses and concentrations used; and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10). (10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as poly(vinylpyrrolidone); amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers described herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use (including rhuPH20) are described in US 2005 / 0260186 and US 2006 / 0104968. In some embodiments, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0692] Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0693] The formulation described herein may also contain more than one active ingredient essential for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0694] The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules) prepared by, for example, cohesive techniques or interfacial polymerization; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed.) (1980).

[0695] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, in the form of molded articles, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (US 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0696] Formulations intended for in vivo administration are typically sterile. For example, sterility can be readily achieved through filtration using a sterile filter membrane. In one embodiment, the formulation is isotonic.

[0697] Treatment methods and compositions

[0698] Any of the anti-human A-β protein antibodies according to the present invention can be used for therapeutic purposes.

[0699] In one aspect, an anti-human A-β protein antibody according to the invention is provided for use as a medicament. In other aspects, an anti-human A-β antibody according to the invention is provided for the prevention and / or treatment of diseases associated with amyloid formation and / or amyloid plaque formation. In some embodiments, an anti-human A-β protein antibody according to the invention is provided for use as a treatment method. In some embodiments, a method of treating an individual suffering from a disease associated with amyloid formation and / or amyloid plaque formation is provided herein, the method comprising administering to the individual an effective amount of the anti-human A-β protein antibody according to the invention. In some embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those listed below or anti-pTau or anti-α-synuclein antibodies. In further embodiments, an anti-human A-β protein antibody according to the invention is provided herein for inhibiting plaque formation and / or breaking down β-amyloid plaques. In some embodiments, this document provides an anti-human A-β protein antibody according to the invention, used in a method for inhibiting plaque formation and / or breaking down β-amyloid plaques in an individual, the method comprising administering an effective anti-human A-β protein antibody according to the invention to the individual to inhibit plaque formation and / or break down β-amyloid plaques. The “individual” according to any of the above embodiments is preferably a human being.

[0700] In another aspect, this document provides the use of the anti-human A-β antibody according to the invention in the manufacture or preparation of a medicament. In some embodiments, the medicament is used to treat a disease associated with amyloid formation and / or amyloid plaque formation. In some embodiments, the medicament is used as a method of treating a disease associated with amyloid formation and / or amyloid plaque formation, the method comprising administering an effective amount of the medicament to an individual suffering from a disease associated with amyloid formation and / or amyloid plaque formation. In some embodiments, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as those listed below or anti-pTau or anti-α-synuclein antibodies, to the individual. In some embodiments, the medicament is used to inhibit plaque formation and / or break down β-amyloid plaques. In some embodiments, the medicament is used as a method of inhibiting plaque formation and / or breaking down β-amyloid plaques in an individual, the method comprising administering an effective amount of the medicament to the individual to inhibit plaque formation and / or break down β-amyloid plaques. An "individual" according to any of the above embodiments can be a human being.

[0701] In other respects, this document provides methods for treating diseases associated with amyloid production and / or amyloid plaque formation. In some embodiments, the method comprises administering an effective amount of an anti-human A-β protein antibody according to the invention to an individual suffering from a disease associated with amyloid production and / or amyloid plaque formation. In some embodiments, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as those listed below or anti-pTau or anti-α-synuclein antibodies, to the individual. An "individual" according to any of the above embodiments may be a human being.

[0702] In other respects, this document provides methods for inhibiting plaque formation and / or breaking down β-amyloid plaques in an individual. In some embodiments, the method includes administering an effective amount of an anti-human A-β protein antibody according to the invention to the individual to inhibit plaque formation and / or break down β-amyloid plaques. In one embodiment, "individual" refers to a person.

[0703] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-human A-β protein antibodies according to the invention, for example, in any of the above-described treatment methods. In some embodiments, the pharmaceutical formulation comprises any of the anti-human A-β protein antibodies according to the invention, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises any of the anti-human A-β protein antibodies according to the invention, and at least one additional therapeutic agent, for example, an anti-pTau or anti-α-synuclein antibody as given below.

[0704] The antibodies according to the invention can be used alone in therapy or in combination with other agents. For example, the antibodies according to the invention can be administered co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a therapeutic agent that is effective in treating the same or different neurological disorders as those treated with the antibodies according to the invention. Exemplary additional therapeutic agents include, but are not limited to: the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rivastigmine, and tacrine), NMDA receptor antagonists (such as memantine), amyloid β-peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors (statins), ampaquinone, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid β-peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-amyloid β-peptide antibodies. In some embodiments, at least one additional therapeutic agent is selected because of its ability to reduce one or more side effects of the neurological drugs.

[0705] The aforementioned combination therapy encompasses both combined administration (where two or more therapeutic agents are included in the same or separate formulation) and single administration. In the case of single administration, the antibody reported herein may be administered before, simultaneously with, and / or after administration of other therapeutic agents or pharmaceuticals. In some embodiments, administration of the anti-human A-β antibody according to the invention and administration of other therapeutic agents occur within approximately one month of each other, or within approximately one, two, or three weeks of each other, or within approximately one, two, three, four, five, or six days. The antibody according to the invention may also be used in combination with other interventional therapies, such as, but not limited to, radiotherapy, behavioral therapy, or other therapies known in the art suitable for treating or preventing neurological disorders.

[0706] The antibodies (and any other therapeutic agents) according to the invention may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if required for local treatment). Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

[0707] The antibody according to the invention will be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific disease to be treated, the specific mammal to be treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to the medical practitioner. The antibody is not mandatory, but may optionally be formulated in conjunction with one or more preparations currently used for the prevention or treatment of the disease in question. The effective amount of such other preparations depends on the amount of the antibody according to the invention present in the preparation, the type of disease or treatment, and other factors discussed above. These are generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and via any route determined empirically / clinically to be appropriate.

[0708] Lipid-based methods for transporting antibodies or fusion constructs containing antibodies according to the invention across the BBB include, but are not limited to, encapsulating the antibody or fusion construct in liposomes coupled to a monovalent binding entity that binds to a receptor on the vascular endothelium of the BBB (see, for example, US 2002 / 0025313), and coating the monovalent binding entity in low-density lipoprotein particles (see, for example, US 2004 / 0204354) or apolipoprotein E (see, for example, US 2004 / 0131692).

[0709] For the prevention or treatment of disease, the appropriate dose of the antibody according to the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody according to the invention is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg to 10 mg / kg) may be an initial candidate dose administered to the patient, for example, by single or multiple administrations alone or by continuous infusion. Depending on the above factors, a typical daily dose range may be from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, treatment will generally continue until the desired suppression of disease symptoms occurs, depending on the condition. An exemplary dose range for the antibody is from about 0.05 mg / kg to about 10 mg / kg. Therefore, patients can be administered one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof). Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives approximately two to approximately twenty doses, or for example, approximately six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be available. Progression of this therapy can be easily monitored using routine techniques and assays.

[0710] It should be understood that any of the above-described formulations or treatments can be performed using the immunoconjugates reported herein, in place of or attached to the anti-human A-β protein antibody according to the present invention.

[0711] Products

[0712] In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective for treating, preventing, and / or diagnosing the condition, and the container may have a sterile inlet (e.g., the container may be an IV solution bag or vial with a stopper capable of being punctured by a hypodermic needle). At least one active agent in the composition is an antibody according to the invention. The label or packaging insert indicates that the composition is for treating the selected condition. Furthermore, the article may include (a) a first container containing the composition, wherein the composition contains an antibody according to the invention; and (b) a second container containing the composition, wherein the composition contains additional cytotoxic agents or other therapeutic agents. The article in this embodiment may further include a packaging instruction indicating that the composition can be used to treat a specific condition. Alternatively or in addition, the article may further include a second (or third) container containing pharmaceutical buffers such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0713] It should be understood that any of the above-described products may include, as reported herein, an immunoconjugate in place of or attached to the antibody according to the invention.

[0714] ***

[0715] The following figures, sequences, and examples are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the described procedures without departing from the spirit of the invention. Attached Figure Description

[0716] Figure 1. HIC chromatogram of gantinglumonab.

[0717] Figure 2 shows the HIC chromatogram of mAb 663.

[0718] Figure 3 shows the HIC chromatogram of mAb 675.

[0719] Figure 4. In vitro A-β plaque decoration using gantinguishumab.

[0720] Figure 5. In vitro A-β plaque decoration using mAb 675.

[0721] Figure 6. In vitro A-β plaque decoration using mAb 663.

[0722] Figure 7. In vitro A-β plaque decoration using mAb 651.

[0723] Figure 8. In vivo A-β plaque decoration results.

[0724] Figure 9. In vivo A-β plaque decoration results.

[0725] Figure 10 Pharmacokinetic results.

[0726] Sequence Description

[0727] SEQ ID NO - Description

[0728]

[0729] Example

[0730] Example 1

[0731] Materials and General Methods

[0732] General information on the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of the antibody chains are numbered and cited according to their sequence numbers, as per Kabat's (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0733] Recombinant DNA technology

[0734] Use standard methods to manipulate DNA, as described in Sambrook, J. et al., Molecular Cloning: Alaboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Use molecular biology reagents according to the manufacturer's instructions.

[0735] Gene synthesis

[0736] The desired gene segment is prepared from oligonucleotides synthesized chemically. Long gene segments with single restriction endonuclease cleavage sites are assembled by annealing and ligating oligonucleotides (including PCR amplification), and subsequently cloned via the designated restriction sites. The DNA sequence of the subcloned gene fragment is confirmed by DNA sequencing. The synthesized gene fragment is sequenced according to the given specifications from Geneart (Regensburg, Germany).

[0737] DNA sequencing

[0738] The DNA sequence was determined by double-strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

[0739] DNA and protein sequence analysis and sequence data management

[0740] Use GCG (Genetics Computer Group, Madison, Wisconsin) software version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 for sequence creation, mapping, analysis, annotation, and description.

[0741] expression carrier

[0742] To express the antibody according to the invention, expression plasmids for transient expression (e.g., in HEK293 cells) can be applied based on cDNA tissue with or without the CMV-intron A promoter or based on genomic tissue with the CMV promoter.

[0743] In addition to the antibody expression cassette, the vector also contains:

[0744] -Origin of replication, which allows the plasmid to be replicated in E. coli, and

[0745] The β-lactamase gene confers ampicillin resistance in Escherichia coli.

[0746] The transcription unit of an antibody gene is composed of the following elements:

[0747] The unique restriction site at the -5' end,

[0748] -Immediate early enhancer and promoter from human cytomegalovirus,

[0749] -Intron A sequence in the case of cDNA tissue,

[0750] -Derived from the 5' untranslated region of the human antibody gene,

[0751] -Immunoglobulin heavy chain signal sequence

[0752] - The corresponding antibody chain encodes nucleic acid, either as cDNA or as a genome exon-intron structure.

[0753] -The 3' untranslated region containing the polyadenylation signal sequence, and

[0754] The unique restriction site at the -3' end.

[0755] A fusion gene encoding an antibody chain is generated via PCR and / or gene synthesis, and the fusion gene is assembled using known recombination methods and techniques, for example, by ligating the corresponding nucleic acid segments using unique restriction sites in the appropriate vector. The nucleic acid sequence of the subcloned organism is verified by DNA sequencing. For transient transfection, a large quantity of plasmids (Nucleobond AX, Macherey-Nagel) is prepared from the transformed *E. coli* culture using plasmid formulations.

[0756] Cell culture technology

[0757] Standard cell culture techniques were used, as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J., and Yamada, KM (ed.), John Wiley & Sons, Inc.

[0758] Transient transfection in the HEK293-F system

[0759] Antibodies are generated through transient expression. Therefore, transfection can be performed using the HEK293-F system (Invitrogen) with the appropriate plasmid, following the manufacturer's instructions. In short, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle™ 293 expression medium (Invitrogen) in shake flasks or stirred fermentation tubes are transfected with the appropriate expression plasmid and a mixture of 293fectin™ or fectin (Invitrogen). For 2 L shake flasks (Corning), HEK293-F cells are seeded at a density of 1.0*1E6 cells / mL in 600 mL and incubated at 120 rpm and 8% CO2. On the second day, cells were transfected at a density of approximately 1.5*1E6 cells / mL using approximately 42 mL of a mixture of the following: A) 20 mL of Opti-MEM medium (Invitrogen) containing 600 μg total plasmid DNA (1 μg / mL) and B) 20 mL of Opti-MEM medium supplemented with 1.2 mL of 293 fectin or fectin (2 μL / mL). Glucose solution was added during fermentation according to glucose consumption. The supernatant containing secreted antibodies was harvested after 5–10 days, and the antibodies were either purified directly from the supernatant or frozen and stored.

[0760] Protein assay

[0761] The protein concentrations of purified antibodies and derivatives were determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence, as described by Pace et al., Protein Science 4 (1995) 2411-1423.

[0762] Antibody concentration determination in supernatant

[0763] The concentration of antibodies in cell culture supernatant was estimated by immunoprecipitation using protein A agarose beads (Roche Diagnostics GmbH, Mannheim, Germany). Therefore, 60 μL of protein A agarose beads were washed three times in TBS-NP40 (50 mM Tris buffer, pH 7.5, supplemented with 150 mM NaCl and 1% Nonidet-P40). Subsequently, 1–15 mL of cell culture supernatant was added to the pre-equilibrated protein A agarose beads in TBS-NP40. After incubating at room temperature for 1 hour, the beads were washed once with 0.5 mL of TBS-NP40 on an Ultrafree-MC filter column (Amicon), twice with 0.5 mL of 2x phosphate-buffered saline (2x PBS, Roche Diagnostics GmbH, Mannheim, Germany), and briefly four times with 0.5 mL of 100 mM sodium citrate buffer (pH 5.0). The bound antibody was eluted by adding 35 μl of NuPAGE® LDS sample buffer (Invitrogen). Half of the sample was either mixed with NuPAGE® sample reducing agent or left unreduced and heated at 70°C for 10 minutes. Therefore, 5-30 μl was applied to 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (non-reducing SDS-PAGE using MOPS buffer and reducing SDS-PAGE using MES buffer with NuPAGE® antioxidant electrophoresis buffer additive (Invitrogen)) and stained with Coomassie blue.

[0764] The concentration of antibodies in cell culture supernatants was quantitatively measured using affinity HPLC. In short, cell culture supernatants containing antibodies binding to protein A were applied to an Applied Biosystems Poros A / 20 column in 200 mM KH₂PO₄, 100 mM sodium citrate, pH 7.4, and eluted with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted antibodies were quantified by UV absorbance and peak area integration. Purified standard IgG1 antibodies were used as standards.

[0765] Alternatively, the concentrations of antibodies and derivatives in the cell culture supernatant can be measured using a Sandwich-IgG-ELISA. In short, the StreptaWell High Bind Streptavidin A-96-well microtiter plate (RocheDiagnostics GmbH, Mannheim, Germany) is used with 100 μL / well of 0.1 μg / mL biotinylated anti-human IgG capture molecule F(ab')2.<h-Fcγ> BI (Dianova) was coated for 1 hour at room temperature or overnight at 4 °C, followed by three washes with 200 μL / well of PBS and 0.05% Tween (PBST, Sigma). Then, 100 μL / well of cell culture supernatant containing the corresponding antibody in a PBS (Sigma) dilution series was added to each well, and the wells were incubated at room temperature on a shaker for 1–2 hours. The wells were then washed three times with 200 μL / well of PBST and 100 μl of 0.1 μg / mL F(ab')2.<hFcγ> POD (Dianova) was used as the detection antibody, and the bound antibody was detected by incubation on a shaker at room temperature for 1–2 hours. Unbound detection antibody was removed by washing three times with 200 μL / well PBST. The bound detection antibody was detected by adding 100 μL / well ABTS and then incubating. Absorbance was measured at a measurement wavelength of 405 nm (reference wavelength 492 nm) on a Tecan Fluor spectrometer.

[0766] Preparative antibody purification

[0767] The antibody was purified from the filtered cell culture supernatant according to a standard protocol. Briefly, the antibody was applied to a Protein A agarose column (GE Healthcare) and washed with PBS. Elution of the antibody was achieved at pH 2.8, followed immediately by neutralization. The aggregated protein was separated from the monomeric antibody by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM histidine buffer (containing 150 mM NaCl) (pH 6.0). The monomeric antibody fractions were pooled, concentrated (if necessary) using a centrifuge such as a MillIPORE Amicon Ultra (30 MWCO), and frozen and stored at -20°C or -80°C. Fractions of the sample were provided for subsequent protein analysis and analytical characterization, for example, by SDS-PAGE, size exclusion chromatography (SEC), or mass spectrometry.

[0768] SDS-PAGE

[0769] Use the NuPAGE® pre-prepared gel system (Invitrogen) according to the manufacturer's instructions. Specifically, use 10% or 4-12% NuPAGE® Novex® Bis-TRIS pre-prepared gel (pH 6.4) and NuPAGE® MES (reducing gel with NuPAGE® antioxidant electrophoresis buffer additive) or MOPS (non-reducing gel) electrophoresis buffer.

[0770] CE-SDS

[0771] Antibody purity and integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Therefore, 5 μl antibody solutions were prepared according to the manufacturer's instructions using the HT Protein Express kit for CE-SDS analysis, and the analysis was performed on the LabChip GXII system using the HT Protein Express chip. Data were analyzed using LabChip GX software.

[0772] Analytical size exclusion chromatography

[0773] Size exclusion chromatography (SEC) was performed by HPLC to determine the aggregated and oligomeric states of the antibody. Briefly, the protein A purified antibody was applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄ / K₂HPO₄ buffer (pH 7.5) on a Dionex Ultimate® system (Thermo Fischer Scientific), or to a Superdex 200 column in 2 x PBS on a Dionex HPLC system (GE Healthcare). The eluted antibody was quantified by UV absorbance and peak area integration. BioRad gel filtration standard 151-1901 was used as a standard.

[0774] mass spectrometry

[0775] This section describes the characterization of the bispecific antibody, focusing on its proper assembly. The expected primary structure was analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact antibody and, in the specific case, the deglycosylated / restricted LysC-digested antibody.

[0776] Antibodies were deglycosylated with N-glycosidase F in phosphate or Tris buffer at a protein concentration of 1 mg / ml at 37°C for up to 17 hours. 100 μg of deglycosylated antibody in Tris buffer (pH 8) for restriction LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestion was performed at room temperature for 120 hours or at 37°C for 40 minutes. Samples were desalted by HPLC on a Sephadex G25 column (GE Healthcare) prior to mass spectrometry. Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0777] Chemical degradation test

[0778] The samples were divided into three equal parts and reburied in 20 mM His / His*HCl, 140 mM NaCl, pH 6.0, or PBS, and stored at 40°C (His / NaCl) or 37°C (PBS). The control samples were stored at -80°C.

[0779] After the incubation period, the relative activity concentration (BIAcore), aggregation (SEC), and fragmentation of the samples were analyzed (capillary electrophoresis or SDS-PAGE) and compared with the untreated control.

[0780] thermal stability

[0781] Samples were prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine chloride, 140 mM NaCl, pH 6.0, transferred to 384-well optical plates by centrifugation via a 0.4 μm filter, and covered with paraffin oil. The hydrodynamic radius was repeatedly measured by dynamic light scattering on a DynaPro plate reader (Wyatt) while the samples were heated from 25 °C to 80 °C at a rate of 0.05 °C / min.

[0782] Alternatively, the samples were transferred to a 10 μL microcuvette array, and static light scattering data and fluorescence data after 266 nm laser excitation were recorded using an Optim1000 instrument (Avacta Inc.), while they were heated from 25 °C to 90 °C at a rate of 0.1 °C / min.

[0783] The aggregation initiation temperature is defined as the temperature at which the hydrodynamic radius (DLS) or the temperature at which the scattered light intensity (Optim1000) begins to increase.

[0784] Alternatively, the sample was transferred in a 9 μL multi-tube array. The array was heated from 35 °C to 90 °C at a constant rate of 0.1 °C / min using an Optim1000 instrument (AvactaAnalytical Inc.). The instrument continuously recorded the intensity of the scattered light from the 266 nm laser, recording data points approximately every 0.5 °C. The light scattering intensity was plotted relative to temperature. The aggregation initiation temperature (T0) is also included. agg The temperature at which the intensity of scattered light begins to increase is defined as the temperature at which the intensity of scattered light begins to increase.

[0785] The melting temperature is defined as the inflection point on the curve of fluorescence intensity versus wavelength.

[0786] mice

[0787] Using B6.Cg-Fcgrt, a hemizygous transgene lacking the mouse FcRn-chain gene but targeting the human FcRn-chain gene. tm1Dcr Tg(FCGRT)276Dcr mice (muFcRn- / - huFcRn tg + / -, strain 276) were used for pharmacokinetic studies. Mice were housed under specific pathogen-free conditions. Mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) (female, 4–10 weeks old, weighing 17–22 g at administration). All animal experiments were approved by the Government of Upper Bavaria, Germany (License No. 55.2-1-54-2532.2-28-10) and conducted in AAALAC-accredited animal facilities in accordance with EU guidelines for the care and use of laboratory animals. Animals were housed in standard cages and had free access to food and water throughout the study.

[0788] Pharmacokinetic studies

[0789] A single dose of the antibody was administered intravenously via the lateral tail vein at a dose level of 10 mg / kg. Mice were randomly divided into three groups of six mice each, covering nine serum collection time points (0.08, 2, 8, 24, 48, 168, 336, 504, and 672 hours post-administration). Each mouse underwent two retroorbital blood samplings under mild anesthesia with isoflurane™ (CP-Pharma GmbH, Burgdorf, Germany); a third blood sample was collected at euthanasia. Blood was collected into serum tubes (Microvette 500Z-Gel, Sarstedt, Nümbrecht, Germany). After incubation for 2 hours, the samples were centrifuged at 9.300 g for 3 minutes to obtain serum. After centrifugation, serum samples were frozen at -20°C until analysis.

[0790] PK Analysis

[0791] Pharmacokinetic parameters were calculated using WinNonlin™ 1.1.1 (Pharsight, CA, USA) via non-compartmental analysis.

[0792] In short, due to the non-linear reduction of the antibody, the area under the curve (AUC) 0-inf The values ​​were calculated using the logarithmic trapezoidal method and extrapolated to infinity using the apparent terminal rate constant λz, from the concentration observed at the last time point.

[0793] Plasma clearance is calculated as dose rate (D) divided by AUC. 0-inf The apparent terminal half-life (T1 / 2) is derived from the equation T1 / 2 = ln2 / λz.

[0794] Example 2

[0795] Expression and purification

[0796] Antibodies were generated as described in the General Materials and Methods section above.

[0797] The antibody was purified from the supernatant using a combination of protein A affinity chromatography and size exclusion chromatography. The obtained product was characterized for identity (by mass spectrometry) and analytical properties such as purity (by CE-SDS), monomer content, and stability.

[0798] As described in the General Methods section, the expected primary structure was analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact antibody and the deglycosylated / fibrinolytically digested or alternatively deglycosylated / restricted LysC-digested antibody.

[0799] Other analytical methods (such as thermal stability, mass spectrometry, and functional assessment) are only used after protein A and SEC purification.

[0800] Example 3

[0801] In vitro binding to Aβ1-40 fibers was determined by ELISA.

[0802] The binding of the antibody to fibrillated Aβ was measured by ELISA. Briefly, Aβ(1-40) in PBS was coated onto Maxisorb plates at 7 μg / mL at 37°C for 3 days to generate fibrillated Aβ, and then dried at room temperature for 3 hours. The plates were blocked at room temperature for 1 hour with 1% Crotein C and 0.1% RSA (in PBS) (blocking buffer), followed by a single wash with washing buffer. Up to 100 nM of antibody or control was added to the blocking buffer and incubated overnight at 4°C. After four washes, the construct was detected by adding anti-human IgG-HRP (Jackson Immunoresearch) diluted 1:10,000 in blocking buffer (1 RT), followed by six washes and incubation in TMB (Sigma). The absorbance was read at 450 nm after stopping the color development with 1 N HCl.

[0803] Example 4

[0804] Natural human β-amyloid plaques from brain slices of Alzheimer's disease patients were stained by indirect immunofluorescence using the antibody according to the invention.

[0805] The staining ability of antibodies against native human β-amyloid plaques can be tested using indirect immunofluorescence via immunohistochemical analysis. This demonstrates specificity and sensitivity for staining genuine human β-amyloid plaques. Frozen sections of unfixed tissue from the temporal lobe cortex obtained post-mortem from Alzheimer's disease-positive patients were labeled using indirect immunofluorescence. The binding of bispecific antibodies was detected using a two-step incubation, revealed by affinity-purified goat anti-human (GAH555) IgG (H+L) (Molecular Probes) conjugated to Alexa 555 dye. Controls may include unrelated human IgG1 antibodies (Sigma) and separate secondary antibodies, both of which should yield negative results.

[0806] Example 5

[0807] In vivo β-amyloid plaque decoration in a mouse model of Alzheimer's disease using antibodies according to the present invention

[0808] The ability of the antibody to immunomodulate β-amyloid plaques in vivo was tested in APP / PS2 double transgenic mice (a mouse model of AD-associated amyloidosis) (Richards, J. Neuroscience, 23 (2003) 8989-9003). This allowed for the assessment of brain penetration and binding to amyloid-β plaques. The antibody was administered at different doses, and after 6 days, the animals were perfused with phosphate-buffered saline, and the brains were frozen on dry ice and cryosectioned.

[0809] The presence of antibodies binding to β-amyloid plaques was assessed using unfixed frozen sections at room temperature via single-labeled indirect immunofluorescence with 15 μg / ml of goat anti-human IgG (H+L) (GAH555) (Molecular Probes) conjugated to Alexa555 dye for 1 hour. Counterstaining of amyloid plaques was performed by incubation at room temperature with 0.5 μg / ml of mouse monoclonal antibody BAP-2 conjugated to Alexa 488 against Aβ for 1 hour. Slides were mounted with fluorescent mounting medium (S3023 Dako) and imaged using confocal laser microscopy.

[0810] Example 6

[0811] FcRn and heparin affinity chromatography

[0812] Preparation of FcRn affinity columns

[0813] FcRn expression in HEK293 cells

[0814] FcRn was transiently expressed in HEK293 cells by transfecting them with two plasmids containing coding sequences for FcRn and β-2-microglobulin. Transfected cells were cultured in shake flasks at 36.5°C, 120 rpm (shake amplitude 5 cm), 80% humidity, and 7% CO2. Cells were diluted to a density of 3–4 × 10⁵ cells / ml every 2–3 days.

[0815] For transient expression, a 14 L stainless steel bioreactor was started at 36.5 °C, pH 7.0 ± 0.2, pO2 35% (aerated with N2 and air, total gas flow rate 200 ml min⁻¹), with a culture volume of 8 L and a stirrer speed of 100–400 rpm. When the cell density reached 20 × 10⁵ cells / ml, 10 mg of plasmid DNA (equimolar amounts of both plasmids) was diluted in 400 ml of Opti-MEM (Invitrogen). 20 ml of 293fectin (Invitrogen) was added to the mixture, and the mixture was incubated at room temperature for 15 min, followed by transfer to a fermenter. Starting the next day, the cells were fed continuously: feed solution was added at a rate of 500 ml per day, with glucose added as needed to maintain a level above 2 g / L. Seven days post-transfection, the supernatant was harvested at 4000 rpm for 90 min using a 1 L oscillating centrifuge. The supernatant (13 L) was removed by a Sartobran P filter (0.45 μm + 0.2 μm, Sartorius) and the FcRn β-2-microglobulin complex was purified from it.

[0816] Biotinylation of Fc receptors in newborns

[0817] Dissolve / dilute 3 mg of FcRn β-2-microglobulin complex in 5.3 mL of 20 mM sodium dihydrogen phosphate buffer containing 150 mM sodium chloride, and add to 250 μL of PBS and one tablet of complete protease inhibitor (ULTRA tablet, Roche Diagnostics GmbH). Biotinylate FcRn using a biotinylation kit from Avidity, following the manufacturer's instructions (Bulk BIRA, Avidity LLC). Allow the biotinylation reaction to proceed overnight at room temperature.

[0818] To remove excess biotinylated FcRn, dialyze it overnight at 4°C against 20 mM MES buffer (containing 140 mM NaCl, pH 5.5) (buffer A).

[0819] Coupling with streptavidin agarose

[0820] To couple with streptavidin agarose, 1 mL of streptavidin agarose (GE Healthcare, United Kingdom) was added to the biotinylated and dialyzed FcRn β-2-microglobulin complex and incubated overnight at 4°C. The FcRn β-2-microglobulin complex-derived agarose was packed into a 4.6 mm x 50 mm column (Repligen). The column was stored in 80% buffer A and 20% buffer B (20 mM Tris(hydroxymethyl)aminomethane, pH 8.8, 140 mM NaCl).

[0821] Chromatography using FcRn affinity columns and pH gradients

[0822] condition:

[0823] Column dimensions: 50 mm x 4.6 mm

[0824] Sample loading: 30 μg sample

[0825] Buffer A: 20 mM MES containing 140 mM NaCl, adjusted to pH 5.5.

[0826] Buffer B: 20 ​​mM Tris / HCl, containing 140 mM NaCl, adjusted to pH 8.8.

[0827] Apply 30 μg of sample to an FcRn affinity column equilibrated with buffer A. After a 10-minute wash in 20% buffer B at a flow rate of 0.5 mL / min, elute for over 70 minutes using a linear gradient of 20% to 70% buffer B. Detection is performed using UV absorption at 280 nm. Regenerate the column with 20% buffer B for 10 minutes after each run.

[0828] To calculate the relative retention time, according to (Bertoletti-Ciarlet, A. et al., Mol. Immunol.46 (2009) 1878-1882), standard samples (anti-Her3 antibodies (SEQ ID NO: 52 and 53)) were oxidized with 0.02% peroxide for 18 hours at the start of the series and after every 10 sample injections.

[0829] In short, the antibody (9 mg / mL) in 10 mM sodium phosphate buffer (pH 7.0) was mixed with H2O2 to a final concentration of 0.02% and incubated at room temperature for 18 hours. To quench the reaction, the sample was thoroughly dialyzed into pre-cooled 10 mM sodium acetate buffer (pH 5.0).

[0830] The relative retention time is calculated using the following formula:

[0831] t_(rel,i)=(t_i-t_peak2) / (t_peak3-t_peak2)

[0832] For the definition of peaks, see Figure 1 in WO 2018 / 197533.

[0833] Chromatography using heparin affinity columns and pH gradients

[0834] condition:

[0835] Column dimensions: 50 mm x 5.0 mm

[0836] Sample loading: 20-50 μg sample

[0837] Buffer A: 50 mM Tris, pH 7.4

[0838] Buffer B: 50 mM Tris, pH 7.4, 1000 mM NaCl

[0839] Protein samples in 20–50 μg of low-salt buffer (≤ 25 mM ionic strength) were applied to a 5.0 x 50 mm TSKgel Heparin-5PW glass column (Tosoh Bioscience, Tokyo / Japan), pre-equilibrated with buffer A at room temperature. Elution was performed over 32 minutes at a flow rate of 0.8 mg / mL using a linear gradient of 0–100% buffer B. Detection was performed using UV absorbance at 280 nm. Each injection series began with retention time standards (anti-pTau antibody; SEQ ID NO: 50 and 51) used to calculate the relative retention time according to the following formula:

[0840] t_(rel,i)=t_i / t_pTau

[0841] (trel,i: relative retention time of peak i; ti: retention time of peak i; tpTau: retention time of anti-pTau antibody peak).

[0842] ***

[0843] Although the invention has been described in considerable detail above by way of example and illustration for the purpose of clarity, such description and examples should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. An antibody that binds to a human A-beta protein, wherein the antibody comprises a heavy chain variable domain (VH) and a light chain variable domain, the heavy chain variable domain and the light chain variable domain comprising CDRs selected from the group consisting of: (1) (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (2) (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 89; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (3) (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91; or (4) (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 89; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

91.

2. The antibody of claim 1, wherein the antibody has one or more of the following properties: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 89; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91; or (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 89; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

91. - specifically binds to a human A-beta protein of SEQ ID NO: 45; - has a glycosylation site in heavy chain CDR2 with a sugar occupancy of at least 95% as determined by CE-SDS; - is free of species having any non-glycosylated glycosylation sites; - has an EC50 value of 0.5 nM or less for a human A-beta protein of SEQ ID NO: 45; - binds to A-beta plaques in vitro and does not bind to non-A-beta protein molecules in brain samples at a staining concentration of at most and including 1 pg / mL; - binds to A-beta plaques in vivo stronger compared to gantenerumab; - has an in vivo relative occupancy in plaques of the cortex and hippocampus exceeding 0.2 as determined by one-way ANOVA; - having a thermal stability (DLS T agg / DLS T m ) of more than 68°C.

3. The antibody of any one of claims 1 to 2, wherein the antibody is a monoclonal antibody.

4. The antibody of any one of claims 1 to 3, wherein the antibody is an antibody fragment that binds to a human A-beta protein.

5. The antibody of any one of claims 1 to 4, wherein the antibody comprises: (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 84; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 80; or (c) a VH sequence as defined in (a) and a VL sequence as defined in (b).

6. The antibody of any one of claims 1 to 5, wherein the antibody comprises the VH sequence of SEQ ID NO: 84 and the VL sequence of SEQ ID NO:

80.

7. The antibody of any one of claims 1 to 6, wherein the antibody is: a) a full-length antibody of the human IgGl subclass, b) a full-length antibody of the human IgG4 subclass, c) a full-length antibody of the human IgGl subclass with the mutations L234A, L235A and P329G, d) a full-length antibody of the human IgGl subclass with the mutations L234A, L235A and P329G in both heavy chains and with the mutations T366W and S354C in one heavy chain and T366S, L368A, Y407V and Y349C in the respective other heavy chain, e) a full-length antibody of the human IgGl subclass with the mutations L234A, L235A and P329G in both heavy chains and with the mutations T366W and Y349C in one heavy chain and T366S, L368A, Y407V and S354C in the respective other heavy chain, f) a full-length antibody of the human IgG4 subclass with the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, g) a full-length antibody of the human IgG4 subclass with the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, h) a full-length antibody of the human IgG1 subclass with the mutations L234A, L235A, P329G, I253A, H310A and H435A in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, i) a full-length antibody of the human IgG1 subclass with the mutations L234A, L235A, P329G, I253A, H310A and H435A in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, j) a full-length antibody of the human IgG1 subclass with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains and the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain, k) a full-length antibody of the human IgG1 subclass with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains and the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain, or l) a full-length antibody of the human IgG1 subclass with the mutations L234A, L235A, P329G, H310A, H433A and Y436A in both heavy chains and the mutations i) T366W and ii) S354C or Y349C in one heavy chain and the mutations i) T366S, L368A and Y407V and ii) Y349C or S354C in the respective other heavy chain, or m) one of a) to l) and without a C-terminal lysine residue.

8. The antibody of any one of claims 1 to 7, wherein the antibody comprises: a heavy chain comprising the heavy chain variable domain of SEQ ID NO: 84 and the heavy chain constant region of SEQ ID NO: 01 ; and a light chain comprising the light chain variable domain of SEQ ID NO: 80 and the light chain kappa constant domain of SEQ ID NO:

29.

9. The antibody of any one of claims 1 to 7, wherein the antibody comprises: a heavy chain comprising the heavy chain variable domain of SEQ ID NO: 84 and the heavy chain constant region of SEQ ID NO: 01, in which a pyroglutamic acid (pE) residue replaces a glutamine (Q) residue as the first N-terminal amino acid residue in the heavy chain variable domain; and a light chain comprising the light chain variable domain of SEQ ID NO: 80 and the light chain kappa constant domain of SEQ ID NO:

29.

10. The antibody of any one of claims 1 to 9, wherein the antibody is a multispecific antibody.

11. A composition of isolated nucleic acid molecules, wherein each of the isolated nucleic acid molecules of the composition encodes one chain of an antibody of any one of claims 1 to 10.

12. A host cell comprising the composition of nucleic acid molecules of claim 11.

13. A method of producing an antibody that binds to a human A-beta protein, the method comprising the steps of: - culturing the host cell of claim 12 in a culture medium, - recovering the antibody from the host cell or / and the culture medium, and - purifying the antibody using one or more chromatography steps.

14. A pharmaceutical composition comprising: the antibody of any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

15. The antibody of any one of claims 1 to 10 or the pharmaceutical composition of claim 14 for use as a medicament.

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