Anti-a-β protein antibodies, methods and uses thereof

Abstract

To provide antibodies against the human A-β protein (anti-A-β protein antibodies), methods for their production, pharmaceutical compositions containing these antibodies, and uses thereof.SOLUTION: The present invention provides 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 (VL) comprising CDRs selected from specific amino acid sequences.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Approximately 70% of all cases of dementia are due to Alzheimer's disease (AD), which is associated with selective damage to brain regions and neural circuits essential for cognition. AD is characterized by neurofibrillary tangles, especially in pyramidal neurons of the hippocampus, and numerous amyloid plaques that contain primarily dense cores and defused halos of amyloid deposits.

[0003] Extracellular neuritic plaques contain primarily large amounts of fibrillar peptides called "amyloid", "amyloid beta", "A-beta", "Abeta4", "beta-A4" or "Abeta". See Selkoe, Ann. Rev. Cell Biol. 10 (1994) 373-403; Koo, Proc. Natl. Acad. Sci. USA 96 (1999) 9989-9990; U.S. Patent No. 4,666,829; Glenner BBRC 12 (1984) 1131). This amyloid is derived from the "Alzheimer's Precursor Protein / P-Amyloid Precursor Protein" (APP). APP is an integral membrane glycoprotein (see Sisodia, Proc. Natl. Acad. Sci. USA 89 (1992) 6075) that is endoproteolytically cleaved within the AP sequence by the plasma membrane protease, α-secretase (see Sisodia (1992), Joe. cit.). Furthermore, further secretase activity, in particular β-secretase and γ-secretase activity, leads to the extracellular release of amyloid-β (Aβ) containing either 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 should be noted that A-beta protein has several naturally occurring forms, the human forms being referred to as Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43 as above. The most prominent form, Aβ42, has the following amino acid sequence (starting from the N-terminus): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV VIA (SEQ ID NO: 45). Aβ41, Aβ40, Aβ39 are missing the C-terminal amino acids A, IA and VIA, respectively. Aβ43 contains an additional threonine residue at the C-terminus of the above sequence.

[0005] The time required to nucleate Aβ40 fibrils has been shown to be significantly longer than that required to nucleate Aβ42 fibrils (see, e.g., Lansbury, Jr., PT and Harper, JD, Ann. Rev. Biochem. 66 (1997) 385-407). As reviewed by Wagner (J. Clin. Invest. 104 (1999) 1239-1332), Aβ42 is more frequently found in association with neuritic plaques and appears to be more fibrillogenic in vitro. It has also been suggested that Aβ42 serves as a "seed" in the nucleation-dependent polymerization of ordered non-crystalline Aβ peptides (see, e.g., Jarrett, Cell 93 (1993) 1055-1058). Altered APP processing and / or the generation of extracellular plaques containing proteinaceous deposits is known from Alzheimer's disease, but also from subjects suffering from other neurological and / or neurodegenerative disorders, including, among others, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Creutzfeldt-Jakob disease, HIV-associated dementia and motor neuropathy.

[0006] So far, only limited medical intervention schemes have been described for amyloid-related diseases. For example, cholinesterase inhibitors such as galantamine, rivastigmine or donepezil have been argued to be beneficial in Alzheimer's patients with only mild to moderate disease. However, adverse events due to the cholinergic action of these drugs have been reported. Although these cholinergic enhancement treatments provide some symptomatic benefit, the therapeutic response is unsatisfactory for the majority of treated patients. Significant cognitive improvement is estimated to occur in only about 5% of treated patients, and there is little evidence that treatment significantly changes the course of this progressive disease.

[0007] As a result, there remains a great clinical need for more effective treatments, particularly treatments that can halt or slow the progression of the disease.

[0008] In addition, NMDA receptor antagonists such as memantine are used. However, adverse events are reported due to pharmacological activity. Moreover, such treatment with these NMDA receptor antagonists can be considered as merely a symptomatic approach, and cannot be considered as a disease modifying approach.

[0009] Furthermore, immunomodulatory approaches have been proposed for treating amyloid-related disorders.WO 99 / 27944 discloses a conjugate that includes a portion of A-β peptide and a carrier molecule, whereby the carrier molecule should enhance immune response.Another active immunization approach is described in WO 00 / 72880, where A-β fragments are also used to induce immune response.

[0010] Furthermore, passive immunization approaches with general anti-A-beta antibodies have been proposed in WO 99 / 27944 or WO 01 / 62801, and specific humanized antibodies against portions of A-beta are described in WO 02 / 46237, WO 02 / 088306 and WO 02 / 088307. WO 00 / 77178 describes antibodies that bind to the transition state adopted by β-amyloid during hydrolysis. WO 03 / 070760 discloses antibody molecules that recognize two discontinuous amino acid sequences on the A-beta peptide.

[0011] WO 2014 / 033074 relates to a blood-brain barrier shuttle that binds to a receptor on the blood-brain barrier and a method of using the same. Blood-brain barrier drug delivery of an IgG fusion protein using a transferrin receptor monoclonal antibody has been reported by Pardridge, W., et al. (Exp. Opin. Drug Deliv. 12 (2015) 207-222). Yu, YJ et al. (Sci. Translat. Med. 6 (2014) 261ra154-261ra154). They reported that a therapeutic bispecific antibody crosses the blood-brain barrier in non-human primates. Disaggregation of amyloid plaques in the brains of Alzheimer's disease transgenic mice treated with daily subcutaneous administration of a tetravalent bispecific antibody targeting the transferrin receptor and Aβ amyloid peptide was reported by Sumbria, RK, et al. (Mol. Pharm. 10 (2013) 3507-3513). Niewoehner, J., et al. (Neuron 81 (2014) 49-609) reported increased brain penetration and efficacy of therapeutic antibodies using a monovalent molecular shuttle.

[0012] WO 2016 / 207240 reported anti-transferrin receptor antibodies with off-rates designed for the human transferrin receptor and their use as blood-brain barrier shuttle modules.

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

[0014] WO 2007 / 068429 reported antibodies against amyloid beta having glycosylation in the variable region. A purified antibody molecule preparation, characterized in that at least one antigen binding site contains a glycosylated asparagine (Asn) in the variable region (VH) of the heavy chain. WO 2007 / 068429 reported a mixture of antibodies containing one or two glycosylated antigen binding sites with a glycosylated asparagine (Asn) in the variable region (VH) of the heavy chain, i.e. a mixture of antibody isoforms containing glycosylated Asn in the variable region (VH) of the heavy chain.

[0015] WO 2017 / 055540 reported trivalent bispecific antibodies against human A-beta and the human transferrin receptor, methods for their preparation, pharmaceutical compositions containing these antibodies, and uses thereof.

[0016] European Patent No. 2368907 reported anti-Aβ antibodies and their uses.

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

[0018] Reported herein are antibodies against human A-beta protein (anti-A-beta protein antibodies), methods for their production, pharmaceutical compositions containing these antibodies and their uses.

[0019] The antibodies according to the invention are variants of the anti-A-beta antibody gantenerumab. They have improved technical and biological properties compared to their parent antibodies. The improvements include, inter alia, improved production properties such as improved production titers, improved production yields and improved process robustness.

[0020] The present invention is based, at least in part, on the discovery that the properties of gantenerumab were improved by introducing specific mutations into the heavy chain CDR2 and by shortening the heavy chain CDR3. Without being bound by this theory, it is believed that the introduced modifications resulted in the improved properties by reducing the sugar occupancy heterogeneity in the Fab and removing deamidation hotspots, thereby reducing the aggregation tendency of the parent antibody gantenerumab.

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

[0022] The anti-A-beta protein antibody according to the invention has substantial and up to 100% carbohydrate occupancy of the glycosylation sites in the heavy chain CDR2.

[0023] The anti-A-beta protein antibodies according to the invention have improved properties, inter alia, with regard to target binding, developmental properties, IHC / plaque binding and PK behavior.

[0024] The present invention encompasses at least the following embodiments.

[0025] 1. An antibody that binds to human A-beta protein, said antibody having the following characteristics: - specifically binds to human A-beta protein of SEQ ID NO: 45; - have a glycosylation site in the heavy chain CDR2 with at least 95% sugar occupancy as determined by CE-SDS; - does not contain species with any non-glycosylated glycosylation sites; - an EC50 value of the human A-β protein of SEQ ID NO: 45 of 0.5 nM or less; - binds to A-β plaques in vitro and does not bind to non-A-β protein molecules in brain samples at a staining concentration of 1 μg / mL or less; -Binds to A-β plaques in vivo more strongly than gantenerumab; - relative occupancy of in vivo plaques in the cortex and hippocampus >0.2 as determined by one-way anova; Thermal stability above -68℃ (DLS T agg / DLS T m ) An antibody having one or more of the following:

[0026] 2. An antibody that binds to human A-beta protein, comprising a CDR selected from: (1) (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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) 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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91 and a light chain variable domain comprising:

[0027] 3. The antibody of embodiment 2, having the following characteristics: - specifically binds to human A-beta protein of SEQ ID NO: 45; - have a glycosylation site in the heavy chain CDR2 with at least 95% sugar occupancy as determined by CE-SDS; - does not contain species with any non-glycosylated glycosylation sites; - an EC50 value of the human A-β protein of SEQ ID NO: 45 of 0.5 nM or less; - binds to A-β plaques in vitro and does not bind to non-A-β protein molecules in brain samples at a staining concentration of 1 μg / mL or less; -Binds to A-β plaques in vivo more strongly than gantenerumab; - in vivo relative occupancy of plaques in the cortex and hippocampus >0.2 as determined by one-way anova; Thermal stability above -68℃ (DLS T agg / DLS T m ) An antibody having one or more of the following:

[0028] 4. The antibody according to any one of embodiments 1 to 3, which is a monoclonal antibody.

[0029] 5. The antibody according to any one of embodiments 1 to 4, which is a humanized or chimeric antibody.

[0030] 6. An antibody according to any one of embodiments 1 to 5, which is an antibody fragment that binds to human A-β protein.

[0031] 7. An antibody according to any one of embodiments 1 to 6, (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 defined in (a) and a VL sequence defined in (b); An antibody comprising:

[0032] 8. The antibody of any one of embodiments 1 to 7, comprising a VH sequence of SEQ ID NO: 84 and a VL sequence of SEQ ID NO: 80.

[0033] 8a. An antibody according to any one of embodiments 1 to 7, comprising a VH sequence of SEQ ID NO: 84 having a pyroglutamic acid (pE) residue instead of a glutamine (Q) residue as the first N-terminal amino acid residue, and a VL sequence of SEQ ID NO: 80.

[0034] 9. An antibody that specifically binds to human A-beta protein, comprising the VH sequence of SEQ ID NO: 84 and the VL sequence of SEQ ID NO: 80.

[0035] 10. An antibody according to any one of embodiments 1 to 9, a) a full-length antibody of human subclass IgG1; b) a full-length antibody of human subclass IgG4; c) a full-length antibody of human subclass IgG1 with the mutations L234A, L235A and P329G; d) a full-length antibody of human subclass IgG1 with the mutations L234A, L235A and P329G in both heavy chains, and with the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain; e) a full-length antibody of human subclass IgG1 with the mutations L234A, L235A and P329G in both heavy chains, with the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain; f) a full-length antibody of human subclass IgG4 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 human subclass IgG4 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 human subclass IgG1 with the mutations L234A, L235A, P329G, I253A, H310A and H435A in both heavy chains, with 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 human subclass IgG1 with the mutations L234A, L235A, P329G, I253A, H310A and H435A in both heavy chains, with 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 human subclass IgG1 with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains, and with 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 human subclass IgG1 with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains, and with 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 human subclass IgG1 having 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) An antibody which is one of a) to l) and does not contain a C-terminal lysine residue.

[0036] 10a. The antibody of embodiment 10, which mediates the uptake of Aβ-coated beads by iPSC-derived microglia in vitro.

[0037] 10b. The antibody of embodiment 10a having an Fc region of c), d), e), h), i), j), k) or l).

[0038] 11. The antibody of any one of embodiments 1 to 10b, which binds to human A-beta protein of SEQ ID NO: 45 with an affinity of less than or equal to 0.4 nM as determined / measured by surface plasmon resonance.

[0039] 12. The antibody according to any one of embodiments 1 to 11, which is a full-length antibody of human subclass IgG1.

[0040] 13. An antibody according to any one of embodiments 1 to 12, comprising i) a heavy chain comprising a heavy chain variable domain of SEQ ID NO: 84 and a heavy chain constant region 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 kappa constant domain of SEQ ID NO: 29.

[0041] 13a. The antibody of any one of claims 1 to 12, comprising a heavy chain comprising a heavy chain variable domain of SEQ ID NO: 84 having a pyroglutamic acid (pE) residue instead of a glutamine (Q) residue as the first N-terminal amino acid residue and a heavy chain constant region of SEQ ID NO: 01, and a light chain comprising a light chain variable domain of SEQ ID NO: 80 and a light chain kappa constant domain of SEQ ID NO: 29.

[0042] 14. The antibody according to any one of embodiments 1 to 13a, which is a multispecific antibody.

[0043] 15. The antibody of any one of embodiments 1 to 14, which is a multispecific antibody comprising at least one binding site that binds to human A-beta protein and at least one binding site / second non-human A-beta protein target that does not bind to human A-beta protein.

[0044] 16. The antibody according to any one of embodiments 14 to 15, wherein 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.

[0045] 17. The antibody of any one of embodiments 14 to 16, wherein the multispecific antibody binds to both of the following: i) human A-beta protein and human transferrin receptor, or ii) human A-β protein and human TREM2 protein, or iii) human A-β protein and human CD20 protein, or iv) human A-β protein and human α-synuclein protein, or v) human A-beta protein and human phospho-tau protein, or vi) Human A-beta protein and human glucocerebrosidase. 18. An antibody according to any one of embodiments 1 to 17, i) a first binding site comprising a heavy chain variable domain of SEQ ID NO: 84 and a light chain variable domain of SEQ ID NO: 80; and ii) a second binding site selected from: a) a heavy chain variable domain of SEQ ID NO: 68 and a light chain variable domain of SEQ ID NO: 72, or b) a heavy chain variable domain of SEQ ID NO: 54 and a light chain variable domain of SEQ ID NO: 55, or c) a heavy chain variable domain of SEQ ID NO: 56 and a light chain variable domain of SEQ ID NO: 57, or d) a heavy chain variable domain of SEQ ID NO: 58 and a light chain variable domain of SEQ ID NO: 59, or e) a heavy chain variable domain of SEQ ID NO: 60 and a light chain variable domain of SEQ ID NO: 61, or f) a heavy chain variable domain of SEQ ID NO: 62 and a light chain variable domain of SEQ ID NO: 63, or g) a heavy chain variable domain of SEQ ID NO: 64 and a light chain variable domain of SEQ ID NO: 65, or h) a heavy chain variable domain of SEQ ID NO: 66 and a light chain variable domain of SEQ ID NO: 67, or i) a heavy chain variable domain of SEQ ID NO: 92 and a light chain variable domain of SEQ ID NO: 93, or j) a heavy chain variable domain of SEQ ID NO: 94 and a light chain variable domain of SEQ ID NO: 95, or k) a heavy chain variable domain of SEQ ID NO: 96 and a light chain variable domain of SEQ ID NO: 97, or l) a heavy chain variable domain of SEQ ID NO: 98 and a light chain variable domain of SEQ ID NO: 99, or m) a heavy chain variable domain of SEQ ID NO: 100 and a light chain variable domain of SEQ ID NO: 101, or n) a heavy chain variable domain of SEQ ID NO: 102 and a light chain variable domain of SEQ ID NO: 103, or o) a heavy chain variable domain of SEQ ID NO: 104 and a light chain variable domain of SEQ ID NO: 105, or p) a heavy chain variable domain of SEQ ID NO: 106 and a light chain variable domain of SEQ ID NO: 107, or q) a heavy chain variable domain of SEQ ID NO: 108 and a light chain variable domain of SEQ ID NO: 109, or r) a heavy chain variable domain of SEQ ID NO: 110 and a light chain variable domain of SEQ ID NO: 111, or s) a heavy chain variable domain of SEQ ID NO: 112 and a light chain variable domain of SEQ ID NO: 113, or t) a heavy chain variable domain of SEQ ID NO: 114 and a light chain variable domain of SEQ ID NO: 115, or u) a heavy chain variable domain of SEQ ID NO: 116 and a light chain variable domain of SEQ ID NO: 117, or v) a heavy chain variable domain of SEQ ID NO: 118 and a light chain variable domain of SEQ ID NO: 119; The antibody is a bispecific antibody comprising:

[0046] 18a. An antibody according to any one of embodiments 1 to 18, which specifically binds to human A-β protein, or specifically binds to human A-β protein and human TREM2.

[0047] 18b. The antibody of any one of embodiments 1 to 18a, which is a bivalent antibody.

[0048] 18c. The antibody of any one of embodiments 14 to 18b, which is a bivalent, trivalent or tetravalent antibody.

[0049] 18d. The antibody of any one of embodiments 14 to 18c, which is a bivalent, bispecific antibody.

[0050] 18e. The antibody of embodiment 18d, which is a bivalent, bispecific antibody in CrossMab format.

[0051] 18f. An antibody according to any one of embodiments 14 to 18e, which binds to human A-beta protein and human TREM2 protein.

[0052] 18g. The antibody of any one of embodiments 14 to 18e, which binds to human A-β protein and human transferrin receptor 1.

[0053] 18h. The antibody of any one of embodiments 14 to 18c, which is a trivalent bispecific antibody.

[0054] The antibody of embodiment 18h, which is a trivalent bispecific antibody in 18i.BS format or TCB format.

[0055] 18j. The antibody of any one of embodiments 14-18c and 18g-18h, which binds to human A-beta protein and human transferrin receptor 1.

[0056] 18k. The antibody of any one of embodiments 14 to 18c, which is a tetravalent bispecific antibody.

[0057] 18l. The antibody of embodiment 18k, which is a tetravalent bispecific antibody in a 2+2-format.

[0058] 18m. An antibody according to any one of embodiments 14 to 18c and 18k to 18l, which binds to human A-beta protein and human TREM2 protein.

[0059] 18n. The antibody of any one of embodiments 14-18c and 18k-18l, which binds to human A-beta protein and human transferrin receptor 1.

[0060] 18o. The antibody of any one of embodiments 14-18c, which is a trivalent trispecific antibody and binds to human A-beta protein, human transferrin receptor 1, and a third non-human A-beta and non-human transferrin receptor 1 target.

[0061] 18p. The antibody of any one of embodiments 14 to 18c and 18o, which is a trivalent trispecific antibody and binds to human A-beta protein, human transferrin receptor 1 and human TREM2.

[0062] The antibody of any one of embodiments 18o to 18p, which is a trivalent trispecific antibody in the 18q.BS format.

[0063] 18r. The antibody of any one of embodiments 18o to 18p, which is a trivalent trispecific antibody in TCB format.

[0064] 19. An immunoconjugate comprising an antibody according to any one of embodiments 1 to 18r and a cytotoxic agent.

[0065] 20. An isolated nucleic acid molecule encoding an antibody according to any one of embodiments 1 to 18r.

[0066] 21. A composition of isolated nucleic acid molecules, wherein each of the isolated nucleic acid molecules of the composition encodes one chain of an antibody according to any one of embodiments 1 to 18r.

[0067] 22. A host cell comprising the nucleic acid molecule of embodiment 20 or the composition of the nucleic acid molecule of embodiment 21.

[0068] 23. A method for producing an antibody that binds to human A-beta protein, comprising culturing a host cell of embodiment 22 in a culture medium under conditions suitable for expression of the antibody.

[0069] 24. The method of embodiment 23, further comprising recovering the antibody from the host cell or / and culture medium.

[0070] 25. The method of any one of embodiments 23 to 24, further comprising purifying the antibody after recovery from the cells and / or culture medium using one or more chromatography steps.

[0071] 26. An antibody produced by the method according to any one of embodiments 23 to 25.

[0072] 27. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 18r or an immunoconjugate of embodiment 19 and a pharma- ceutically acceptable carrier.

[0073] 28. The pharmaceutical composition of embodiment 27, further comprising an additional therapeutic agent.

[0074] 28a. The pharmaceutical composition of embodiment 28, wherein the additional therapeutic agent is an anti-TREM2 antibody.

[0075] 28b. A pharmaceutical composition according to any one of embodiments 27 to 28a, for the treatment of Alzheimer's disease.

[0076] 28c. A pharmaceutical composition according to any one of embodiments 27 to 28a, for the treatment of Parkinson's disease.

[0077] 29. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use as a medicament.

[0078] 29a. The antibody of embodiment 29, wherein the medicament is for the treatment of Alzheimer's disease.

[0079] 29b. The antibody of embodiment 29, wherein the medicament is for the treatment of Parkinson's disease.

[0080] 30. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in the treatment of a disease in an individual having a disease associated with amyloid formation in the brain.

[0081] 31. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in the treatment of a disease in an individual having a disease associated with amyloid plaque formation in the brain.

[0082] 32. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in the treatment of an amyloid disease or disorder in the brain.

[0083] 33. The antibody according to any one of embodiments 30 to 32, wherein the disease is selected from the group consisting of dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeldt-Jakob disease, Hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Parkinson's disease, HIV-associated dementia, ALS or neuronal disorders associated with aging.

[0084] 34. The antibody of any one of embodiments 30 to 32, wherein the disease is Alzheimer's disease.

[0085] 34a. The antibody of any one of embodiments 30 to 32, wherein the disease is Parkinson's disease.

[0086] 35. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in inhibiting the formation of amyloid plaques in the brain.

[0087] 36. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in delaying the formation of amyloid plaques in the brain.

[0088] 37. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in the prevention of amyloid formation in the brain.

[0089] 38. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in the prevention of amyloid plaque formation in the brain.

[0090] 39. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in reducing amyloid plaques in the brain.

[0091] 40. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in removing amyloid plaques in the brain.

[0092] 41. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in disrupting amyloid plaques in the brain.

[0093] 42. An antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c, for use in inhibiting the formation of amyloid plaques in the brain.

[0094] 43. The antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for use in disrupting amyloid plaques in the brain.

[0095] 44. Use of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c, for the manufacture of a medicament.

[0096] 45. The use according to embodiment 44, wherein the medicament is for treating a disease associated with amyloid formation in the brain of an individual.

[0097] 46. ​​The use according to embodiment 44, wherein the medicament is for treating a disease associated with amyloid plaque formation in the brain of an individual.

[0098] 47. The use according to embodiment 44, wherein the medicament is for treating an amyloid disease or disorder in the brain.

[0099] 48. The use according to embodiment 47, wherein the amyloid disease is selected from the group consisting of dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeldt-Jakob disease, Hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Parkinson's disease, HIV-associated dementia, ALS or neuronal disorders associated with aging.

[0100] 49. The use according to any one of embodiments 47 or 48, wherein the medicament is for the treatment of Alzheimer's disease.

[0101] 49a. The use according to any one of embodiments 47 or 48, wherein the medicament is for the treatment of Parkinson's disease.

[0102] 50. The use according to embodiment 44, wherein the medicament is for inhibiting the formation of amyloid plaques in the brain.

[0103] 51. The use according to embodiment 44, wherein the medicament is for slowing the formation of amyloid plaques in the brain.

[0104] 52. The use according to embodiment 44, wherein the medicament is for preventing amyloid formation in the brain.

[0105] 53. The use according to embodiment 44, wherein the medicament is for preventing amyloid plaque formation in the brain.

[0106] 54. The use according to embodiment 44, wherein the medicament is for reducing amyloid plaques in the brain.

[0107] 55. The use according to embodiment 44, wherein the medicament is for removing amyloid plaques in the brain.

[0108] 56. The use according to embodiment 44, wherein the medicament is for disrupting amyloid plaques in the brain.

[0109] 57. The use according to embodiment 44, wherein the medicament is for inhibiting the formation of amyloid plaques in the brain.

[0110] 58. The use according to embodiment 44, wherein the medicament is for disrupting amyloid plaques in the brain.

[0111] 59. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for the treatment of a disease in an individual having a disease associated with amyloid formation in the brain.

[0112] 60. Use of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c, for the treatment of a disease in an individual having a disease associated with amyloid plaque formation in the brain.

[0113] 61. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for the treatment of an amyloid disease or disorder in the brain.

[0114] 62. The use of the antibody according to embodiment 61, wherein the amyloid disease is selected from the group consisting of dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeldt-Jakob disease, Hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Parkinson's disease, HIV-associated dementia, ALS or neuronal disorders associated with aging.

[0115] 63. The use of an antibody according to any one of embodiments 61 to 62, wherein the medicament is for the treatment of Alzheimer's disease.

[0116] 63a. The use of an antibody according to any one of embodiments 61 to 62, wherein the medicament is for the treatment of Parkinson's disease.

[0117] 64. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c for inhibiting the formation of amyloid plaques in the brain.

[0118] 65. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c for delaying the formation of amyloid plaques in the brain.

[0119] 66. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for the prevention of amyloid formation in the brain.

[0120] 67. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c, for the prevention of amyloid plaque formation in the brain.

[0121] 68. Use of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c for reducing amyloid plaques in the brain.

[0122] 69. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c for removing amyloid plaques in the brain.

[0123] 70. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c for disrupting amyloid plaques in the brain.

[0124] 71. Use of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c for inhibiting the formation of amyloid plaques in the brain.

[0125] 72. Use of the antibody according to any one of embodiments 1 to 18r, or the immunoconjugate according to embodiment 19, or the pharmaceutical composition according to any one of embodiments 27 to 28c for disrupting amyloid plaques in the brain.

[0126] 73. A method for treating an individual having amyloid plaques in the brain, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c.

[0127] 74. A method for treating an individual having amyloid formation in the brain, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c.

[0128] 75. A method for treating an individual having or predicted to develop amyloid plaque formation in the brain, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c.

[0129] 76. The method of any one of embodiments 73-75, further comprising administering to the individual an additional therapeutic agent.

[0130] 76a. The method of embodiment 76, wherein the additional therapeutic agent is an anti-TREM2 antibody.

[0131] 77. A method for treating a disease associated with amyloid formation in an individual, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c, for treating a disease associated with amyloid formation.

[0132] 78. A method for treating a disease associated with amyloid plaque formation in the brain in an individual, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c, for treating a disease associated with amyloid plaque formation.

[0133] 79. A method for treating an amyloid disease or disorder in the brain of an individual, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c, to treat the disease or disorder in the brain.

[0134] 80. The method of treatment according to embodiment 79, wherein the amyloid disease or disorder in the brain is selected from the group consisting of dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeldt-Jakob disease, Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type, Parkinson's disease, HIV-associated dementia, ALS or neuronal disorders associated with aging.

[0135] 81. A method of treatment according to any one of embodiments 79 to 80, wherein the disease is Alzheimer's disease.

[0136] 81a. A method of treatment according to any one of embodiments 79 to 80, wherein the disease is Parkinson's disease.

[0137] 82. A method for treating a disease associated with amyloid formation in an individual, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 18r, or an immunoconjugate according to embodiment 19, or a pharmaceutical composition according to any one of embodiments 27 to 28c, for treating a disease associated with amyloid formation.

[0138] In addition to the various embodiments depicted and claimed, the subject matter of the present disclosure is also directed to other embodiments having other combinations of features disclosed or claimed herein. Thus, the specific features presented herein can 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 features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject matter of the present disclosure to the disclosed embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0139] The present invention is based, at least in part, on the discovery that the properties of the antibody gantenerumab were improved by introducing specific mutations into the heavy chain CDR2 and by shortening the heavy chain CDR3. Without being bound by this theory, it is believed that the introduced modifications led to the improved properties by reducing the sugar occupancy heterogeneity in the Fab and removing deamidation hotspots, thereby reducing the aggregation tendency of the parent antibody gantenerumab.

[0140] The present invention is based, at least in part, on the discovery that the light chain of the parent antibody gantenerumab does not require modification to restore binding affinity and specificity when combined with a modified heavy chain according to the invention.

[0141] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0142] As used herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as "Kabat numbering." Specifically, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domain CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3, which is further clarified herein by referring to the "Kabat EU index numbering" in this case).

[0143] Knobs-into-holes dimerization modules and their use in antibody engineering are described in Carter P.; Ridgway JBB; Presta LG: Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73(1).

[0144] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is provided in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0145] Methods and techniques useful for carrying out the invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND, and Hames, BD, ed., 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, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0146] The use of recombinant DNA techniques allows the production of derivatives of nucleic acids. Such derivatives can be modified, for example, by substitution, alteration, replacement, deletion or insertion, at individual or several nucleotide positions. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be easily 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).

[0147] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those of skill in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably.

[0148] The term "about" refers to a range of ±20% of the numerical value that follows. In one embodiment, the term "about" refers to a range of ±10% of the numerical value that follows. In one embodiment, the term "about" refers to a range of ±5% of the numerical value that follows.

[0149] The term "amyloid plaques" refers to aggregates of misfolded proteins that form in the spaces between nerve cells. These abnormally structured proteins are thought to play a central role in Alzheimer's disease. Amyloid plaques develop first in areas of the brain involved in memory and other cognitive functions.

[0150] The term "determining" as used herein also encompasses the terms measuring and analyzing.

[0151] The term "comprising" also includes the term "consisting of."

[0152] The term "anti-(human) A-beta protein antibody" refers to an antibody that can bind human A-beta protein with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting human A-beta protein.

[0153] It should be noted that human A-beta protein has several naturally occurring forms, the human forms being called Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43. The most prominent form, Aβ42, has the amino acid sequence of SEQ ID NO: 45. Aβ41, Aβ40, Aβ39 lack the C-terminal amino acids A, IA and VIA, respectively. Aβ43 contains an additional threonine residue at the C-terminus of SEQ ID NO: 45. In one preferred embodiment, the antibody according to the invention specifically binds to human A-beta protein having the amino acid sequence of SEQ ID NO: 45.

[0154] "Central nervous system" or "CNS" refers to the complex of nervous tissue that controls bodily functions and includes the brain and spinal cord.

[0155] The term "antibody" is used broadly herein and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and fragments thereof, so long as they exhibit the desired human A-beta protein binding activity.

[0156] As detailed above, the modified antibodies according to the invention may be bispecific or multispecific antibodies. In a particular embodiment, the antibodies according to the invention are bispecific antibodies in one of the following formats: -Full-length antibodies with domain swapping (CrossMab format): A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment: a) only the CH1 and CL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises the VL and CH1 domains and the heavy chain of the first Fab fragment comprises the VH and CL domains); b) only the VH and VL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises the VH and CL domains and the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or c) the CH1 and CL domains are replaced with each other and the VH and VL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises the VH and CH1 domains and the heavy chain of the first Fab fragment comprises the VL and CL domains); and The second Fab fragment comprises a light chain comprising a VL and a CL domain, and a heavy chain comprising a VH and a CH1 domain; A full-length antibody with domain swapping comprises a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, both CH3 domains being complementarily engineered by respective amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain; - Full length antibodies with an additional heavy chain C-terminal binding site (BS format): Multispecific IgG antibodies, including: a) a full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of full-length heavy chains specifically binds to a first antigen, or one binds to the first antigen and the other binds to a second antigen, and b) one additional Fab fragment, which is fused to the C-terminus of one of the heavy chains of the full-length antibody, and the binding site of the additional Fab fragment specifically binds to a second antigen, or, if the full-length antibody is bound to a first and a second antigen, the additional Fab fragment binds to a third antigen; Additional Fab fragments that specifically bind to a second antigen i) contain domain crossovers such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are replaced with each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are replaced with each other, or ii) are single chain Fab fragments; The full-length antibody comprises a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, both CH3 domains being complementarily engineered by respective amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain; -Full-length antibodies with two additional heavy chain C-terminal binding sites (2+2-format): Multispecific IgG antibodies, including: a) a full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein a binding site formed by each pair of full-length heavy chain and full-length light chain specifically binds to a first antigen; and b) two additional Fab fragments, one additional Fab fragment fused to each C-terminus of the heavy chain of the full-length antibody, the binding sites of the additional Fab fragments specifically binding to a second antigen; -Full length antibody with additional heavy chain N-terminal portion (TCB format): Multispecific IgG antibodies, including: a) a full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of full-length heavy chains specifically binds to a first antigen, or one binds to the first antigen and the other binds to a second antigen, and b) one additional Fab fragment, wherein the additional Fab fragment is inserted between one of the Fab fragments and the Fc region of the full-length antibody, and wherein the binding site of the additional Fab fragment specifically binds to a second antigen or, if the full-length antibody binds to a first and a second antigen, the additional Fab fragment binds to a third antigen; Additional Fab fragments that specifically bind to a second antigen contain domain crossovers such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are replaced with each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are replaced with each other; -1-arm single chain format (=1-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light kappa domain) -Light / heavy chain combination (variable light domain + light constant domain + peptide linker + variable heavy domain + CH1 + hinge + CH2 + CH3 with knob mutation) -heavy chain (variable heavy domain + CH1 + hinge + CH2 + CH3 with hole mutation); -2-arm single chain format (=2-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light / Heavy Chain 1 Combination (Variable Light Domain + Light Constant Domain + Peptide Linker + Variable Heavy Domain + CH1 + Hinge + CH2 + CH3 with Hole Mutation) - Light chain / heavy chain 2 combination (variable light domain + light chain constant domain + peptide linker + variable heavy domain + CH1 + hinge + CH2 + CH3 with knob mutation); -Common light chain bispecific formats (=common light chain bispecific antibodies): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light domain) -Heavy Chain 1 (VH domain + CH1 + hinge + CH2 + CH3 with hole mutation) - Heavy chain 2 (variable heavy domain + CH1 + hinge + CH2 + CH3 with knob mutation).

[0157] As used herein, the term "replaced by each other" with respect to corresponding heavy and light chain domains refers to the domain crossover described above. Thus, when CH1 and CL domains are "replaced by each other", this term refers to the domain crossover described under item (i) and the resulting heavy and light chain domain sequences. Thus, when VH and VL are "replaced by each other", this term refers to the domain crossover described under item (ii), and when CH1 and CL domains are "replaced by each other" and VH and VL domains are "replaced by each other", this term refers to the domain crossover described under item (iii).

[0158] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the same antigen as the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); DutaFab (bispecific Fab) and multispecific antibodies formed from antibody fragments.

[0159] "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 specified, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity can be measured by common methods known in the art, including those described herein.

[0160] The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to the lysis of target cells by the antibodies reported herein in the presence of effector cells, a function mediated by Fc receptor binding. ADCC can be measured by treating a preparation of CD19-expressing red blood 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) or purified effector cells from buffy coats, such as monocytes or NK (natural killer) cells. Target cells are labeled with 51Cr and then incubated with the antibody. The labeled cells are incubated with effector cells and the supernatant is analyzed for released 51Cr. Controls include incubation of target endothelial cells with effector cells but without antibody. The ability of antibodies to trigger early steps in mediating ADCC is examined by measuring binding of the antibodies to cells expressing Fcγ receptors, such as cells recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which constitutively express FcγRIIIA).

[0161] A "multispecific antibody" refers to an antibody that has binding specificities for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies or DutaFab) or combinations thereof (e.g., full-length antibody plus additional / fused one or more Fv, scFv or Fab fragments). Engineered antibodies with two, three or more (e.g., four) functional antigen-binding sites have been reported (see, e.g., US2002 / 0004587A1).

[0162] The term "binding (to an antigen)" refers to the binding of an antibody to its cognate antigen. Binding may be determined in an in vitro assay. In certain embodiments, binding is measured in a binding assay in which an antibody is bound to a surface and binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). The affinity of binding is defined by the terms ka (rate constant for the association of the antibody from the antibody / antigen complex), kd (dissociation constant), and KD (kd / ka). Thus, binding refers to a specific and detectable interaction between an antibody and its cognate antigen, for example, a binding affinity (KD) of 1E-4M or less. "Specifically binds" refers to a binding affinity (KD) of 1E-8M or less, in some embodiments of 1E-13 to 1E-8M, and in some embodiments of 1E-13 to 1E-9M.

[0163] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0164] The "class" of an antibody refers to the type of constant domain or region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called μ and μ, respectively.

[0165] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor or anticancer agents disclosed below.

[0166] The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of cells induced by an antibody according to the invention in the presence of complement. CDC can be measured by treatment of CD19-expressing human endothelial cells with an antibody according to the invention in the presence of complement. The cells can be labeled with calcein. CDC is found if the antibody induces lysis of 20% or more of the target cells at a concentration of 30 μg / ml. Binding to the complement factor C1q can be measured by ELISA. In such an assay, as a rule, an ELISA plate is coated with a range of concentrations of the antibody according to the invention to which purified human C1q or human serum is added. C1q binding is detected by an antibody against C1q followed by a peroxidase-labeled conjugate. Detection of binding (maximum binding Bmax) is measured as the optical density at 405 nm (OD405) for the peroxidase substrate ABTS® (2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonate (6)]).

[0167] "Effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the class of antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0168] Fc receptor binding-dependent effector functions can be brought about by the interaction of the Fc region of an antibody with the Fc receptor (FcR), a specialized cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cellular targets (e.g., tumor cells) coated with the corresponding antibodies via antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, Annu. Rev. 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.

[0169] Cross-linking of receptors for the Fc region of IgG antibodies (FcγR) triggers a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as clearance of immune complexes and control of antibody production. In humans, three classes of FcγR have been characterized: - FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modification of the Fc region IgG at at least one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbering according to the EU index of Kabat) reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 into IgG1 and IgG4 reduces binding to FcγRI by 10. 32-fold reduction and abolished human monocyte responses to antibody-sensitized red blood cells (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624); - FcγRII (CD32) binds complexed IgG with moderate 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 be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, FcγRIIB appears to function to further suppress immunoglobulin production and isotype switching, for example to the IgE class. On macrophages, FcγRIIB acts to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B form may help suppress the activation of these cells through the binding of IgE to its distinct receptor. Reduced binding to FcγRIIA is seen, for example, in antibodies comprising an IgG Fc region having a mutation in at least one of amino acid residues E233 to G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the EU index as per Kabat); - FcγRIII (CD16) binds IgG with moderate to low affinity and exists as two types. FcγRIIIA is found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to FcγRIIIA is seen, for example, in antibodies that contain an IgG Fc region with mutations in 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 (numbering according to the Kabat EU index).

[0170] Mapping of the binding sites on human IgG1 for Fc receptors, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0171] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0172] The term "Fc receptor" as used herein refers to an activating receptor characterized by the presence of a cytoplasmic ITAM sequence associated with the receptor (see, e.g., Ravetch, JV and Bolland, S., Annu. Rev. Immunol. 19 (2001) 275-290). Such receptors are FcγRI, FcγRIIA and FcγRIIIA. The term "no FcγR binding" indicates that at an antibody concentration of 10 μg / ml, the binding of the antibody to NK cells is 10% or less than that observed for the anti-OX40L antibody LC.001 reported in WO 2006 / 029879.

[0173] IgG4 shows reduced FcR binding, whereas antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, and 234, 235, 236 and 237 Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are residues that also reduce FcR binding when altered (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 0307434). In a particular embodiment, the antibody according to the invention is of the IgG1 or IgG2 subclass and comprises the mutations PVA236, GLPSS331, L234A / L235A or P329G / L234A / L235A. In a particular embodiment, the antibody reported herein is of the IgG4 subclass and comprises the mutation L235E. In a particular embodiment, the antibody according to the invention further comprises the mutation S228P.

[0174] The term "Fc region polypeptide (of human origin)" refers to the C-terminal region of an immunoglobulin heavy chain (of human origin) containing at least a portion of the hinge region, the CH2 domain and the CH3 domain. In a particular embodiment, a human IgG heavy chain Fc region polypeptide extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. In a preferred embodiment, the Fc region polypeptide comprises the amino acid sequence of SEQ ID NO: 05 or is a variant thereof. However, the C-terminal lysine (Lys447) of the Fc region polypeptide or the complete antibody heavy chain may or may not be present.

[0175] "Antibody Fc region" is a term well known to those skilled in the art and is defined based on papain cleavage of an antibody. In a specific embodiment, the Fc region is a human Fc region. An "Fc region (of human origin)" comprises two heavy chain Fc region polypeptides (of human origin) covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0176] The antibody according to the present invention comprises as Fc region, in a particular embodiment, an Fc region derived from human origin. In a particular embodiment, the Fc region comprises all parts 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. The effect of the antibody on the complement system depends on the particular conditions, but the binding to C1q is caused by a defined binding site in the Fc region. Such binding sites are known in the state of the art and are described, for example, in 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 European Patent No. 0307434. Such binding sites include, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the Kabat EU index; unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is the EU numbering system, also called the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. Antibodies of the subclasses IgG1, IgG2 and IgG3 usually exhibit complement activation, C1q binding and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q and does not activate C3.

[0177] The term "FcRn" refers to the human neonatal Fc receptor. FcRn functions to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. FcRn is a heterodimeric protein consisting of two polypeptides: the 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and the 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 1:2 stoichiometry, with one IgG 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 endosomes at acidic pH (pH<6.5) and IgG is released at the neutral cell surface (pH of about 7.4). The pH sensitivity of the interaction facilitates FcRn-mediated protection of IgG pinocytosed into the cell from intracellular degradation by binding to receptors in the acidic environment of the endosome. FcRn then facilitates recycling of IgG to the cell surface and subsequent release into the bloodstream upon exposure of the FcRn-IgG complex to the extracellular neutral pH environment.

[0178] The term "FcRn-binding portion of an Fc region" refers to a portion of an antibody heavy chain polypeptide extending from about EU position 243 to EU position 261, and from about EU position 275 to EU position 293, and from about EU position 302 to EU position 319, and from about EU position 336 to EU position 348, and from about EU position 367 to EU position 393, and from EU position 408, and from about EU position 424 to EU position 440. In one embodiment, one or more of the following amino acid residues according to the EU numbering of Kabat are altered: F243, P244, P245, P246, P247, P248, P249, P250, P251, P252, P253, P254, P255, P256, P257, P258, P259, P260, P261, P262, P263, P264, P265, P266, P267, P268, P269, P270, P271, P272, P273, P274, P275, P275, P276, P277, P278, P279, P280, P281, P282, P283, P284, P285, P285, P286, P287, P288, P289, P290, P291, P292, P293, P294, P295, P296, P297, P298, P299, P300, P301, P302, P303, P304, P305, P306, P307, P308, P309, P3 P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N2 86, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307 , V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S3 37, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369 , F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G3 85, 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 numbering).

[0179] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences generally appear in a VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0180] 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 comprising a variable domain and a constant domain, and ii) two full-length antibody heavy chains, each comprising a variable domain, a first constant domain, a hinge region, a second constant domain and a third constant domain. A full-length antibody may comprise further domains, such as additional scFvs, or scFabs or domain-swapped Fabs conjugated to the C-terminus of one or more of the chains of the full-length antibody, preferably one or more of the heavy chains.

[0181] The terms "host cell" and "host cell line" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the original transformed cell and the progeny derived from the original transformed cell, regardless of the number of passages. The progeny may not be completely identical to the parent cell, and may contain mutations. As used herein, included are progeny of mutants that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0182] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In a particular embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In a particular embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0183] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to a non-human antibody and all or substantially all of the FRs correspond to a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0184] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that comprises stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Typically, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0185] HVR includes: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising 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).

[0186] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0187] The term "derived from" denotes that an amino acid sequence is derived from a parent amino acid sequence by introducing an alteration at at least one position. The amino acid sequence so derived differs from the corresponding parent amino acid sequence at at least one corresponding position (numbering according to Kabat EU index for antibody Fc regions). In certain embodiments, the amino acid sequence derived from the parent amino acid sequence differs by 1-15 amino acid residues at the corresponding positions. In certain embodiments, the amino acid sequence derived from the parent amino acid sequence differs by 1-10 amino acid residues at the corresponding positions. In certain embodiments, the amino acid sequence derived from the parent amino acid sequence differs by 1-6 amino acid residues at the corresponding positions. Similarly, the derived amino acid sequence has a high amino acid sequence identity to its parent amino acid sequence. In certain embodiments, the amino acid sequence derived from the parent amino acid sequence has 80% or higher amino acid sequence identity. In certain embodiments, the amino acid sequence derived from the parent amino acid sequence has 90% or higher amino acid sequence identity. In certain embodiments, the amino acid sequence derived from the parent amino acid sequence has 95% or higher amino acid sequence identity. In one preferred embodiment, the amino acid sequence derived from the parent amino acid sequence has 98% or higher amino acid sequence identity.

[0188] The term "(human) Fc region polypeptide" refers to an amino acid sequence identical to a "native" or "wild-type" (human) Fc region polypeptide. The term "variant (human) Fc region polypeptide" refers to an amino acid sequence that is derived from a "native" or "wild-type" (human) Fc region polypeptide by virtue of at least one "amino acid alteration." A "variant (human) Fc region" is composed of two Fc region polypeptides, whereby both may be variant (human) Fc region polypeptides, or one is a (human) Fc region polypeptide and the other is a variant (human) Fc region polypeptide.

[0189] In certain embodiments, the human Fc-region polypeptide has the amino acid sequence of, or is a variant of, a human IgG1 Fc-region polypeptide, or has the amino acid sequence of, or is a variant of, a human IgG2 Fc-region polypeptide, or has the amino acid sequence of, or is a variant of, a human IgG3 Fc-region polypeptide, or has the amino acid sequence of, or is a variant of, a human IgG4 Fc-region polypeptide. In certain 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 certain embodiments, the Fc-region polypeptide comprises / has about 1 to about 10 amino acid mutations. In certain embodiments, the Fc-region polypeptide comprises / has about 1 to about 5 amino acid mutations.

[0190] In certain embodiments, the Fc region polypeptide has the amino acid sequence of a human IgG1 Fc region polypeptide with a PGLALA mutation and a knob mutation (SEQ ID NO: 12), optionally with a deletion of the C-terminal lysine residue. In certain embodiments, the Fc region polypeptide has the amino acid sequence of a human IgG1 Fc region with a PGLALA mutation and a knob-cis or whole-cis mutation (SEQ ID NO: 16 or SEQ ID NO: 15), optionally with a deletion of the C-terminal lysine residue.

[0191] In certain embodiments, the Fc-region polypeptide has at least about 80% sequence identity to the human Fc-region polypeptide of SEQ ID NO:05 or SEQ ID NO:17. In certain embodiments, the Fc-region polypeptide has at least about 90% sequence identity to the human Fc-region polypeptide of SEQ ID NO:05 or SEQ ID NO:17. In certain embodiments, the Fc-region polypeptide has at least about 95% sequence identity to 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 to the human Fc-region polypeptide of SEQ ID NO:05 or SEQ ID NO:17.

[0192] In certain embodiments, the Fc-region polypeptide has at least about 80% sequence identity to the human Fc-region polypeptide of SEQ ID NO:05 or SEQ ID NO:17. In certain embodiments, the Fc-region polypeptide has at least about 90% sequence identity to the human Fc-region polypeptide of SEQ ID NO:05 or SEQ ID NO:17. In certain embodiments, the Fc-region polypeptide has at least about 95% sequence identity to 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 to 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.

[0193] Variant Fc-region polypeptides derived from a parent (human) Fc-region polypeptide of SEQ ID NO:05 or SEQ ID NO:17 are further defined by the amino acid changes they contain compared to the parent or wild-type sequence. Thus, for example, the term P329G refers to an Fc-region polypeptide derived from a (human) Fc-region polypeptide and having a proline to glycine mutation at amino acid position 329 relative to the human Fc-region polypeptide of SEQ ID NO:05 or SEQ ID NO:17 (numbering according to Kabat).

[0194] The human IgG1 Fc region polypeptide has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 05), optionally with an additional lysine residue (K) added to the C-terminus.

[0195] The Fc region polypeptide derived from human IgG1 Fc region having the mutations L234A and L235A (LALA mutation) has the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 06), optionally with an additional lysine residue (K) added to the C-terminus.

[0196] The Fc region polypeptide derived from a human IgG1 Fc region having Y349C, T366S, L368A and Y407V mutations (knob-cis-mutations) has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 07), optionally with an additional lysine residue (K) added to the C-terminus.

[0197] The Fc region polypeptide derived from human IgG1 Fc region having S354C and T366W (whole-cis-mutation) mutations has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 08), optionally with an additional lysine residue (K) added to the C-terminus.

[0198] The Fc region polypeptide derived from a human IgG1 Fc region having the L234A, L235A mutations and the Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 09), optionally with an additional lysine residue (K) added to the C-terminus.

[0199] The Fc region polypeptide derived from human IgG1 Fc region having L234A, L235A, S354C, and T366W mutations has the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10), optionally with an additional lysine residue (K) added to the C-terminus.

[0200] The Fc region polypeptide derived from a human IgG1 Fc region having a P329G mutation (PG mutation) has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11), optionally with an additional lysine residue (K) added to the C-terminus.

[0201] The Fc region polypeptide derived from a human IgG1 Fc region having the L234A, L235A and P329G mutations (LALAPG mutations) has the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 12), optionally with an additional lysine residue (K) added to the C-terminus.

[0202] The Fc region polypeptide derived from a human IgG1 Fc region having a P329G mutation and Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13), optionally with an additional lysine residue (K) added to the C-terminus.

[0203] The Fc region polypeptide derived from human IgG1 Fc region having the P329G mutation and the S354C and T366W mutations has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 14), optionally with an additional lysine residue (K) added to the C-terminus.

[0204] The Fc region polypeptide derived from human IgG1 Fc region having the L234A, L235A, P329G, and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15), optionally with an additional lysine residue (K) added to the C-terminus.

[0205] The Fc region polypeptide derived from human IgG1 Fc region having L234A, L235A, and P329G mutations and S354C and T366W mutations has the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16), optionally with an additional lysine residue (K) added to the C-terminus.

[0206] The human IgG4 Fc region polypeptide has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 17).

[0207] The Fc region polypeptide derived from a human IgG4 Fc region having S228P and L235E mutations has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 18).

[0208] The Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E and P329G mutations has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 19).

[0209] The Fc region polypeptide derived from a human IgG4 Fc region having S354C and T366W mutations has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 20).

[0210] The Fc region polypeptide derived from a human IgG4 Fc region having Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 21).

[0211] The Fc region polypeptide derived from human IgG4 Fc region having S228P, L235E, S354C, and T366W mutations has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 22).

[0212] The Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E, and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 23).

[0213] The Fc region polypeptide derived from a human IgG4 Fc region having a P329G mutation has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 24).

[0214] The Fc region polypeptide derived from a human IgG4 Fc region having P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 25).

[0215] The Fc region polypeptide derived from human IgG4 Fc region having P329G, S354C, and T366W mutations has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 26).

[0216] The Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E, P329G, and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 27).

[0217] The Fc region polypeptide derived from human IgG4 Fc region having S228P, L235E, P329G, and S354C, T366W mutations has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 28).

[0218] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0219] An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., size-exclusion chromatography or ion exchange or reverse-phase HPLC) analytical methods. For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0220] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment.

[0221] An "isolated nucleic acid encoding an anti-human A-beta protein antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single plasmid or in separate plasmids.

[0222] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0223] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0224] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation, which variants are generally present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the invention can be produced 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, including, but not limited to, such methods and other exemplary methods for producing monoclonal antibodies described herein.

[0225] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0226] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, which contain information regarding the indications, uses, dosages, administration, concomitant therapy, contraindications and / or warnings regarding such therapeutic product.

[0227] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0228] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be described as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0229] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredients contained in the preparation is in a form such that it is effective, and which does not contain ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0230] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0231] As used herein, the term "plasmid" refers to a nucleic acid molecule capable of propagating another nucleic acid that they contain. The term includes plasmids as self-replicating nucleic acid structures and plasmids integrated into the genome of a host cell into which they are introduced. Certain plasmids are capable of directing the expression of the nucleic acids that they contain. Such plasmids are referred to herein as "expression plasmids."

[0232] The term "recombinant antibody" as used herein refers to all antibodies (chimeric, humanized and human) that are prepared, expressed, generated or isolated by recombinant means. It includes antibodies isolated from host cells such as NS0, HEK, BHK or CHO cells or from animals (e.g., mice) that are transgenic for 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 the recombinant antibody are derived from and related to human germline VH and VL sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0233] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention aimed at altering the natural history of the individual being treated, and may be performed for prophylaxis or during the clinical pathology process. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, amelioration or remission of the disease, and remission or improvement of prognosis. In certain embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0234] The term "valency" as used in this application refers to the presence of a specified number of binding sites in an (antibody) molecule. Thus, the terms "bivalent", "trivalent" and "tetravalent" refer to the presence of two binding sites, three binding sites and four binding sites, respectively, in an (antibody) molecule.

[0235] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to its antigen. The variable domains of an antibody's heavy and light chains (VH and VL, respectively) generally have a similar structure, with each domain containing four framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt, TJ et al. Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), page 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of an antibody that binds the antigen. See, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628. The pairing of an antibody heavy chain variable domain with an antibody light chain variable domain (cognate) forms a binding site.

[0236] The term "variant" refers to a molecule having an amino acid sequence that differs from that of the respective parent molecule. Typically, such molecules have one or more changes (mutations), insertions or deletions. In a particular embodiment, an antibody according to the invention comprises at least a portion of a non-naturally occurring Fc region. Such molecules have less than 100% sequence identity with the parent antibody. In a particular embodiment, the variant antibody has an amino acid sequence that has from about 75% to less than 100%, particularly from about 80% to less than 100%, particularly from about 85% to less than 100%, particularly from about 90% to less than 100%, particularly from about 95% to less than 100% amino acid sequence identity with the amino acid sequence of the parent antibody. In a preferred embodiment, the parent and variant antibodies differ in 1 (single), 2, 3, 5, 7 or 10 amino acid residues.

[0237] Amyloid hypoplasia Amyloid plaques form when pieces of a protein called β-amyloid aggregate. β-amyloid is produced when a much larger protein called amyloid precursor protein (APP) is broken down. APP is composed of 771 amino acids and is cleaved by two enzymes to produce β-amyloid. The large protein is cleaved first by β-secretase and then by γ-secretase to produce β-amyloid pieces that can be composed of 38, 40 or 42 amino acids. β-amyloid composed of 42 amino acids is chemically "stickier" than other lengths and therefore more likely to form plaques. Studies have shown that three genetic abnormalities associated with early stage Alzheimer's disease each alter the function of γ-secretase in a way that leads to increased production of Aβ42.

[0238] It is not clear how β-amyloid causes toxic damage to nerve cells, but some studies suggest that it may break into fragments and release free radicals that then attack neurons. Another theory is that β-amyloid forms pores in nerve membranes, which leads to an unregulated influx of calcium that can cause nerve cell death. Regardless of the exact pathological process by which β-amyloid causes nerve damage, the result is that neurons die.

[0239] Plaques are formed that consist of a mixture of these degenerating neurons and β-amyloid aggregates. These plaques cannot be broken down and removed by the body, so they gradually build up in the brain. This accumulation of amyloid leads to amyloidosis, which is thought to be a contributing factor in several neurodegenerative diseases.

[0240] Amyloid plaques form one of the two defining features of Alzheimer's disease, the other being neurofibrillary tangles. β-amyloid is also believed to be responsible for the formation of these tangles, which also damage neurons and cause dementia symptoms. Technically, a person may present with all the hallmarks of Alzheimer's disease, but if a brain biopsy or positron emission tomography does not reveal the presence of amyloid plaques or neurofibrillary tangles, then a diagnosis of Alzheimer's disease is not made.

[0241] Description of the embodiments of the present invention Gantenerumab is a fully human IgG1 antibody that binds with subnanomolar affinity to a conformational epitope of Aβ consisting of both N-terminal and central amino acids. It preferentially binds to the fibrillar form of the protein. The therapeutic principle of this antibody is to act centrally to disassemble and degrade amyloid plaques by recruiting microglia and activating phagocytosis. It prevents new plaque formation. Gantenerumab 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 oligomeric Aβ42-mediated inhibitory effects on long-term potentiation in rat brain. In APP / PS1 transgenic mice, gantenerumab binds to brain Aβ and reduces small plaques by recruiting microglia, preventing new plaque formation. Gantenerumab did not alter systemic levels of Aβ, suggesting that clearance of soluble Aβ was not impeded. Gantenerumab in the SCarlet RoAD, a phase 3, multicenter, randomized, double-blind, placebo-controlled trial, demonstrated target engagement, resulting in plaque clearance and reduced levels of phosphorylated tau in spinal fluid (see, e.g., Sumner, IL, et al., Front. Neurosci. 12 (2018) article 254; https: / / www.alzforum.org / therapeutics / gantenerumab).

[0242] Gantenerumab has two groups of glycosylation sites: the first group encompasses the glycosylation site in each Fab within the heavy chain CDR2, and the second group encompasses the glycosylation site in each Fc region at Asn297 (numbering according to Kabat).

[0243] The glycosylation pattern, i.e., sugar occupancy, of the glycosylation sites differs between both groups of glycosylation sites. The glycan occupancy of each glycosylation site in the Fc region is uniform and comparable to each other as well as to other recombinantly produced antibodies. In contrast, the glycan occupancy of the glycosylation sites in the Fab is diverse, i.e., both glycosylation sites are glycosylated, or only one of these glycosylation sites is glycosylated, or none of these glycosylation sites is glycosylated. Thus, recombinantly produced gantenerumab is obtained from the production cells as a mixture of three different glycosylation isoforms. Since the glycan occupancy and glycosylation pattern affect the pharmacokinetic properties as well as plaque binding, it is necessary to remove the non-glycosylated isoform. The different glycoforms can be distinguished and separated using chromatography, such as, for example, hydrophobic interaction chromatography (HIC).

[0244] Here, the inventors found that the properties of gantenerumab were improved by introducing specific mutations into the heavy chain CDR2 and by shortening the heavy chain CDR3. Without being bound by this theory, it is believed that the introduced modifications led to the improved properties by reducing the sugar occupancy heterogeneity in the Fab and removing deamidation hotspots, thereby reducing the aggregation tendency of the parent antibody gantenerumab.

[0245] Furthermore, the inventors have found that the light chain of the parent antibody gantenerumab does not require modification to retain binding affinity and specificity when combined with a modified heavy chain according to the invention.

[0246] Thus, aspects of the present invention are antibodies against human A-beta protein (anti-A-beta protein antibodies), methods for their production, pharmaceutical compositions containing these antibodies and uses thereof.

[0247] The antibodies according to the invention are variants of the anti-A-beta antibody gantenerumab. They have improved technical and biological properties compared to their parent antibodies. The improvements include, inter alia, improved production titers, improved production yields, improved glycosylation uniformity, reduced aggregation tendency and improved process robustness.

[0248] The anti-A-beta protein antibody according to the invention has substantial and up to 100% carbohydrate occupancy of the glycosylation sites in the heavy chain CDR2.

[0249] The anti-A-beta protein antibodies according to the invention have improved properties, inter alia, with regard to target binding, developmental properties, IHC / plaque binding and PK behavior.

[0250] The present invention is based, at least in part, on the discovery that complete removal of glycosylation sites in the Fab of gantenerumab resulted in reduced plaque binding or increased clearance, respectively. Therefore, modifications that result in reduced or even eliminated glycosylation of the Fab glycosylation sites of gantenerumab are disadvantageous and need to be prevented.

[0251] Four different variants of gantenerumab with altered Fab glycan occupancy have been generated.

[0252] Two variants with altered heavy chains (numbering according to Kabat): -Full Fab glycosylation by introducing the heavy chain S53T mutation (mAb675); -Complete elimination of Fab glycosylation by introducing the heavy chain N52Q mutation (mAb663); Two variants with altered heavy and light chains (numbering according to Kabat): - complete Fab glycosylation by introducing a heavy chain S53T mutation and compensation of the heavy chain modification by introducing a light chain M95H mutation (mAb651); - Complete elimination of Fab glycosylation by introducing a heavy chain N52Q mutation and compensation of the heavy chain modification by introducing a light chain M95H mutation (mAb638).

[0253] The four variants of gantenerumab are either fully Fab glycosylated or fully Fab aglycosylated. Except for mAb651, all variants have the same in vitro binding affinity for human A-beta protein. [Table 1]

[0254] The different glycosylation levels have been confirmed by hydrophilic interaction chromatography (HIC), as shown in Figures 1 to 3.

[0255] FIG. 1 shows partial glycosylation of the parent antibody gantenerumab, which has the sequence NAS in the heavy chain CDR (two peaks at alignment times of approximately 22-24 s).

[0256] FIG. 2 shows complete aglycosylation of the variant antibody mAb663 with sequence QAS in the heavy chain CDRs (one peak at an alignment time of about 22 seconds).

[0257] FIG. 3 shows complete glycosylation of the variant antibody mAb675 with sequence QAS in the heavy chain CDR (one peak at an alignment time of about 23 seconds).

[0258] The antibodies according to the invention as well as gantenerumab have been produced on a small scale and the by-product distribution has been analyzed after a first purification step using Protein A affinity chromatography and a second purification step using preparative size exclusion chromatography. The results are shown in the table below. [Table 2]

[0259] The four variants have been further characterized with respect to their binding properties and specificity, and the qualitative results are shown in the table below. [Table 3]

[0260] Each characteristic is described in more detail below.

[0261] A-β plaque decoration in vitro The parent antibody, gantenerumab, shows strong specific plaque binding and some background, non-specific binding, especially at a concentration of 1 μg / ml (FIG. 4).

[0262] MAb 675 ("142") exhibits strong specific plaque binding with no detectable background, non-specific binding (Figure 5).

[0263] MAb 663 ("143") shows low specific plaque binding and some background, non-specific binding, especially at a concentration of 1 μg / ml (FIG. 6).

[0264] MAb 651 ("144"), even at a concentration of 1 μg / ml, shows little specific plaque binding and no background or nonspecific binding (FIG. 7).

[0265] MAb 638 shows low specific plaque binding and some background, non-specific binding, especially at a concentration of 1 μg / ml.

[0266] Thus, mAb675 unexpectedly exhibited equal specific plaque binding when compared to gantenerumab, but much less background, nonspecific binding. Thus, fully glycosylated glycosylation sites in the Fab were found to reduce background, nonspecific binding in vitro at a concentration of 1 μg / mL.

[0267] The staining results are summarized in qualitative form in the table below. [Table 4-1] [Table 4-2]

[0268] A-β plaque decoration in vivo Male APP / PS2 mice (9 months old, n=3 per group) were administered 20 mg / kg of gantenerumab and the four variants, respectively. Seven days after injection, antibody distribution was determined in brain sections (detection antibody: goat anti-human IgG antibody conjugated to Alexa555 (Invitrogen (#A21433)); co-staining for A-β protein: Amylo Glo, Biosensis Cat. No. TR-300-AG; image settings: 30x magnification). The results are shown in Figures 8 and 9.

[0269] As a result, mAb675 ("142" in Figures 8 and 9) unexpectedly showed improved plaque binding in vivo when compared to gantenerumab. Thus, fully glycosylated glycosylation sites within the Fab were found to increase A-β plaque binding in vivo.

[0270] Pharmacokinetics The pharmacokinetics of four gantenerumab variants were determined in rats and mice.

[0271] mAb663, which lacks Fab glycosylation and has an unmodified light chain variable domain, was found to exhibit an increased plasma clearance rate in rats. All other variants exhibit plasma clearance rates similar to gantenerumab.

[0272] The results are shown in Figure 10.

[0273] Unexpectedly, mAb638, mAb675 and mAb651 have lower clearance rates than gantenerumab (historical data). It must be pointed out that differences in plasma clearance rates do not explain the differences in plaque decoration of mAb638.

[0274] The single-dose pharmacokinetic (SDPK) properties of gantenerumab variants are being further evaluated using a combination of FcRn and heparin affinity chromatography.

[0275] The combination of FcRn affinity chromatography and heparin affinity chromatography allows to define FcRn and heparin affinity chromatography column retention time thresholds and therefore a second dimension retention time region in which antibodies with slow clearance, i.e. long systemic circulation half-life, can be identified. This combination therefore allows, inter alia, the selection of antibodies with long systemic circulation half-life.

[0276] For characterization, the retention times on the FcRn affinity chromatography column and the heparin affinity chromatography column are normalized based on the retention times of the reference antibodies on the respective columns. This defines a relative retention time region that mainly contains antibodies with slow clearance. More specifically, this region is defined by a relative retention time on the FcRn affinity chromatography column of less than 1.78 (using an oxidized (H2O2-treated) anti-Her3 antibody preparation as the reference antibody) and a relative retention time on the heparin affinity chromatography column of less than 0.87 (using an anti-pTau antibody as the reference antibody). For details of this method, please refer to WO 2018 / 197533. [Table 5]

[0277] thermal stability The thermal stability of mAb675, mAb663 and mAb638 was determined by dynamic light scattering. The aggregation temperature (T agg ) and melting temperature (T m ) has been determined by application of a heat lamp. Unexpectedly, all three variants tested show improved values ​​compared to the parent gantenerumab. [Table 6]

[0278] Stability assessment MAb675, mAb663 and mAb638 were subjected to heat stress by incubation at elevated temperatures in different buffer systems.

[0279] mAb638 has been found to exhibit substantial monomer loss upon incubation in phosphate buffered saline.

[0280] Unexpectedly, however, all three gantenerumab variants showed improved stability with respect to target binding compared to the parent gantenerumab. [Table 7]

[0281] Considering all data (100% uniform Fab glycooccupancy, removal of deamidation sites, wild-type light chain), mAb675 was identified as the most advantageous gantenerumab variant with improved properties.

[0282] Exemplary Anti-Human A-beta Protein Antibodies According to the Invention Therefore, the present invention encompasses at least the following embodiments.

[0283] In one aspect, the invention provides an antibody that binds to human A-beta protein.

[0284] In one aspect, the invention provides an isolated antibody that binds to human A-beta protein.

[0285] In one aspect, the invention provides an antibody that specifically binds to human A-beta protein.

[0286] In certain embodiments of all aspects and embodiments of the invention, the anti-A-beta antibodies according to the invention have one or more of the following properties: - specifically binds to human A-beta protein of SEQ ID NO: 45; - have a glycosylation site in the heavy chain CDR2 with at least 95% sugar occupancy as determined by CE-SDS; - does not contain species with any non-glycosylated glycosylation sites; - an EC50 value of the human A-β protein of SEQ ID NO: 45 of 0.5 nM or less; - binds to A-β plaques in vitro and does not bind to non-A-β protein molecules in brain samples at a staining concentration of 1 μg / mL or less; -Binds to A-β plaques in vivo more strongly than gantenerumab; - relative occupancy of in vivo plaques in the cortex and hippocampus >0.2 as determined by one-way anova; Thermal stability above -68℃ (DLS T agg / DLS T m ) An antibody having one or more of the following:

[0287] In one aspect, the invention provides an anti-A-beta protein antibody that comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from the following: (1) (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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) 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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91.

[0288] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from: (1) (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; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; or (2) (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; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; or (3) (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; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; or (4) (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; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87.

[0289] In a preferred embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO:87.

[0290] In another aspect, the invention provides an antibody comprising at least one VL CDR sequence, at least two VL CDR sequences, or all three VL CDR sequences selected from: (1) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (2) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (3) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91; or (4) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a 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:91.

[0291] In another embodiment, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from: (1) (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; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; or (2) (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; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; or (3) (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; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; or (4) (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; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from: (1) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (2) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83; or (3) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91; or (4) (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a 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:91.

[0292] In another aspect, the present invention provides an anti-A-beta protein antibody comprising: (1) (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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) 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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91.

[0293] In another aspect, an anti-A-beta protein antibody according to the invention comprises one or more of the VH CDR sequences of SEQ ID NO: 84 or SEQ ID NO: 88. In another embodiment, an anti-A-beta protein antibody comprises one or more of the VL CDR sequences of SEQ ID NO: 80 or SEQ ID NO: 90. In another embodiment, an anti-A-beta antibody comprises (1) the VH CDR sequence of SEQ ID NO: 84 and the VL CDR sequence of SEQ ID NO: 80; or (2) the VH CDR sequence of SEQ ID NO: 88 and the VL CDR sequence of SEQ ID NO: 80; or (3) the VH CDR sequence of SEQ ID NO: 84 and the VL CDR sequence of SEQ ID NO: 90; or (4) the VH CDR sequence of SEQ ID NO: 88 and the VL CDR sequence of SEQ ID NO: 90.

[0294] In a preferred embodiment, the anti-A-beta protein antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 84 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 80.

[0295] In one embodiment, an anti-A-beta 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 framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 84. In one embodiment, an anti-A-beta 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 framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 84. In one embodiment, an anti-A-beta protein antibody comprises the three heavy chain CDR amino acid sequences of a VH domain of SEQ ID NO: 84 and a framework having at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 84. In another embodiment, an anti-A-beta protein antibody comprises the three heavy chain CDR amino acid sequences of a VH domain of SEQ ID NO: 84 and a framework having at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 84.

[0296] In one embodiment, an anti-A-beta 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 framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 80. In one embodiment, an anti-A-beta protein antibody comprises three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 80 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 80. In one embodiment, the anti-A-beta protein antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 80 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 80. In another embodiment, the anti-A-beta protein antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 80 and a framework of at least, particularly at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 80.

[0297] In one embodiment, the anti-A-beta 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, as well as a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80. In a preferred embodiment, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:84 and the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:80.

[0298] In one embodiment, the anti-A-beta 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 an 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 to the amino acid sequence of SEQ ID NO: 80; the antibody specifically binds to human A-beta protein. In a preferred embodiment, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 84 and the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 80.

[0299] In another embodiment, the anti-A-beta 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 to the amino acid sequence of SEQ ID NO: 84. In one embodiment, the anti-A-beta protein antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-A-beta protein antibody comprising the sequence retains the ability to bind to human A-beta protein. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 84. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-A-beta protein antibody comprises a VH sequence of SEQ ID NO: 84, including post-translational modifications of the sequence. In certain embodiments, 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 embodiment, an anti-A-beta protein antibody is provided, the antibody comprising a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80. In one embodiment, the anti-A-beta protein antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 80. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-A-beta protein antibody comprising the sequence retains the ability to bind to human A-beta protein. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 80.In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-A-beta protein antibody comprises a VL sequence of SEQ ID NO: 80, including post-translational modifications of that sequence. In certain embodiments, the 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.

[0300] In another aspect, an anti-A-beta protein antibody is provided, the antibody comprising a VH sequence of any of the aspects provided above and a VL sequence of 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.

[0301] In a further embodiment of the invention, the anti-A-beta protein antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-A-beta protein antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 antibody or other antibody class or isotype as defined herein. [Table 8-1] [Table 8-2] [Table 8-3]

[0302] In certain embodiments, amino acid sequence variants of the antibodies according to the invention are contemplated. 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, but are not limited to, deletions of residues from, and / or insertions into, and / or substitutions of residues within the amino acid sequences of the antibodies according to the invention. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, so long as the final construct has the desired characteristics (e.g., antigen binding).

[0303] In a particular embodiment of all aspects and embodiments according to the invention, the heavy chain variable domain of the antibody according to the invention comprises as the first residue a pyroglutamic acid (pE) residue instead of a glutamine (Q) residue.

[0304] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in the table below under the heading of "preferred substitutions". More substantial changes are provided in the table below under the heading of "exemplary substitutions" and as further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 9-1] [Table 9-2]

[0305] Amino acids can be classified according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0306] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0307] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing have altered (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0308] Alterations (e.g., substitutions) may be made, for example, in HVRs to improve antibody affinity. Such alterations may be made in HVR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196), and / or residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from the secondary libraries is 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 genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0309] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody according to the invention to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the HVRs. Such changes may, for example, be outside of antigen contact residues within the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0310] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085. In this method, a residue or target group of residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) is identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at the positions of amino acids that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they contain the desired properties.

[0311] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., in the case of ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0312] b) Fc region glycosylation variants In certain embodiments, antibodies according to the invention are altered to increase or decrease the extent to which the antibody Fc region is glycosylated. Addition or deletion of glycosylation sites to an antibody according to the invention can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0313] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Typically, natural antibodies produced by mammalian cells contain branched, biantennary oligosaccharides that are usually attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32. The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in the antibodies described herein may be performed to generate antibody variants with specific improved properties.

[0314] In certain embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan of Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region, although Asn297 may also be located ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to slight sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication Nos. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication Nos. 2003 / 0115614; 2002 / 0164328; 2004 / 0093621; 2004 / 0132140; 2004 / 0110704; 2004 / 0110282; 004 / 0109865; WO 2003 / 085119; WO 2003 / 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, which are deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545; US Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (e.g., 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).

[0315] Further provided are antibody variants with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Pat. No. 6,602,684; and U.S. Pat. App. Pub. No. 2005 / 0123546. Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0316] c) Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody according to the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0317] In certain embodiments, antibody variants are contemplated herein that have some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important, but where certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 of Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492, page 464. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., 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); U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA, and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18 (2006: 1759-1769).

[0318] Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc region variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, such as the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0319] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0320] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0321] In some embodiments, modifications are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0322] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is involved in 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) have been described in US Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc region variants include substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0323] For other examples of Fc region variants, see also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0324] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be created, for example, as described in U.S. Pat. No. 7,521,541.

[0325] e) Antibody derivative In certain embodiments, the antibodies according to the invention may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one polymer is attached, the polymers can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0326] In certain embodiments, a complex of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In certain embodiments, the non-proteinaceous moiety is a carbon nanotube (Kam, N. Wet al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells but that heat the non-proteinaceous moiety to a temperature that kills cells proximal to the antibody-non-proteinaceous moiety.

[0327] Recombinant methods and compositions Antibodies according to the invention may be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. The invention provides one or more isolated nucleic acid molecules encoding an anti-human A-beta antibody according to the invention. Such nucleic acid molecules may encode an amino acid sequence comprising the VL of an antibody according to the invention and / or an amino acid sequence constituting the VH of the antibody (e.g., the light chain and / or the heavy chain of an antibody according to the invention). In certain embodiments, one or more plasmids (e.g., expression plasmids) comprising such nucleic acid molecules are provided. In certain embodiments, host cells comprising such nucleic acid molecules are provided. In a preferred embodiment, the host cell comprises (e.g., has been transformed with): (1) a plasmid comprising a first nucleic acid molecule encoding an amino acid sequence comprising the VL of the antibody and a second nucleic acid molecule encoding an amino acid sequence comprising the VH of the antibody, or (2) a first plasmid comprising a nucleic acid molecule encoding an amino acid sequence comprising the VL of the antibody and a second plasmid comprising a nucleic acid molecule encoding an amino acid sequence comprising the VH of the antibody. In certain embodiments, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell). In certain embodiments, a method of producing an anti-human A-beta protein antibody is provided, comprising culturing a host cell comprising one or more nucleic acid molecules encoding an antibody as provided above under conditions suitable for expression of the antibody, and, optionally, recovering the antibody from the host cell (or host cell culture medium).

[0328] For recombinant production of anti-human A-beta protein antibodies, for example, nucleic acid molecules encoding the antibodies as 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 easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody).

[0329] Suitable host cells for cloning or expressing nucleic acid molecules encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in appropriate fractions and may be further purified.

[0330] In addition to prokaryotes, eukaryotic organisms such as filamentous fungi and yeast are suitable as cloning or expression hosts for antibody-encoding vectors, including bacterial and yeast strains that have been "humanized" in their glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0331] Suitable host cells for the expression of (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that may be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0332] Plant cell cultures can also be utilized as hosts. See, for example, U.S. Patent No. 5,959,177, U.S. Patent No. 6,040,498, U.S. Patent No. 6,420,548, U.S. Patent No. 7,125,978, and U.S. Patent No. 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). (商標) See, for example, US Pat. No. 6,213,945, which is incorporated herein by reference in its entirety.

[0333] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney lines (e.g., 293 cells or 293 cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74, baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., Mather, JP et al., Annals of NY Acad. Sci. 383 (1982) 44-68), MRC5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cells 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.

[0334] Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the anti-human A-beta protein antibodies according to the present invention are useful for detecting the presence of A-beta protein in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue.

[0335] In certain embodiments, an anti-human A-beta protein antibody according to the present invention is provided for use in a method of diagnosis or detection. In certain embodiments, a method for detecting the presence of A-beta protein in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-human A-beta protein antibody according to the present invention under conditions permissive for binding of the anti-human A-beta protein antibody to the A-beta protein, and detecting whether a complex is formed between the anti-human A-beta protein antibody and the A-beta protein. Such a method may be an in vitro or in vivo method.

[0336] In certain embodiments, a labeled anti-human A-beta protein antibody according to the present invention is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent, chromophoric, electron-dense, chemiluminescent and radioactive labels) and moieties that are indirectly detected, for example, by enzymatic reaction or molecular interaction (e.g., enzymes or ligands). Exemplary labels include the radioisotopes 32P, 14C, 125I, 3H, and 131I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosidase, galact ... These include, but are not limited to, glycoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0337] Pharmaceutical preparations Pharmaceutical formulations of anti-human A-β antibodies according to the present invention are prepared by mixing such antibodies having the desired purity with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed.) (1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as low molecular weight (less than about 10 residues) polypeptides, 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., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®, Baxter International, Inc.). Some exemplary sHASEGPs, including rhuPH20, and methods of use are described in US 2005 / 0260186 and US 2006 / 0104968. In certain embodiments, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0338] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine acetate buffer.

[0339] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the purpose intended.

[0340] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed.) (1980).

[0341] Sustained release preparations may also be prepared.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. US3773919), copolymers of L-glutamic acid and γ-L-ethyl glutamate, 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.

[0342] Formulations to be used for in vivo administration are generally sterile. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes. In one embodiment, the formulation is isotonic.

[0343] Therapeutic Methods and Compositions Any of the anti-human A-beta protein antibodies according to the invention may be used in therapeutic methods.

[0344] In one aspect, an anti-human A-beta protein antibody according to the invention is provided for use as a medicament. In a further aspect, an anti-human A-beta antibody according to the invention is provided for use in the prevention and / or treatment of a disease associated with amyloidogenesis and / or amyloid plaque formation. In a particular embodiment, an anti-human A-beta protein antibody according to the invention is provided for use in a method of treatment. In a particular embodiment, an anti-human A-beta protein antibody according to the invention is provided for use in a method of treating an individual having a disease associated with amyloidogenesis and / or amyloid plaque formation, comprising administering to the individual an effective amount of an anti-human A-beta protein antibody according to the invention. In a particular embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those listed below, or an anti-pTau or anti-alpha-synuclein antibody. In a further embodiment, an anti-human A-beta protein antibody according to the invention is provided for use in inhibiting plaque formation and / or the breakdown of beta-amyloid plaques. In a particular embodiment, there is provided herein an anti-human A-beta protein antibody according to the invention for use in a method of inhibiting formation of plaques and / or disruption of β-amyloid plaques in an individual, comprising administering to the individual an effective amount of an anti-human A-beta protein antibody according to the invention to inhibit formation of plaques and / or disrupt β-amyloid plaques. An "individual" according to any of the above embodiments is preferably a human.

[0345] In a further aspect, there is provided herein the use of an anti-human A-beta antibody according to the invention in the manufacture or preparation of a medicament. In certain embodiments, the medicament is for treating a disease associated with amyloidogenesis and / or amyloid plaque formation. In certain embodiments, the medicament is for use in a method of treating a disease associated with amyloidogenesis and / or amyloid plaque formation, comprising administering an effective amount of the medicament to an individual having a disease associated with amyloidogenesis and / or amyloid plaque formation. In certain 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 an anti-pTau or anti-alpha synuclein antibody. In certain embodiments, the medicament is for inhibiting the formation of plaques and / or the breakdown of β-amyloid plaques. In certain embodiments, the medicament is for use in a method of inhibiting the formation of plaques and / or the breakdown of β-amyloid plaques in an individual, comprising administering to the individual an amount of the medicament effective to inhibit the formation of plaques and / or to break down β-amyloid plaques. An "individual" according to any of the above embodiments may be a human.

[0346] In a further aspect, provided herein is a method for treating a disease associated with amyloidogenesis and / or amyloid plaque formation. In certain embodiments, the method comprises administering to an individual having a disease associated with amyloidogenesis and / or amyloid plaque formation an effective amount of an anti-human A-beta protein antibody of the present invention. In certain 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 an anti-pTau or anti-alpha-synuclein antibody. An "individual" according to any of the above embodiments may be a human.

[0347] In a further aspect, provided herein is a method of inhibiting plaque formation and / or disrupting β-amyloid plaques in an individual. In certain embodiments, the method comprises administering to the individual an effective amount of an anti-human A-β protein antibody according to the invention to inhibit plaque formation and / or disrupt β-amyloid plaques. In one embodiment, the "individual" is a human.

[0348] In a further aspect, provided herein is a pharmaceutical formulation comprising any of the anti-human A-beta protein antibodies according to the invention, e.g., for use in any of the above-mentioned therapeutic methods. In certain embodiments, the pharmaceutical formulation comprises any of the anti-human A-beta protein antibodies according to the invention and a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical formulation comprises any of the anti-human A-beta protein antibodies according to the invention and at least one additional therapeutic agent, e.g., as set forth below, or an anti-pTau or anti-alpha-synuclein antibody.

[0349] The antibody according to the present invention can be used alone or in combination with other agents in treatment.For example, the antibody according to the present invention can be co-administered with at least one additional therapeutic agent.In certain embodiments, the additional therapeutic agent is a therapeutic agent that is effective in treating the same or different neurological disorder that the antibody according to the present invention is used to treat. Exemplary additional therapeutic agents include, but are not limited to, the various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, lovastigmine, and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, gamma-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPAR gamma agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid beta peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-amyloid beta peptide antibodies. In certain embodiments, at least one additional therapeutic agent is selected for its ability to reduce one or more side effects of the neurological drug.

[0350] Such combination therapy as described above encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of the antibody reported herein can occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In certain embodiments, administration of the anti-human A-beta antibody according to the present invention and administration of the additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five or six days of each other. The antibody according to the present invention can also be used in combination with other interventional therapies, such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and suitable for the neurological disorder being treated or prevented.

[0351] The antibodies (and any additional therapeutic agents) according to the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0352] The antibodies according to the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of the antibodies according to the invention present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and by any route of administration as described herein, or about 1-99% of the dosages described herein, or in any dosage and by any route empirically / clinically determined to be appropriate.

[0353] Lipid-based methods of transporting an antibody according to the invention or a fusion construct comprising an antibody according to the invention across the BBB include, but are not limited to, encapsulating the antibody or fusion construct in a liposome coupled to a monovalent binding entity that binds to a receptor on the vascular endothelium of the BBB (see, e.g., US2002 / 0025313) and coating low density lipoprotein particles (see, e.g., US2004 / 0204354) or apolipoprotein E (see, e.g., US2004 / 0131692) with a monovalent binding entity.

[0354] For the prevention or treatment of disease, the appropriate dosage 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 being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous 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 suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg-15 mg / kg (e.g., 0.5 mg / kg to 10 mg / kg) of antibody can be an initial candidate dose for administration to the patient, whether by one or more individual administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. In repeated administrations over several days or more, depending on the symptoms, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example every week or every three weeks (e.g., so that the patient receives from about 2 to about 20, or for example about 6 doses of the antibody). An initial higher loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0355] It will be understood that any of the above formulations or methods of treatment may be practiced using an immunoconjugate as described herein instead of, or in addition to, an anti-human A-beta protein antibody according to the invention.

[0356] manufactured goods In another aspect according to the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The article of manufacture comprises a container and a label or package insert inserted into or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition by itself or in combination with another composition effective for treating, preventing and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody according to the invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may comprise (a) a first container in which a composition comprising an antibody according to the invention is contained; and (b) a second container in which a composition comprising an additional cytotoxic or other therapeutic agent is contained. The article of manufacture in this embodiment may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0357] It will be understood that any of the above articles of manufacture may include an immunoconjugate as described herein instead of, or in addition to, an antibody according to the invention.

[0358] The following figures, sequences and examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention. [Brief description of the drawings]

[0359] [Figure 1] HIC chromatogram of gantenerumab. [Diagram 2] HIC chromatogram of mAb663. [Diagram 3] HIC chromatogram of mAb675. [Figure 4] In vitro A-β plaque decoration by gantenerumab. [Diagram 5] In vitro A-β plaque decoration by mAb675. [Figure 6] In vitro A-β plaque decoration by mAb663. [Figure 7] In vitro A-β plaque decoration by mAb651. [Figure 8] Results of A-β plaque decoration in vivo. [Figure 9] Results of A-β plaque decoration in vivo. [Figure 10] Pharmacokinetic results.

[0360] Array Description [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] EXAMPLES

[0361] Example 1 Materials and General Methods General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains are numbered and referenced according to the numbering according to Kabat (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0362] Recombinant DNA Technology Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturers' instructions.

[0363] Gene synthesis The desired gene segments were prepared from oligonucleotides made by chemical synthesis. Long gene segments flanked by single restriction endonuclease cleavage sites were constructed by annealing and ligating oligonucleotides, including PCR amplification, and then cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered from Geneart (Regensburg, Germany) according to the given specifications.

[0364] DNA sequencing DNA sequences were determined by double-stranded sequencing, which was performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

[0365] DNA and protein sequence analysis and sequence data management The GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 were used for sequence construction, mapping, analysis, annotation and illustration.

[0366] Expression vector For expression of the antibodies according to the invention, expression plasmids for transient expression (e.g. in HEK293 cells) can be applied, either based on a cDNA construction with or without the CMV-intron A promoter or on a genomic construction with the CMV promoter.

[0367] In addition to the antibody expression cassette, the vector contains: an origin of replication allowing replication of this plasmid in E. coli, -β-lactamase gene that confers ampicillin resistance in E. coli Contains:

[0368] The transcription unit for an antibody gene contains the following elements: - Unique restriction site at the 5' end - the immediate early gene and promoter from human cytomegalovirus, - intron A sequence in the case of a cDNA construction, - a 5'-untranslated region derived from a human antibody gene, - immunoglobulin heavy chain signal sequence, - nucleic acids encoding each antibody chain, either as cDNA or in a genomic exon-intron configuration; - a 3' untranslated region carrying a polyadenylation signal sequence, - Unique restriction site at the 3' end It consists of:

[0369] Fusion genes encoding antibody chains are generated by PCR and / or gene synthesis and assembled by known recombinant methods and techniques, e.g., by joining matching nucleic acid segments using unique restriction sites in each vector. Subcloned nucleic acid sequences are confirmed by DNA sequencing. For transient transfection, large quantities of plasmid are prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

[0370] Cell culture technology Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc. are used.

[0371] Transient transfection in the HEK293-F system Antibodies were produced by transient expression. Therefore, transfection with the respective plasmids was performed using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle™ 293 Expression Medium (Invitrogen) in either shake flasks or stirred fermenters were transfected with a mix of the respective expression plasmids and 293fectin™ or fectin (Invitrogen). For 2L shake flasks (Corning), HEK293-F cells were seeded at a density of 1.0*1E6 cells / mL in 600 mL and incubated at 120 rpm and 8% CO2. The next day, at a cell density of approximately 1.5*1E6 cells / mL, cells are transfected with a mixture of approximately 42 mL of 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 293fectin or fectin (2 µL / mL). Glucose solution is added during the course of fermentation according to glucose consumption. The supernatant containing the secreted antibody is harvested after 5-10 days and the antibody is either purified directly from the supernatant or the supernatant is frozen and stored.

[0372] Protein determination Protein concentrations of purified antibodies and derivatives were determined by determining the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science 4 (1995) 2411-1423.

[0373] Determination of antibody concentration in the supernatant The concentration of antibodies in cell culture supernatants was estimated by immunoprecipitation with protein A agarose beads (Roche Diagnostics GmbH, Mannheim, Germany). Therefore, 60 μL of protein A agarose beads were washed three times with TBS-NP40 (150 mM Tris buffer, pH 7.5 supplemented with 50 mM NaCl and 1% Nonidet-P40). Then, 1–15 mL of cell culture supernatant was applied to the protein A agarose beads pre-equilibrated in TBS-NP40. After 1 h of incubation at room temperature, the beads were briefly washed once with 0.5 mL TBS-NP40, twice with 0.5 mL 2x phosphate-buffered saline (2x PBS, Roche Diagnostics GmbH, Mannheim, Germany) and four times with 0.5 mL 100 mM Na-citrate buffer (pH 5.0) on an Ultrafree-MC-filter column (Amicon). Bound antibodies were eluted by adding 35 μL of NuPAGE® LDS Sample Buffer (Invitrogen). Half of the sample was combined with NuPAGE® Sample Reducing Agent or left unreduced, respectively, and heated at 70° C. for 10 min. 5–30 μl was then applied to 4–12% NuPAGE® Bis-Tris SDS-PAGE gels (Invitrogen) (containing MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidant Running Buffer Additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.

[0374] The concentration of antibodies in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies that bind 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 antibody was used as a standard.

[0375] Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96-well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) were filled with 100 μL / well of biotinylated anti-human IgG capture molecule F(ab')2<h-Fcγ> Plates were coated with BI (Dianova) at 0.1 μg / mL for 1 h at room temperature or overnight at 4°C, followed by washing 3 times with 200 μL / well PBS, 0.05% Tween (PBST, Sigma). 100 μL / well of a dilution series of each antibody-containing cell culture supernatant in PBS (Sigma) was then added to the wells and incubated for 1-2 h on a shaker at room temperature. Wells were washed 3 times with 200 μL / well PBST, and bound antibodies were deconvoluted by adding 100 μL F(ab')2 at 0.1 μg / mL as detection antibody by incubating for 1-2 h on a shaker at room temperature.<hFcγ> Detection was performed using POD (Dianova). Unbound detection antibody was removed by washing three times with 200 μL / well PBST. Bound detection antibody was detected by adding 100 μL ABTS / well followed by incubation. Absorbance determination was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).

[0376] Preparative antibody purification Antibodies were purified from the filtered cell culture supernatants with reference to standard protocols. Briefly, the antibodies were applied to a Protein A Sepharose column (GE healthcare) and washed with PBS. Elution of the antibodies was achieved at pH 2.8 and immediately neutralized. Aggregated proteins were separated from monomeric antibodies by size-exclusion chromatography (Superdex 200, GE Healthcare) in PBS or 20 mM histidine buffer (pH 6.0) containing 150 mM NaCl. Monomeric antibody fractions were pooled, concentrated (if necessary) using, for example, MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrators, frozen, and stored at -20°C or -80°C. Part of the sample was submitted for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, size-exclusion chromatography (SEC) or mass spectrometry.

[0377] SDS-PAGE The NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions, specifically, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4), and NuPAGE® MES (reducing gels, containing NuPAGE® antioxidant running buffer additive) or MOPS (non-reducing gels) running buffer.

[0378] CE-SDS Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Therefore, 5 μl of antibody solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according to the manufacturer's instructions and analyzed on a LabChip GXII system using the HT Protein Express Chip. Data was analyzed using LabChip GX Software.

[0379] Analytical Size Exclusion Chromatography Size exclusion chromatography (SEC) for the determination of antibody aggregation and oligomeric state was performed by HPLC chromatography. Briefly, Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4 / K2HPO4 buffer (pH 7.5) on a Dionex Ultimate® system or to a Superdex 200 column (GE Healthcare) in 2×PBS on a Dionex HPLC-System. Eluted antibodies were quantified by UV absorbance and peak area integration. BioRad Gel Filtration Standard 151-1901 served as the standard.

[0380] mass spectrometry In this section, the characterization of bispecific antibodies is described with emphasis on their correct assembly. The predicted primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of deglycosylated intact antibodies and, in a special case, of deglycosylated / limited LysC digested antibodies.

[0381] Antibodies were deglycosylated with N-glycosidase F in phosphate or Tris buffer at a protein concentration of 1 mg / ml for up to 17 h at 37° C. Restriction LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestion was performed with 100 μg of deglycosylated antibody in Tris buffer (pH 8) for 120 h at room temperature or 40 min at 37° C., respectively. Prior to mass spectrometry, samples were desalted by HPLC on a Sephadex G25 column (GE Healthcare). Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0382] Chemical Decomposition Test Samples were divided into three aliquots, rebuffered in 20 mM His / His*HCl, 140 mM NaCl, pH 6.0 or PBS, respectively, and stored at 40° C. (His / NaCl) or 37° C. (PBS). Control samples were stored at −80° C.

[0383] After the incubation was completed, samples were analyzed for relative activity concentration (BIAcore), aggregation (SEC) and fragmentation (capillary electrophoresis or SDS-PAGE) and compared to untreated controls.

[0384] thermal stability Samples were prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine chloride, 140 mM NaCl, pH 6.0, centrifuged through a 0.4 μm filter plate, transferred to an optical 384-well plate, and overlaid with paraffin oil. Hydrodynamic radii were repeatedly measured by dynamic light scattering on a DynaPro plate reader (Wyatt) as samples were heated from 25°C to 80°C at a rate of 0.05°C / min.

[0385] Alternatively, samples were transferred to a 10 μL microcuvette array and heated from 25°C to 90°C at a rate of 0.1°C / min while static light scattering data, as well as fluorescence data upon excitation with a 266 nm laser, were recorded using an Optim1000 instrument (Avacta Inc.).

[0386] The onset of aggregation temperature is defined as the temperature at which the hydrodynamic radius (DLS) or scattered light intensity (Optim1000) begins to increase.

[0387] Alternatively, samples were transferred to a 9 μL multicuvette array. The multicuvette array was heated at a constant rate of 0.1° C. / min from 35° C. to 90° C. in an Optim1000 instrument (Avacta Analytical Inc.). The instrument continuously records the scattered light intensity of a 266 nm laser with data points approximately every 0.5° C. The light scattering intensity was plotted against temperature. The aggregation onset temperature (T agg) is defined as the temperature at which the scattered light intensity begins to increase.

[0388] The melting point is defined as the inflection point on a graph of fluorescence intensity versus wavelength.

[0389] mouse B6.Cg-Fcgrt, which lacks the mouse FcRn chain gene but is hemizygous transgenic for the human FcRn chain gene tm1Dcr Tg(FCGRT)276Dcr mice (muFcRn- / -huFcRn tg+ / -, line 276) were used for the 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 the time of dosing). All animal experiments were approved by the Government of Upper Bavaria, Germany (permit number 55.2-1-54-2532.2-28-10) and were performed in an AAALAC-accredited animal facility in accordance with the European Union Normative for Care and Use of Experimental Animals. Animals were housed in standard cages and had free access to food and water throughout the entire study.

[0390] Pharmacokinetic studies A single dose of antibody was injected iv via a lateral tail vein at a dose level of 10 mg / kg. Mice were divided into three groups of six mice each to cover a total of nine serum collection time points (0.08, 2, 8, 24, 48, 168, 336, 504 and 672 hours post-dosing). Each mouse was subjected to two retro-orbital bleeds performed under light anesthesia with Isoflurane™ (CP-Pharma GmbH, Burgdorf, Germany) and a third blood sample was collected at the time of euthanasia. Blood was collected in serum tubes (Microvette 500Z-Gel, Sarstedt, Numbrecht, Germany). After 2 h of incubation, samples were centrifuged at 9.300 g for 3 min to obtain serum. After centrifugation, serum samples were stored frozen at -20°C until analysis.

[0391] PK analysis Pharmacokinetic parameters were calculated by noncompartmental analysis using WinNonlin™ 1.1.1 (Pharsight, CA, USA).

[0392] Briefly, the area under the curve (AUC) was calculated using the logarithmic trapezoidal method due to the nonlinear decrease in the antibody. 0-inf ) values ​​were calculated and extrapolated to infinity using the apparent terminal rate constant, λz, using an extrapolation from the concentration observed at the last time point.

[0393] Dose rate (D) vs. AUC 0-inf Plasma clearance was calculated as the plasma concentration of the drug divided by the plasma concentration of the drug. The apparent elimination half-life (T1 / 2) was derived from the equation T1 / 2 = ln2 / λz.

[0394] Example 2 Expression and purification Antibodies were generated as described above in the general materials and methods section.

[0395] The antibody was purified from the supernatant by a combination of Protein A affinity and size exclusion chromatography. The resulting product was characterized for attributes by mass spectrometry and analytical properties such as purity, monomer content and stability by CE-SDS.

[0396] The predicted primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact antibody and the deglycosylated / plasmin digested antibody or alternatively the deglycosylated / restricted LysC digested antibody as described in the general methods section.

[0397] Additional analytical methods (eg, thermostability, mass spectrometry and functional assessment) were applied only after Protein A and SEC purification.

[0398] Example 3 Determination of binding to Aβ1-40 fibrils in vitro by ELISA Binding of antibodies to fibrillar Aβ is measured by ELISA assay. Briefly, Aβ(1-40) is coated on Maxisorb plates at 7 μg / mL in PBS for 3 days at 37° C. to generate fibrillar Aβ, then dried at room temperature for 3 hours. Plates are blocked with 1% Crotein C and 0.1% RSA in PBS (blocking buffer) for 1 hour at room temperature, then washed once with washing buffer. Antibodies or controls are added at concentrations up to 100 nM in blocking buffer and incubated overnight at 4° C. After 4 washing steps, constructs are detected by addition of anti-human-IgG-HRP (Jackson Immunoresearch) at a 1:10,000 dilution in blocking buffer (1 RT), followed by 6 washes and incubation in TMB (Sigma). After stopping color development with 1N HCl, absorbance is read at 450 nm.

[0399] Example 4 Staining of natural human β-amyloid plaques from brain sections of Alzheimer's disease patients by indirect immunofluorescence using the antibody according to the invention Antibodies can be tested for their ability to stain native human β-amyloid plaques by immunohistochemical analysis using indirect immunofluorescence. Specific and sensitive staining of true human β-amyloid plaques can be demonstrated. Cryostat sections of unfixed tissue from the temporal cortex obtained postmortem from patients positively diagnosed with Alzheimer's disease are labeled by indirect immunofluorescence. A two-step incubation is used to detect bound bispecific antibodies revealed by affinity purified goat anti-human (GAH555) IgG(H+L) conjugated to Alexa555 dye (Molecular Probes). Controls can include an irrelevant human IgG1 antibody (Sigma) and secondary antibodies alone, all of which should give negative results.

[0400] Example 5 In vivo β-amyloid plaque decoration by antibodies according to the invention in a mouse model of Alzheimer's disease The antibodies were tested for their ability to immuno-decorate β-amyloid plaques in vivo in APP / PS2 double transgenic mice (Richards, J. Neuroscience, 23 (2003) 8989-9003), a mouse model of AD-associated amyloidosis. This allowed assessment of the extent of brain penetration and binding to amyloid-β plaques. Antibodies were administered at different doses, and 6 days later the animals were perfused with phosphate-buffered saline and the brains were frozen on dry ice and prepared for cryosectioning.

[0401] The presence of antibodies bound to β-amyloid plaques was assessed on unfixed cryostat sections by either single-label indirect immunofluorescence using goat anti-human IgG (H+L) conjugated to Alexa555 dye (GAH555) (Molecular Probes) at a concentration of 15 μg / ml for 1 h at room temperature. Counterstaining of amyloid plaques can be performed by incubation with BAP-2, a mouse monoclonal antibody against Aβ conjugated to Alexa488 at a concentration of 0.5 μg / ml for 1 h at room temperature. Slides are embedded in fluorescent mounting medium (S3023 Dako) and imaged by confocal laser scanning microscopy.

[0402] Example 6 Affinity chromatography of FcRn and heparin Preparation of FcRn affinity column Expression of FcRn in HEK293 cells FcRn was transiently expressed by transfection of two plasmids containing the coding sequences of FcRn and β-2-microglobulin into HEK293 cells. The transfected cells were cultured in shaker flasks at 36.5°C, 120 rpm (shaker amplitude 5 cm), 80% humidity and 7% CO2. The cells were diluted every 2-3 days to a density of 3-4*105 cells / ml.

[0403] For transient expression, a 14 l stainless steel bioreactor was started with a culture volume of 8 l at 36.5 °C, pH 7.0 ± 0.2, pO2 35% (gassing with N2 and air, total gas flow 200 ml min-1) and agitator 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, which was then incubated at room temperature for 15 min before being transferred to the fermenter. From the next day, the cells were fed in continuous mode. Feed solution was added at a rate of 500 ml / day and glucose was added as necessary to keep the level higher than 2 g / l. The supernatant was harvested 7 days after transfection using a swing-head centrifuge with 1 l bucket at 4000 rpm for 90 min. The supernatant (13 L) was removed by a Sartobran P filter (0.45 μm+0.2 μm, Sartorius) and the FcRnβ-2-microglobulin complex was purified therefrom.

[0404] Biotinylation of neonatal Fc receptors 3 mg of FcRnβ-2-microglobulin complex was dissolved / diluted in 5.3 mL of 20 mM sodium dihydrogen phosphate buffer containing 150 mM sodium chloride and added to 250 μL of PBS and 1 tablet complete protease inhibitor (complete ULTRA Tablets, Roche Diagnostics GmbH). FcRn was biotinylated using a biotinylation kit from Avidity according to the manufacturer's instructions (Bulk BIRA, Avidity LLC). The biotinylation reaction was carried out overnight at room temperature.

[0405] The biotinylated FcRn was dialyzed overnight at 4° C. against 20 mM MES buffer containing 140 mM NaCl, pH 5.5 (buffer A) to remove excess biotin.

[0406] Coupling to streptavidin sepharose For conjugation to streptavidin sepharose, 1 mL of streptavidin sepharose (GE Healthcare, United Kingdom) was added to the biotinylated and dialyzed FcRnβ-2-microglobulin complex and incubated overnight at 4° C. The FcRnβ-2-microglobulin complex-derivatized sepharose was packed into a 4.6 mm×50 mm chromatography 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).

[0407] Chromatography using FcRn affinity columns and pH gradients conditions: Column dimensions: 50mm x 4.6mm Loading: 30μg of sample Buffer A: 20 mM MES, 140 mM NaCl, pH adjusted to 5.5 Buffer B: 20 ​​mM Tris / HCl, 140 mM NaCl, adjusted to pH 8.8

[0408] 30 μg of sample was applied to an FcRn affinity column equilibrated with buffer A. After a 10 min washing step in 20% buffer B at a flow rate of 0.5 mL / min, elution was performed with a linear gradient of 20% to 70% buffer B over 70 min. UV light absorption at a wavelength of 280 nm was used for detection. After each run the column was regenerated with 20% buffer B for 10 min.

[0409] For the calculation of relative retention times, a standard sample (anti-Her3 antibody (SEQ ID NO: 52 and 53) oxidized for 18 hours with 0.02% hydrogen peroxide according to (Bertoletti-Ciarlet, A., et al., Mol. Immunol. 46 (2009) 1878-1882) was run at the start of the sequence and after each of the 10 sample injections.

[0410] Briefly, antibodies (9 mg / mL) in 10 mM sodium phosphate pH 7.0 were mixed with H2O2 to a final concentration of 0.02% and incubated for 18 h at room temperature. To quench the reaction, samples were extensively dialyzed into pre-chilled 10 mM sodium acetate buffer pH 5.0.

[0411] The relative retention time was calculated according to the following formula: t_(rel,i)=(t_i-t_peak2) / (t_peak3-t_peak2)

[0412] For peak definitions, please see Figure 1 of WO 2018 / 197533.

[0413] Chromatography using heparin affinity columns and pH gradients conditions: Column dimensions: 50mm x 5.0mm Loading: 20-50μg sample Buffer A: 50 mM TRIS pH 7.4 Buffer B: 50 mM TRIS pH 7.4, 1000 mM NaCl

[0414] Protein samples of 20-50 μg in low-salt buffer (ionic strength of ≦25 mM) were applied to a TSKgel Heparin-5PW Glass column, 5.0 × 50 mm (Tosoh Bioscience, Tokyo / Japan) pre-equilibrated with buffer A at room temperature. Elution was performed with a linear gradient of 0-100% buffer B over 32 min at a flow rate of 0.8 mg / mL. UV light absorption at a wavelength of 280 nm was used for detection. Every injection sequence was initiated with retention time standards (anti-pTau antibody; SEQ ID NOs: 50 and 51) which were used to calculate relative retention times according to the following formula: t_(rel,i)=t_i / t_pTau (trel,i: relative retention time of peak i; ti: retention time of peak i; tpTau: retention time of the anti-pTau antibody peak).

[0415] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the illustrations and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. An antibody that binds to a human A-beta protein, comprising a CDR selected from: （１） (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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) 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; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; (d) 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) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; (b) 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) 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) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

91. and a light chain variable domain comprising:

2. 10. The antibody of claim 1, having the following characteristics: - specifically binds to the human A-beta protein of SEQ ID NO: 45; - have a glycosylation site in said heavy chain CDR2 with a carbohydrate occupancy of at least 95% as determined by CE-SDS; - does not include species with any non-glycosylated glycosylation sites; - SEQ ID NO: 45 has an EC50 value for said human A-beta protein of 0.5 nM or less; - binds to A-β plaques in vitro and does not bind to non-A-β protein molecules in brain samples at a staining concentration of 1 μg / mL or less; - Binds to A-β plaques in vivo more avidly than gantenerumab; - relative in vivo occupancy of plaques in the cortex and hippocampus >0.2 as determined by one-way Anova; Thermal stability above -68°C (DLS T ａｇｇ / DLS T ｍ ) An antibody having one or more of the following:

3. 3. The antibody of claim 1 or 2, which is a monoclonal antibody.

4. The antibody according to any one of claims 1 to 3, which is an antibody fragment that binds to human A-β protein.

5. The antibody according to any one of claims 1 to 4, (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 defined in (a) and a VL sequence defined in (b); An antibody comprising:

6. An antibody according to any one of claims 1 to 5, comprising a VH sequence of SEQ ID NO: 84 and a VL sequence of SEQ ID NO:

80.

7. The antibody according to any one of claims 1 to 6, wherein the antibody comprises: a) a full-length antibody of human subclass IgG1; b) a full-length antibody of human subclass IgG4; c) a full-length antibody of human subclass IgG1 with the mutations L234A, L235A and P329G; d) a full-length antibody of human subclass IgG1 with the mutations L234A, L235A and P329G in both heavy chains, and with the mutations T366W and S354C in one heavy chain and the mutations T366S, L368A, Y407V and Y349C in the respective other heavy chain; e) a full-length antibody of human subclass IgG1 with the mutations L234A, L235A and P329G in both heavy chains, with the mutations T366W and Y349C in one heavy chain and the mutations T366S, L368A, Y407V and S354C in the respective other heavy chain; f) a full-length antibody of human subclass IgG4, having 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 human subclass IgG4, carrying 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 human subclass IgG1 with the mutations L234A, L235A, P329G, I253A, H310A and H435A in both heavy chains, and with 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 human subclass IgG1 with the mutations L234A, L235A, P329G, I253A, H310A and H435A in both heavy chains, with 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 human subclass IgG1 with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains, and with 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 human subclass IgG1 with the mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains, and with 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 human subclass IgG1 having 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) An antibody which is one of a) to l) and which does not contain a C-terminal lysine residue.

8. 8. The antibody of claim 1, comprising a heavy chain comprising a heavy chain variable domain of SEQ ID NO: 84 and a heavy chain constant region of SEQ ID NO: 01, and a light chain comprising a light chain variable domain of SEQ ID NO: 80 and a light chain kappa constant domain of SEQ ID NO:

29.

9. 8. The antibody of claim 1 , comprising a heavy chain comprising a heavy chain variable domain of SEQ ID NO: 84 having a pyroglutamic acid (pE) residue instead of a glutamine (Q) residue as the first N-terminal amino acid residue and a heavy chain constant region of SEQ ID NO: 01 , and a light chain comprising a light chain variable domain of SEQ ID NO: 80 and a light chain kappa constant domain of SEQ ID NO:

29.

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

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

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

13. 1. A method for producing an antibody that binds to human A-β protein, comprising: - culturing a host cell according to claim 12 in a culture medium, - recovering the antibody from the host cells and / or the culture medium, and - purifying said antibody using one or more chromatography steps The method includes:

14. A pharmaceutical composition comprising the antibody of any one of claims 1 to 10 and a pharma- ceutically acceptable carrier.

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

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